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					Procise™/Procise cLC
Protein Sequencing System
Service Troubleshooting Manual
January 1997
PE Applied Biosystems




                                                            This product is not for use
                                                            in diagnostic procedures.




Aquapore, Brownlee, INHERIT, MicroBlotter, MicroCoat, MPLC, NEWGUARD, OPC, Perkin-Elmer, POLYPORE, Precipi-
tette, ProBlott, ProSorb, ProSort, ProSpin, SeqEd, SPHERI10, SPHERI5, SynthAssist, and VeloSep are registered trademarks
of The Perkin-Elmer Corporation. ABI PRISM, ABI, Amplicover, Anitron, Applied Biosystems, AutoAssembler, BaseSprinter,
Biobytes, CATALYST, FastPhoramidite, GeneAssist, GeneScan, Genotyper, HLP, Hot Start, Masterpiece, ONESTEP, PCR-
MATE, PDQ, Phosphalink, PROCISE, ProFocus, Sequence Navigator, StockMarks, Stretch, and Synergy are trademarks of
The Perkin-Elmer Corporation.
All other trademarks are the sole property of their respective owners.


©Copyright 1996, The Perkin-Elmer Corporation
PE Applied Biosystems




                          Contents
                        1 About this manual                                                  1-1
                              Models covered                                                 1-1
                              Using this manual                                              1-1

                        2 Troubleshooting                                                    2-1
                          Troubleshooting Table                                              2-1
                          Troubleshooting Delivery Problems using Fluid Sensor Data Files   2-37
                              Fluid Sensor Data Files                                       2-37
                              Recording Fluid Sensor data                                   2-37
                              Valve Status File                                             2-38
                              Opening Fluid Sensor & Valve Status Files                     2-39
                              Opening Sensor Data Files using Teachtext                     2-39
                              Opening Fluid Sensor Data Files using Excel 5                 2-40
                              Interpreting Fluid Sensor Data File Information               2-41
                              Examples of Problem Deliveries                                2-42
                              Cartridge Load, Cartridge Outlet & Flask Load Sensors         2-42
                              Transfer Sensor                                               2-44
                              Sample Loop Load & Full Sensors                               2-46
                              Causes of Delivery Problems                                   2-49

                        3 Routine Maintenance                                               3-11
                          Procise Planned Maintenance Checklist                             3-11

                        4 Procedures                                                         4-1
                          Pressure System                                                    4-1
                               The Power-On Selftest                                         4-1
                               Testing the Control Feedback System                           4-1
                               Testing the 0-5 psi Pressure Transducers                      4-1
                               Leak Testing the Pressure Board                               4-2
                               Replacing a Lee Valve on the Pressure Board                   4-2
                               Replacing a Pressure Transducer                               4-3
                               Replacing the Pressure Board                                  4-4
                          Delivery Valve System                                              4-5
                               Testing Liquid FlowRates                                      4-5
                               Testing Gas Flowrates                                         4-6
                               Testing Valveblocks for Leakage to Common Path                4-8
                               Testing Valveblocks for Ruptured Diaphragms                   4-9
                               Testing 3-way Valves                                         4-11
                               Replacing Valveblocks                                        4-11



January 1997                                                                                       i
PE Applied Biosystems




                             Replacing Teflon Lines in the Waste Manifold           4-12
                        Vacuum Assist System                                        4-14
                             Adjusting the Vacuum Switch                            4-14
                             Setting the Pressure Switch                            4-14
                             Cleaning the Clippard Valves                           4-15
                        Reaction Cartridge                                          4-16
                             Cleaning the Reaction Cartridge Glass Blocks           4-16
                        Conversion Flask                                            4-17
                             Cleaning the Conversion Flask                          4-17
                             Removing a stuck Conversion Flask Vial                 4-18
                        Optical Fluid Sensors                                       4-20
                             Reporting Sensor Information                           4-20
                             The Sensor & Delivery Test Procedure                   4-20
                        Heaters                                                     4-22
                             Testing the Heater Boards                              4-22
                             Testing for correct Temperature                        4-22
                             Replacing the Thermal Fuse                             4-23
                             Replacing a Heater Cartridge Board                     4-23
                             Replacing the Flask Heater Board                       4-24
                             Replacing the Column Oven Heater board                 4-25
                        Electronics                                                 4-26
                             Digital Controller Board DIP Switch configuration      4-26
                             Jumper configuration                                   4-26
                             Replacing the Digital Controller Board                 4-26
                             Replacing the MEL Card (Firmware)                      4-27
                             Replacing the I/O Board                                4-28
                             Replacing the Distribution Board                       4-29
                             Power Supply Configuration                             4-29
                             Replacing the Power Supply                             4-30
                        Chemistry and Chromatography                                4-31
                             Optimizing the PTH-Standard Separation                 4-31
                             Improving the Shape of the Baseline                    4-32
                             Test Pumping System using Run Gradient Method          4-33
                             Static Pressure Test                                   4-33
                             Specification                                          4-34
                             Dynamic Pressure Monitoring                            4-35
                             Calibrating the 140C FlowRate                          4-36
                             Replacing the Pump Seals (and Cylinders)               4-37
                             Adjusting the 140 Switching Valve                      4-42
                             Replacing the Pump Rotor Seals                         4-42
                             Phosphate Wash of Pump and Column                      4-44
                             Phosphoric Acid Wash of Pump                           4-45
                             785A Detector Dry Cell Test                            4-46



ii                                                                               January 1997
PE Applied Biosystems




                              785A Detector Wavelength Test (Visual)         4-47
                              Testing the Wavelength Drive Mechanism         4-47
                              Replacing the 785A Detector UV lamp            4-48
                              Replacing the 785A Detector Flowcell Windows   4-49
                              Manual Injection Procedure                     4-51
                              Testing the Injector for blockages             4-53
                              Replacing the Injector Rotor Seal              4-54
                          Macintosh Computer                                 4-56
                              Procedures using keystroke commands            4-56
                              Formatting the Hard Drive                      4-56

                        5 Diagnostics                                        5-1
                          Dialog Boxes                                        5-1
                               Procise Dialog Box Messages                    5-1
                               Model 610A Dialog Box Messages                 5-2
                               File already exists                            5-2
                               No data collected for 12 hours                 5-3
                               File error                                     5-3
                               Mac Operating System Dialog Box Messages       5-4
                          The Event Log                                       5-5
                               Opening the Event Log                          5-5
                                Event Log Messages                            5-7
                               On-line Error Messages                         5-8
                               Pass/Fail Messages with preset Criteria       5-11
                               Pass/Fail Messages with adjustable Criteria   5-13
                          Door Panel Visor Lights                            5-14
                               PROCISE Logo                                  5-14
                               READY LED                                     5-14
                               COM LED                                       5-15
                               SEQ LED                                       5-15
                               ERROR LED                                     5-15
                          Powering-up the Procise System                     5-16
                               Power-up sequence                             5-16
                               Procise Power-up Sequence of Events           5-16
                               Macintosh Computer power-up Sequence          5-17
                          System Selftest                                    5-18
                               Components Tested                             5-18
                               Performing the Electrical Test                5-18
                               Self-testing Individual Components            5-19
                          Selftest Descriptions                              5-20
                               CPU Board Tests                               5-20
                               Functional Tests                              5-20
                               Self-Test Results                             5-25


January 1997                                                                        iii
PE Applied Biosystems




                        Leak Test Procedures                     5-26
                             Leak Test Pressurization Paths      5-28




iv                                                            January 1997
PE Applied Biosystems




                        1 About this manual
                          Models covered
                          491, 492, 494, 610A V2.1, 140C, 785A
                          Supplemental manuals                                   Part Number
                          Procise user manual set                                  903079
                          Model 610A user manual                                   901423
                          Model 140C user manual                                   903078
                          Model 140C service manual                                903249
                          Model 785A user manual                                  1000-0547
                          Procise Theory of Operation manual                       904564
                          Procise Service Reference manual                         904565


                          Using this manual
                          This manual is intended for service personnel who have been specifically
                          trained by PE Applied Biosystems. PE Applied Biosystems is not liable for
                          damage or injury that results from use of this manual by unauthorized or
                          untrained parties. Such use may void any applicable warranties (see warranty
                          statement for limitations). Information contained in this manual is
                          proprietary information of PE Applied Biosystems.




November 1996             1 About this manual                                                       1-1
PE Applied Biosystems




                        2 Troubleshooting
                          Troubleshooting Table
                                                     Chemistry & Chromatography
                                                                                                 Chapter/Page
                          Baseline disturbances and anomalies
                          Additional peaks in Blank, Std. & Residue chromatograms anywhere        page 2-12
                          after injection artifact
                          Cycling baseline (low frequency)                                         page 2-6
                          Deflection (small) at consistent retention time in all chromatograms    page 2-10
                          Deflection (off scale) in chromatogram                                  page 2-10
                          Humps or dips on baseline                                               page 2-10
                          Noise (high frequency) on baseline                                       page 2-4
                          Noise (medium frequency) on baseline                                     page 2-7
                          Slope (negative-going) on which early eluting amino acids ride           page 2-9
                          Spikes on baseline                                                       page 2-6
                          Stepping of baseline intermittently in chromatograms                     page 2-5

                          Missing Peaks
                          All Residue cycles resemble a Blank chromatogram. Maybe some            page 2-12
                          aniline. No Event log errors
                          All Residue cycles resemble a Blank chromatogram. Transfer sensor       page 2-13
                          error in Event log
                          Deflection at about 6 minutes. Injection artifact. No other peaks.      page 2-13
                          Injector Full sensor error in Event log
                          Reduced number of peaks elute early in chromatogram. The peaks          page 2-13
                          are broad
                          Flat baseline with no injection artifact. No errors in Event log        page 2-12

                          Poor recovery, standard chromatogram
                          All peak heights reduced and peak width increased                       page 2-14
                          All peak heights reduced                                                page 2-14
                          All peak heights reduced intermittently                                 page 2-14
                          Low Lysine                                                              page 2-14
                          Low Lysine & PE Cys                                                     page 2-14

                          Poor recovery of residue amino acids
                          Low Ser & Thr                                                           page 2-15
                          Low Asn or Gln and high Asp & Glu respectively                          page 2-15
                          Low Glycine                                                             page 2-15
                          Low His & Arg                                                           page 2-15
                          Low Lysine                                                              page 2-14
                          Low Lysine & PE Cys                                                     page 2-14




January 1997              2 Troubleshooting                                                                     2-1
PE Applied Biosystems




                        Low repetitive yield
                        Low repetitive yield, high lag                                            page 2-16
                        Low repetitive yield, no lag                                              page 2-16

                        Artifact peaks
                        DTT-PITC adduct close to Pro                                              page 2-16
                        High aniline                                                              page 2-16
                        High oxidized DTT                                                         page 2-17

                        Retention time problems
                        All peaks, including injection artifact, delayed by same amount           page 2-19
                        General retention time instability during the run                         page 2-17
                        Most peaks miscalled even though peak retention times reasonably          page 2-17
                        stable
                        Only early eluting peaks miscalled                                        page 2-19
                        Retention time shift only in occasional cycles                            page 2-19
                        Standard or First residue retention times do not agree with rest of run   page 2-19

                                                          Event log messages
                        Cartridge load, cartridge outlet, flask load and transfer fluid sensor error messages
                        Fluid never reaches sensor (Average Wet = 0))                           page 2-21
                        Too many bubbles in liquid slug (Average Wet reading ≠ 0)               page 2-23

                        Injector sample loop fluid sensor errors
                        Sample loop full errors and no amino acid peaks                           page 2-25
                        Sample loop full errors with partial injection                            page 2-25
                        Sample loop full and load errors, no injection                            page 2-26

                        Other event log messages generated during a run
                        Argon tank pressure too low                                               page 2-27
                        Cannot reach set temperature                                              page 2-27
                        Communication with HPLC lost                                              page 2-27
                        Event buffer overrun                                                      page 2-27
                        Injector position error and no corresponding Sample loop sensor           page 2-28
                        errors
                        Insufficient data collection memory for cycle                             page 2-28
                        Invalid sensor Dry reading                                                page 2-28
                        Power failure                                                             page 2-29
                        Vacuum assist activated (more than once every 8 hours)                    page 2-29

                        Leak test error messages
                        Actual pressure above 5.5psi                                              page 2-29
                        All leak tests fail                                                       page 2-29
                        Cartridge leak test fails                                                 page 2-30
                        Failing vent test                                                         page 2-29
                        Flask failing vent test                                                   page 2-30
                        Leak test fails because pressure too high                                 page 2-30



2-2                     2 Troubleshooting                                                             January 1997
PE Applied Biosystems




                                               Software and communication problems
                        Lock-ups
                        Frequent Macintosh lock-ups                                           page 2-30
                        Macintosh locks up during data collection                             page 2-31
                        No communication between Macintosh and sequencer                      page 2-31

                        Procise, 610A and Macintosh OS errors
                        “No data has been collected for 12 hours” dialog box message          page 2-35
                        generated
                        610A does not print all cycles                                        page 2-35
                        610A Print Quality Poor                                               page 2-35
                        File already exists error generated by the 610A                       page 2-34
                        File error every time 610A is launched                                page 2-32
                        File missing error when Procise launched                              page 2-34
                        File or disk error every time Procise is launched. Usually [PROCISE   page 2-33
                        error-48]
                        Macintosh operating system consumes excessive RAM                     page 2-36

                                                 Pump and detector error messages
                        785A
                        Detector (785) beeping                                                page 2-36

                        140
                        Pump A fails to fill                                                  page 2-36




January 1997            2 Troubleshooting                                                                 2-3
PE Applied Biosystems




                        Chemistry & Chromatography
                        Baseline disturbances
                        Noise (high frequency) on baseline
                        High frequency noise is usually electronic noise since flow/mixing problems
                        produce a slower baseline response. As UV lamp energy reduces with age,
                        the electronic noise, which is always present at some level, is amplified.
                        Similarly, if a wrong wavelength is monitored at which the lamp energy is less
                        intense, the noise will increase and the baseline profile may change due to
                        different absorbency characteristics of the HPLC solvents. The detector has
                        an internal variable rise time filter. For this application, the rise time must
                        be set to 1.0 seconds. If it is less, the baseline noise will increase.
                        If a small bubble becomes trapped in the flowcell and interferes with the UV
                        transmission, high frequency noise can result but tends to come and go
                        throughout the cycle and the run.
                                   noise HF                                                       Blank 1




                           -1.00




                           -2.00




                                              3.0   6.0       9.0        12.0       15.0       18.0



                        Figure 2-1. High frequency noise (the scale of the diagram has been expanded to
                        exaggerate the noise)

                        Every chromatogram affected to same extent
                         1. The detector UV lamp (P/N 2900-0480) is faulty or has exceeded its
                            useful life (> 1000 hours). Replace the lamp (see Replacing the 785A
                            Detector UV lamp).
                         2. The detector risetime is less than 1.0 seconds. Set it to 1 second.
                         3. The detector wavelength is not 269nm. In this case the noise will be
                            amplified and the baseline profile may change. Set wavelength to
                            269nm.


2-4                     2 Troubleshooting                                                      January 1997
PE Applied Biosystems




                         4. The wavelength is incorrect even though 269nm is displayed on the
                            front panel. Check the wavelength accuracy as described in Testing the
                            detector. If the wavelength is incorrect, check that the wavelength drive
                            mechanism has not jammed (see testing the jammed wavelength drive
                            mechanism). If the detector is powered-down to replace the UV lamp,
                            the wavelength drive mechanism will drive to the home position before
                            driving to the stored (most recently used) wavelength. If the
                            mechanism jams at this time, the actual wavelength will different from
                            the wavelength displayed on the front panel.
                         5. Perform the detector Dry cell test as described in the Testing
                            procedures to isolate the cause of the problem.

                        Not all chromatograms or sections of baseline are affected to the same
                        extent
                        A small bubble is lodged in the flowcell. Ensure that the back-pressure device
                        is correctly installed onto the flowcell outlet line and that there are no leaks
                        before or after the flowcell. Clean the flowcell.
                        Stepping of baseline intermittently in chromatograms
                        As a UV lamp ages, the electrodes burn and the arc becomes less stable. The
                        arc then has a tendency to jump to another location intermittently and later
                        return to its original position. This gives rise to a step on the baseline.
                        Although rare, this condition can also be caused by an unstable electronic
                        component.
                                   stepping                                                         Blank 2




                            1.00




                            0.00




                           -1.00




                           -2.00




                                              3.0   6.0        9.0        12.0       15.0        18.0



                        Figure 2-2. Baseline stepping

                         1. Stepping can be due to an aged (>1000 hours) or faulty lamp. Replace
                            lamp.



January 1997            2 Troubleshooting                                                                     2-5
PE Applied Biosystems




                         2. Faulty electronics or loose connections could be the cause of the
                            problem. Perform the Dry cell test to isolate components.
                        Cycling baseline (low frequency)
                        Cycling is due to slow current variation due to heating and cooling effects at
                        bad connections. It will also occur if environmental signals are not effectively
                        screened out due to incorrect or poor grounding.
                         1. Cycling can be due to an aged (>1000 hours) or faulty lamp. Replace
                            the lamp (replacement procedure).
                         2. All instruments must be plugged into the same power source. For
                            example, if a UPS is being used, all of the instruments should be
                            powered from this one source.
                         3. Check that the detector fan is functioning.
                         4. Check that the signal cable shielding is grounded correctly.
                         5. Faulty electronics or loose connections could be the cause of the
                            problem. Perform the Dry cell test to isolate components.
                        Spikes on baseline
                        Small spikes on the chromatogram as shown in can be caused by line voltage
                        disturbances which would show up in a dry cell test.
                        A similar type of spike will be encountered if the PTH-column loses packing
                        material (silica). This can be associated with a build up of white material in
                        the flowcell which should be flushed with 90% B or methanol. Alternatively,




2-6                     2 Troubleshooting                                                     January 1997
PE Applied Biosystems




                        tiny bubbles due to inadequate back-pressure on the flowcell could be
                        causing the spikes. In either of these two cases, the spikes will only be seen
                        when the pump is running.
                               Procise™-BetaLac                                                 1:Blank 1




                        4.00




                        3.00




                        2.00




                        1.00




                        0.00




                                              3.0   6.0     9.0         12.0       15.0       18.0

                        Figure 2-3. Spikes on baseline

                         1. All instruments must be plugged into the same power source. For
                            example, if a UPS is being used, all of the instruments should be
                            powered from this one source.
                         2. Unstable site power. Plug system into different circuit.
                         3. Small bubble in flowcell. Set detector wavelength to 656nm and look
                            for bubble in flowcell. Ensure that flowcell backpressure device is
                            correctly installed.
                         4. Column losing packing material. Check for precipitate at column
                            outlet. If it is present, replace column and clean flowcell.
                        Noise (medium frequency) on baseline
                        Medium-term noise can be attributed to inefficient mixing, leaking or
                        introduction of air into the system. Normally, a mixing problem will have less
                        impact on retention time variation and will have a characteristic dip at the
                        front end of the chromatogram.
                        A bad seal will cause a retention time shift and baseline abnormalities
                        depending on the severity.




January 1997            2 Troubleshooting                                                              2-7
PE Applied Biosystems




                        Use the Pressure tests and Run Gradient method to help determine the
                        source of the problem.
                                   no mixer                                                Blank 3




                            1.00




                            0.00




                           -1.00




                                              3.0   6.0         9.0   12.0    15.0      18.0




                        Figure 2-4. Dynamic mixer not turning

                        Retention times stable
                         1. Dynamic mixer inoperative. Check 140 1A fuse (located near the
                            terminal strip at the back of the instrument). Ensure that the mixer
                            motor shaft does not slip with respect to the drive magnet.
                         2. The detector UV lamp (P/N 2900-0480) is faulty has exceeded its
                            useful life (> 1000 hours). Replace the lamp (Replacement
                            procedures).
                                   stepping                                                    Blank 5




                            1.00




                            0.00




                           -1.00




                           -2.00




                                              3.0   6.0         9.0   12.0     15.0      18.0




                        Figure 2-5. Leaking pump seal




2-8                     2 Troubleshooting                                                January 1997
PE Applied Biosystems




                        Retention times unstable
                         1. Leaking pump seal. Leak should be evident from pump assembly leak
                            points. Replace all pump seals. Inspect cylinder inner surface. Replace
                            if scored.
                         2. Leaking 140 Rheodyne valve rotor seal. Replace both 140 Rheodyne
                            valve rotor seals.
                         3. Leak elsewhere in system. Perform static leak test. Monitor pressure
                            during a run.
                        Slope (negative-going) on which early eluting amino acids ride
                        The sloping front end shown is normally caused by a UV-absorbing
                        contaminant in solvent A or inadequate column equilibration.
                        The profile of the hump early on in the chromatogram is more consistent
                        with some kind of contaminant in the pumping system. This type of
                        phenomenon is common after replacing a component of the system such as
                        a pump seal and usually disappears on its own over time. To minimize the
                        slope, add sodium phosphate monobasic to buffer A. Also see washing
                        procedures



                                            If the baseline is sloping from the start of the chromatogram
                                            It is possible that the column requires longer equilibration.




                                            If a hump occurs early on in the chromatogram, contaminaion
                                            could be the problem




                                      3.0           6.0               9.0                 12.0              15.0   18.0

                        Figure 2-6. Slope or hump at start of chromatogram

                         1. Pumping system contaminated. Wash column and pumping system
                            (see Phosphate wash of pump and column).
                         2. Column equilibration (time between Prepare Pump step and Load
                            Injector step in Flask cycle) time must be no less than X minutes.
                         3. Ensure that pump is configured to “Fill between runs”.




January 1997            2 Troubleshooting                                                                                 2-9
PE Applied Biosystems




                        Humps or dips on baseline
                        Should the baseline have an abnormal profile, either A or B solvent could
                        be the cause. Normally A is more commonly the cause since it degrades
                        more quickly. Also ensure that the detector flowcell is flush with the
                        monochromator. If it is not, refractive index effects will be exaggerated.
                               Procise™-BetaLac                                                1:Blank 1




                        4.00




                        3.00




                        2.00




                        1.00




                        0.00




                                              3.0   6.0     9.0        12.0       15.0        18.0

                        Figure 2-7. Hump on baseline

                         1. Run the “Run Gradient” method to isolate the pumping system. If
                            problem persists, replace solvents A & B and purge 3 times.
                         2. If “Run Gradient” profile looks normal, clean or replace the conversion
                            flask vial and replace R4 and S4.
                        Deflection (small) at consistent retention time in all chromatograms
                        Such a phenomenon is consistent with a scratched pump cylinder. To
                        confirm this, monitor the pressure as the system is running.
                         1. Inspect the pump cylinders and replace if scored.
                        Deflection (off scale) in chromatogram
                        If a bubble becomes trapped in the flowcell, the absorbency will be too high
                        to be on scale. Generally, bubbles will tend to occur at high concentrations
                        of aqueous solvent and can normally be washed out of the cell by increasing
                        the solvent B concentration to 90%.
                        As long as the back-pressure device is properly installed, the system is very
                        effective at preventing the formation of bubbles and it is not necessary to
                        degas the solvents. To demonstrate this, the resulting chromatogram of an




2-10                    2 Troubleshooting                                                   January 1997
PE Applied Biosystems




                        air injection is shown. Even though the 80µL loop was completely full of air
                        when the injection took place, there is no indication that a large air bubble
                        has become trapped in the flowcell.


                         4.00




                         3.00




                         2.00




                         1.00




                         0.00




                                            3.0     6.0     9.0        12.0       15.0       18.0

                        Figure 2-8. Bubble in flowcell




                          4.00




                          2.00




                          0.00




                        -2.00




                        -4.00

                                            3.0     6.0     9.0        12.0       15.0       18.0

                        Figure 2-9. Air injection

                         1. Check for bubble in flowcell. Set wavelength to 656nm and look into
                            flowcell. The red light will be defracted.
                         2. Check that the back pressure device is properly installed.
                         3. Ensure that the HPLC solvents have no run dry.


January 1997            2 Troubleshooting                                                           2-11
PE Applied Biosystems




                        Additional peaks in Blank, Std. & Residue chromatograms anywhere after
                        injection artifact
                        There is a contaminant in the HPLC solvents, the Flask reagents or in the
                        flask system or pumping system. To isolate the flask from the pumping
                        system, run the “Run Gradient” method.
                         1. Replace the HPLC solvents.
                         2. Replace the flask reagents.
                         3. Clean or replace the conversion flask.
                         4. Replace the 80µL injector loop.
                        Flat baseline with no injection artifact. No errors in Event log
                        Since data collection occurred and there were no errors in the Event log, an
                        injection took place. The problem, therefore, must be due to the pump, A/
                        D or detector.
                         1. Check that UV lamp is lit: Press the UTIL> key on the front panel of the
                            detector. The Reference reading should be between –0.25 and –0.4.
                         2. Check that the pump is not over- or under-pressurizing. If it does, a
                            message will be displayed momentarily and the pump will stop
                            pumping and return to the main menu.
                         3. Valve A has a misaligned sensor. In this case, although the valve moves
                            correctly, it is not sensed as having moved. After a time out period, a
                            valve failure message will appear and the pump will shut down. Ensure
                            that the position sensors are secure and that the flag does not overshoot
                            the sensor.
                        All Residue cycles resemble a Blank chromatogram. Maybe some aniline. No
                        Event log errors
                        Since there are no fluid sensor errors in the Event log, all liquid deliveries
                        and transfers took place. Therefore the conclusion is that R2 vapour did not
                        deliver.
                         1. Check that the R2 Set and Actual pressures are the default values. It is
                            normal for the Actual pressure to float a little higher than the Set
                            pressure while there is no R2 delivery.
                         2. Check that there is R2 in bottle.
                         3. Leak test the R2 bottle and repair any leaks.
                         4. Check that R2 delivery path is clear.




2-12                    2 Troubleshooting                                                  January 1997
PE Applied Biosystems




                        All Residue cycles resemble a Blank chromatogram. Transfer sensor error in
                        Event log
                        The transfer from the cartridge to the flask did not take place. This could be
                        due to a blockage in the transfer line or an incorrect pressure. Also, if the
                        respective cartridge outlet sensor determines that it is sensing liquid when,
                        in fact, it is not, there will be no S2 extraction and transfer.
                         1. Check that regulator pressures are set correctly. If reg #5 is set to “0”,
                            the cartridge leak test may have been aborted before the operating
                            pressure was saved. Never abort a leak test.
                         2. Ensure that the cartridge outlet lines are free from crimps and watch a
                            “Deliver S2, cart sensor” function. You should see liquid reach the
                            cartridge before the “fluid sensed” light turns on. If it turns on
                            immediately as the function is executed, reinitialize the sensors and try
                            again. If there is still a problem, the flushing of the cartridge lines is
                            failing or the sensor is faulty.
                         3. Clear the blockage in the transfer line.
                        Deflection at about 6 minutes. Injection artifact. No other peaks. Injector
                        Full sensor error in Event log
                        There is an injection artifact so the some form of injection took place. The
                        small dry reading of 29 (0.15 seconds) in the sample loop full sensor error
                        indicates that the injector was actuated almost immediately after the “Load
                        injector” function started and before any liquid could reach the sample
                        loop. The result is an air injection. The Sample loop load sensor was
                        incorrectly initialized or residual liquid had not been completely flushed out
                        the injection system prior to the “Load injector” step.




                        Reduced number of peaks elute early in chromatogram. The peaks are
                        broad
                        Because not all the peaks are eluting, there may not be an adequate
                        concentration of organic solvent delivered by the pumping system. This
                        could be due to a leak or because the gradient did not start.
                         1. Check that the cable running between the inject output of the
                            sequencer and the inject input of the pump is connected correctly.


January 1997            2 Troubleshooting                                                           2-13
PE Applied Biosystems




                         2. Check that the correct gradient is being used.
                         3. Check for leaks in the pumping system.
                        All peak heights reduced and peak width increased
                        The column is losing plate count or there is severe contamination in the
                        pumping system.
                         1. Replace the column.
                         2. Wash the column and pumping system with phosphate.
                        All peak heights reduced
                         1. Wrong detector output used. Plug the sequencer signal cable into the
                             COMP output on the detector.
                         2. Ensure that the “Concentrate sample” step time is OK.
                         3. Make up fresh PTH-Standards.
                        All peak heights reduced intermittently
                        If the flask bubbling is too intensive, some liquid can flow to waste through
                        the flask vent valve.
                         1. Ensure that the flask bubble pressure is correct.
                        Low Lysine
                        PTH-Hydroxylysine elutes just after PTH-Val. PTH-Methyllysine elutes just
                        after PTH-Leu. PTH-Succinyllysine elutes midway between DMPTU and
                        PTH-Ala.
                        If the “Concentrate sample” step is too long, much of the acetonitrile will
                        have evaporated leaving behind water. In this environment, the lysine is
                        more inclined to adhere to the glass vial than to remain in solution.
                        Therefore, less will be injected onto the column.
                        Lysine is extremely sensitive to metal contamination and peroxides that can
                        form in solvent A as a result of THF oxidation. Generally speaking, if the
                        lysine is normal height in the PTH-Standard chromatogram (taller than
                        leucine), but not in the residue cycles, the HPLC system and solvents are not
                        responsible for the lysine degradation and more scrutiny should be paid to
                        the reaction cartridge delivery system. Contaminated S2 can also destroy
                        lycine.
                        Low Lysine & PE Cys
                        Authentic PTH-Cys is not usually recovered in sufficient yield to be seen.
                        PTH-dehydroalanine, generated by loss of H2S from the side chain can be
                        seen as the DTT derivative although the recovery of this compound is less
                        with cystine than with serine.
                        Both lycine and cystine are sensitive to metal contamination and peroxide
                        which can form in solvent A over a period of time.



2-14                    2 Troubleshooting                                                  January 1997
PE Applied Biosystems




                         1. Replace solvent A
                         2. Wash the column and pumping system with phosphate or add
                            phosphate to solvent A so that the final concentration is 100mmol.
                        Low Ser & Thr
                        A significant amount of serine dehydrates during cleavage to form
                        dehydroalanine which is very reactive and unstable. The DTT added to R4
                        reacts with this derivative and has a stabilizing effect. This DTT-
                        dehydroanaline derivative, commonly called delta-serine (∆S) elutes
                        between PTH-Ala and PTH-Tyr and can be used to help identify a serine
                        residue.
                        A significant amount of threonine dehydrates during cleavage to form
                        dehydro-alpha-aminoisobutyric acid. This product subsequently reacts with
                        the DTT added to R4 to produce two to four derivatives. These elute midway
                        between PTH-Tyr and PTH-Pro.
                        The most critical step for these amino acids is the pre-conversion dry. There
                        must still be liquid present in the flask at the end of this step. It is best to err
                        on the side of too much liquid. Although this will dilute the R4, there should
                        be plenty of TFA present for conversion to take place.
                        Low Asn or Gln and high Asp & Glu respectively
                        About 10% of PTH-Asn and PTH-Gln are degraded by deamidation to yield
                        PTH-Asp and PTH-Glu respectively in the conversion flask under typical
                        conversion conditions. If deamidation is severe, it is more likely to be due to
                        sample storage or handling.
                        Low Glycine
                        ATZ-Gly converts to PTH-Gly somewhat slowly, the reaction being only 80-
                        85% complete during normal conversion conditions. The remaining 15-
                        20% elutes as PTC-Gly near the end of the solvent front.
                        Check that the flask temperature is indeed 64 °C.
                        Low His & Arg
                        Both His and Arg are positively charged amino acids and, as such, have an
                        affinity for glass. An example of this is the problem encountered when a
                        glass bottle is used to contain the PTH-Standard. If the bottle is clean, the
                        His and Arg will stick to the sides and appear much smaller in the PTH-
                        standard chromatogram. The effect will decrease over time as the surface of
                        the glass becomes coated. The Procise uses a polyethylene R5 bottle for this
                        reason.
                        In the same way, the His and Arg are very difficult to extract from the glass
                        fiber filter, especially if they are allowed to dry onto it. Therefore, the most
                        critical step for His/Arg recovery is the post-cleavage dry cartridge dry step.
                        Reduce this step to see if it improves the His/Arg recovery. However, if this
                        step is too short and an excessive amount of TFA (R3) remains, the sample
                        will wash out.


January 1997            2 Troubleshooting                                                              2-15
PE Applied Biosystems




                        His and Arg will stick to the sides of a new conversion flask as well. If a new
                        flask is installed, it is best to run the PTH-Standard method for three or four
                        cycles to coat the sides of the flask.
                        Low repetitive yield, high lag
                        Lag is due to incomplete coupling or incomplete cleavage of the N-terminal
                        amino acid. Lag is nominally 1.5% of the residue in the previous cycle. In
                        order to determine the lag, you must first subtract the background which
                        increases with the length of the run. In Pulsed-liquid cycles, since the TFA
                        (R3) is metered by a fluid sensor, the base (R2) delivery should be suspected
                        if there are no sensor errors and the R2 delivery/pressurization path should
                        be checked for restrictions. The expected flowrate for R2 measured at the
                        waste line at the back of the instrument during the “Del R2g cart (top)” fxn
                        is 23 sccm @ 1psi. If you do not possess a flow meter, you can use the clicking
                        frequency of the pressure valve to determine whether or not there is a
                        restriction as follows:
                         1. Set the R2 pressure to 0.3 psi.
                         2. Activate fxn#, Del R2 cart (top) and allow it to equilibrate for 1 minute.
                         3. The pressure valve clicking frequency should be.
                        A similar symptom is encountered if the R3 valve is partially stuck open and
                        TFA is constantly leaching into the system. It has the effect of neutralizing
                        the basic environment required for coupling.
                         1. Check the R2 (base delivery).
                         2. Check that both the R3 (acid) both gas and liquid delivery valves
                            prevent seepage when closed.
                         3. Check that the R2 (base) delivery valve prevents seepage when closed.
                        Low repetitive yield, no lag
                        Since there is no lag, coupling and cleavage are OK. In this case, wash out
                        should be suspected. The height of PMTC, DPTU and DPU will be reduced
                        in this case.
                        1. Check that the S2 pressure is set correctly.
                        2. Check that the S2 flowrate is correct.
                        DTT-PITC adduct close to Pro
                        Applied Biosystems does not add DTT to any of the solvents, S1, S2 and S3.
                        Therefore, this adduct should not be seen unless one of the solvents is
                        contaminated. If DTT is added to one of these solvents to improve the
                        recovery of oxidation-sensitive amino acids, it is normal to see this artifact.
                        High aniline
                        It is normal to see aniline at the sub-pmol level.




2-16                    2 Troubleshooting                                                    January 1997
PE Applied Biosystems




                        Aniline elutes between PTH-Asn and PTH-Ser and can interfere with either
                        if working at high sensitivity. To reduce it’s size, ensure that there are no
                        dead volumes in the reaction plumbing where unreacted PITC could get
                        trapped. If the size of the peak is larger for a particular cartridge, suspect the
                        cleanliness of the glass blocks or a cartridge line that is not flush with the end
                        of the ferrule.
                        To get rid of most of the aniline that may have been generated, increase the
                        Post-conversion dry step time. Since the amino acids should be in the stable
                        PTH form at this time, increasing the time of this step will not have a
                        detrimental effect on amino acid recovery. As a general rule, after the flask
                        is visibly dry, continue to dry for a further 90 seconds.




                        Figure 2-10. Incorrectly installed cartridge ferrule

                        High oxidized DTT
                        DTT is added to both R4 (25% TFA in water) and R5 (acetonitrile) during
                        the manufacturing process. DTT is an oxygen scavenger and the oxidized
                        DTT reaction product appears as a peak immediately after the injection
                        artifact. If the DTT is so high that PTH-Asp rides on the shoulder, the R4
                        should be replaced.
                        General retention time instability during the run
                        Since all peaks are eluting late, there could be a leak anywhere in the
                        pumping system since the effective flowrate is reduced.
                         1. Visually inspect the fittings for leaks.
                         2. Check the syringe leak points for liquid.
                         3. Perform the static leak test.
                         4. Once you have determined the source of the leak, replace the suspect
                            component.
                        Most peaks miscalled even though peak retention times reasonably stable
                        Ensure that a suitable reference peak is chosen.
                        The purpose of a reference peak is to allow the 610A to compensate for
                        similar shifts of all peaks in the same direction. Fluctuating laboratory
                        temperature can cause such a phenomenon.
                        The reference peak must be:



January 1997            2 Troubleshooting                                                             2-17
PE Applied Biosystems




                                 •    Present in all residue (sequencing) cycles and the PTH-Standard
                                      cycle.
                                 •    Far from amino acid peaks (± 0.25 minutes).
                                 •    The largest peak if part of a group of non-amino acid peaks.
                        The PTH-Standard mixture currently includes four (4) “peaks” that are not
                        amino acids: DMPTU, DPTU, DPU and PMTC. The suitability of these and
                        other reference peaks is described below:
                        DMPTU is not suitable as a reference peak because it is not produced as a by-
                        product of the N-methylpiperidine chemistry.
                                 Procise™-BetaLac                                                            3:Residue 1




                                                                                                    PMTC
                        200.00




                        100.00




                                                                                             DPTU
                                                    Oxidized DTT




                          0.00


                                                3.0                6.0   9.0     12.0       15.0             18.0


                        DPTU is only useful as a reference peak if it appears larger than the PMTC
                        peak in the residue cycles.
                        PMTC is normally a larger peak than the DPTU in the residue cycles. As
                        such, it makes an ideal candidate for a reference peak.

                        Note                    More than the quoted amount of PMTC should be added to the
                                                PTH-Standard working solution since it tends to sublime during
                                                the flask dry-downs.


                        DPU is the oxidation product of DPTU. It can be used as a reference peak if
                        an adequate amount is generated in each cycle.
                        A suitable Amino Acid can also be used as a reference peak if there is
                        significant background in each cycle. In this case, the peak type code is “rc”.
                        None. If laboratory temperatures are stable and the PTH-column has settled
                        down, there may be no need to use a reference peak at all.


2-18                    2 Troubleshooting                                                                  January 1997
PE Applied Biosystems




                        Only early eluting peaks miscalled
                        If TFA is injected onto the column, it tends to affect the retention times and
                        resolution of the early eluting amino acids. It is essential that the flask is
                        completely dry after the Post-conversion dry step. Also, the tubing inside the
                        flask should be positioned to prevent liquid from becoming caught up.
                        Retention time shift only in occasional cycles
                        Air may be occasionally sucked into the cylinders if there is a leak at the
                        pump inlet manifold. The same symptom will result should a partially
                        blocked solvent filter cause cavitation.
                         1. Check fittings at pump inlet manifold and fill/deliver Rheodyne valve
                            for leaks.
                         2. Sonicate the solvent filters in nitric acid.
                        All peaks, including injection artifact, delayed by same amount




                        Should the above message appear in the Event log together with the delayed
                        chromatogram, the injector failed to turn during the “Load injector” step
                        and instead turned during the subsequent “Inject position” step. Data
                        collection started as soon as liquid was sensed at the Sample loop load sensor
                        (approximately 10 seconds after the “Load injector” step started). However,
                        the gradient does not start until the injector moves to the inject position and
                        opens a mechanical switch, in this case, 30 seconds later. The above scenario
                        is due to a bug in Procise firmware 1.0. The predicted frequency of
                        occurrence is extremely low: 1 in 1500 injections.
                        The chromatogram can also be delayed if the column back pressure varies
                        between cycles at the time of injection. This is because the contents of the
                        sample loop is somewhat compressible. Establish why the pressure is varying
                        and correct the condition. Monitor the pressure during a run to confirm
                        that this is the cause of the problem.
                        Standard or First residue retention times do not agree with rest of run
                        For optimal retention time reproducibility it is important that the column
                        flow and composition dynamics are consistent from cycle to cycle. The
                        standard methods are designed with this in mind. However, should the
                        length of a particular cycle be altered significantly, these conditions may be
                        compromized.




January 1997            2 Troubleshooting                                                             2-19
PE Applied Biosystems




                        For the standard method using a normal 21.5 minute gradient, the pump
                        will stop, refill and wait for an equal amount of time between the Blank,
                        Standard, Residue 1, Residue 2 etc. until it receives the Prep pump
                        command to start equilibration.




                        Figure 2-11. Pump synchronization

                        Table 2-1. Definition of terms

                        Equilibration       The time between the “Prep pump” and “Load injector” steps for which
                                            the column sees initial%B conditions. Ensure that this time is the
                                            same for all Flask cycles.
                        Gradient            Defined in Gradient view. Use same gradient for Blank, Standard and
                                            Residue cycles.
                        Fill                The amount of time that the pump takes to fill after the gradient has
                                            finished. Note: if the subsequent cycle’s “Prep pump” step stops the
                                            current gradient before it is finished, the effective Equilibration time
                                            will be reduced by an amount equal to the Fill time. The Fill time will
                                            be consistent for a particular gradient.
                        Wait                The amount of time that the pump sits idle (no flow) after refilling
                                            between gradients.


                        As demonstrated above, it is easy to maintain consistent gradient and
                        equilibration times. However, the Wait time will vary if a the length of a
                        particular cycle changes. To remove this variable, increase the gradient time
                        by holding the final%B for an extra 10 minutes. This will not increase the
                        run time. It simply means that the current gradient will run until the next
                        cycle’s “Prep pump” step interrupts it and starts the subsequent cycle’s
                        equilibration phase. In this case, instead of the pump waiting for a different
                        length of time, the gradient will be interrupted at a different point. Since the
                        column will be fully equilibrated with 90%B before the interruption occurs,
                        this approach offers more consistent flow dynamics from cycle to cycle.
                         1. Use the same gradient for Blank, Standard and Residue cycles.
                         2. The time between the “Prep pump” and “Load injector” steps must be
                            the same for Blank, Standard and Residue cycles. Increase this time by
                            adding 120 second wait step between “Prep pump” and “Load
                            injection” in all cycles.



2-20                    2 Troubleshooting                                                                 January 1997
PE Applied Biosystems




                         3. Edit the gradient so that the final %B is held for an additional 10
                            minutes.
                        Fluid never reaches sensor (Average Wet = 0))
                        Dry = 716                       Threshold = 1075             Average Wet = 0
                        Dry           Wet               Dry          Wet             Dry          Wet
                        3846          0                 0            0               0            0
                        0             0                 0            0               0            0
                        0             0                 0            0               0            0
                        Load R1 Cart (lg loop)


                        In this case, the Average Wet value is “0” because no readings were detected
                        above the Threshold. There will be no “Wet” values.
                        Empty bottle. As the bottle reaches the empty state, the fluid in the delivery
                        line becomes segmented with argon bubbles. This causes the delivery to slow
                        down until it eventually stops. When installing a full bottle of chemical,
                        ensure that the respective bottle change procedure is run so that the
                        delivery line is backflushed before the line is primed.
                        Incorrect Set pressure. If the Set pressure is too low, the delivery will slow
                        down and may stop altogether. If it is too high, the chemical will be subject
                        to increased outgassing. Check the Set and actual pressures in the Pressures
                        & Temperature screen.

                        Note                If a set pressure is at “0”, it maybe as a result of aborting a leak
                                            test before it is finished. Always allow a leak test to finish or use
                                            the Next Step radio button to reach the end step prematurely.


                        Click on the Default button to use operating pressures established by
                        Applied Biosystems (if this turns the heaters off, turn them back on). Click
                        the Execute button. The pressure and vent valves will actuate so that the
                        Actual bottle pressures reach the Set pressures (non-bottle Actual pressures
                        may remain higher than the Set pressure until an associated function is
                        activated).

                        Note                In V1.00 firmware, the R3 default operating pressure is 1.2psi. The
                                            R3 liquid-phase delivery may be more reliable at 1.5psi. The R3
                                            gas-phase delivery is optimal at 0.8psi.


                        If the Actual bottle pressure does not follow the Set pressure within ± 0.1psi
                        after clicking Execute, there is a problem with the pressure management
                        system. Before replacing parts such as the respective pressure transducer,
                        ensure that the tubing connected to the pressure transducer is unrestricted
                        and free of crimps.
                        Corrupted RAM Reset the sequencer:


January 1997            2 Troubleshooting                                                                     2-21
PE Applied Biosystems




                         1. Shutdown the Macintosh.
                         2. Power-down the sequencer.
                         3. Unplug the MEL card (firmware)
                         4. Power-up the sequencer.
                         5. Power-down the sequencer.
                         6. Plug in the MEL card.
                         7. Power-up the sequencer.
                         8. Reboot the Macintosh.
                        Manual regulator pressure too low. Lower the plumbing plate and check the
                        manual regulator gauge. Ensure that the regulator is set to 5.5psi.
                        Bottle pressure leak. Examine the bottle seal from the respective bottle and
                        replace if there any cracks. Run the automatic leak test. Make repairs as
                        necessary.
                        Restricted pressurization path. The delivery pressures are monitored at the
                        pressure management PCB. Should there be a flow restriction between the
                        PCB and the respective bottle, the effective bottle pressure will be reduced
                        during a delivery. Run the respective Leak test and ensure that the actual
                        pressure reported in the Event log after the venting portion of the test is no
                        greater than 0.1psi. If it exceeds this value, determine the source of the
                        restriction (the checkvalve is a good place to start).
                        Restricted fluid delivery path. If there is no problem with the bottle
                        pressurization, there could be a blockage in the fluid delivery path. First
                        determine whether any other deliveries are affected by examining Event log
                        and sensor data. Concentrate on flow paths that are common to other
                        affected deliveries. If the problem is unique to a single chemical, check the
                        delivery line for crimps or restrictions.
                        Insufficient vacuum assist. Check the vacuum gauge. The vacuum should be
                        no less than 12inchesHg.
                        Restricted waste lines. Ensure the lines from the waste bottle to the fume
                        hood are free of restrictions such as trapped liquid.
                        Valve failure. Run the Electrical test to check the electrical integrity or all the
                        valves. Should a valve continuity failure be reported, check associated
                        electrical connections. If a valve strike failure is reported, the power supply
                        or I/O PCB may be faulty.
                        Examine the corresponding Valve status data for the respective function. If
                        it is incorrect, there is a problem with the CPU PCB.




2-22                    2 Troubleshooting                                                        January 1997
PE Applied Biosystems




                        Valveblock port leakage. If one or more of the valveblock ports are stuck
                        open, delivery problems of one form or another will result. Chemicals can
                        become cross-contaminated and deliveries can be fragmented with air
                        bubbles. Examine sensor data as necessary to determine whether one of the
                        valveblocks is common to all of the delivery problems. Carry out the
                        Valveblock test procedure.
                        Valveblock diaphragm punctured. If a valveblock diaphragm is punctured,
                        liquid can leak into the vacuum system instead of flowing to its intended
                        destination. The symptoms of a diaphragm leak are frequent recharges of
                        the vacuum and discoloration in the vacuum tube connected to the affected
                        valveblock.
                        Restricted flushing path. Before a “Load” function, the plumbing pathway is
                        flushed with high pressure argon to ensure that it is dry. All flushing
                        functions use the high pressure argon supply connected to the respective 3-
                        way valve. The high pressure input to valves #46 & #48 is connected directly
                        to the manual regulator (5.5psi) and for valve #47, it is connected to PMS
                        regulator #. Check for restrictions using the Testing gas deliveries
                        procedure.
                        3-way valve mechanical failure. If the 3-way valve fails to switch from the low
                        pressure input to the high pressure (5.5psi) input during a flush function,
                        the effectiveness of the flushing will be compromized. To determine
                        whether the valve is switching correctly, follow the Testing the 3-way valves
                        procedure.
                        Severe leak. Inspect ports for signs of leakage during the sensor function.
                        You may smell the chemical as it delivers.
                        Too many bubbles in liquid slug (Average Wet reading ≠ 0)
                        Dry = 716                 Threshold = 1075           Average Wet + 2744
                        Dry           Wet         Dry           Wet          Dry          Wet
                        2800          180         8             98           78           165
                        78            183         89            173          78           79
                        84            187         167           25           74           171
                        Load R1 Cart (lg loop)


                        In this case, the fluid is fragmented as it flows through the sensor. This
                        problem will probably be associated with a longer than normal initial “Dry”
                        reading because the fragmentation tends to slow down the delivery. Because
                        the sensor never detects a sufficient number of consecutive wet readings, the
                        function will time out and an error will be posted in the Event log. All 18
                        field for Dry/Wet reading will contain a non-zero value.
                        Incorrect Set pressure (too high). If the Set pressure is too low, the delivery
                        will slow down and may stop altogether. If it is too high, the chemical will be
                        subject to increased outgassing.



January 1997            2 Troubleshooting                                                           2-23
PE Applied Biosystems




                        Check the Set and actual pressures in the Pressures & Temperature screen.
                        Click on the Default button to use operating pressures established by
                        Applied Biosystems (if this turns the heaters off, turn them back on). Click
                        the Execute button. The pressure and vent valves will actuate so that the
                        Actual bottle pressures reach the Set pressures (non-bottle Actual pressures
                        may remain higher than the Set pressure until an associated function is
                        activated).

                        Note                In V1.00 firmware, the R3 default operating pressure is 1.2psi. The
                                            R3 liquid-phase delivery may be more reliable at 1.5psi. The R3
                                            gas-phase delivery is optimal at 0.8psi.


                        If the Actual pressure does not follow the Set pressure within ± 0.1psi after
                        clicking Execute, there is a problem with the pressure management system.
                        Before replacing parts such as the respective pressure transducer, ensure
                        that the tubing connected to the pressure transducer is unrestricted and free
                        of crimps.
                        Fume hood vacuum too high. If there is too much suction on the vent/waste
                        line, gas will be pulled out of solution during a delivery causing the flow to
                        be severely segmented. Ensure that the fume hood vacuum complies with PE
                        Applied Biosystem's guidelines drawn out in the Pre-installation manual.
                        Valveblock port leakage. If one or more of the valveblock ports are stuck
                        open, delivery problems of one form or another will result. Chemicals can
                        become cross-contaminated and deliveries can be fragmented with air
                        bubbles. Examine sensor data as necessary to determine whether one of the
                        valveblocks is common to all of the delivery problems. Carry out the
                        Valveblock test procedure.
                        Instrument has been sitting idle. If the instrument is idle for a long period
                        of time with the default pressure settings, a significant amount of argon is
                        absorbed by the chemicals which will tend to outgas when the chemical is
                        next delivered. To minimize this problem, adjust all of the bottle Set
                        pressures to 1psi before leaving the instrument idle.
                        Valveblock temperature too high. If the valve hold current is too high due to
                        a shorted transistor on the I/O board, the valveblocks will heat up and cause
                        the more volatile chemicals to boil during delivery. Since the cartridge and
                        flask valveblocks are powered independently, this phenomenon will tend to
                        affect all cartridge deliveries or all flask deliveries
                        Instrument at high altitude. Reduce the bottle set pressure to compensate
                        for the reduced atmospheric pressure.




2-24                    2 Troubleshooting                                                           January 1997
PE Applied Biosystems




                        Void volume. Ensure that all ferrules are installed correctly.




                                     Incorrect           Correct

                        Sample loop full errors and no amino acid peaks
                        The small dry reading of 29 (0.15 seconds) in the sample loop full sensor
                        error indicates that the injector was actuated almost immediately after the
                        “Load injector” function started and before any liquid could reach the
                        sample loop. The result is an air injection. The Sample loop load sensor was
                        incorrectly initialized or residual liquid had not been completely flushed out
                        the injection system prior to the “Load injector” step.
                         1. Ensure that PEEK fittings are installed at ports #5 & #6 of the injector.
                         2. Remove the orange Teflon lines from ports #5 & #6 of the injector. Cut
                            off 1 inch from each. If this makes the lines too short, they will have to
                            be replaced. Route the tubing connecting the injector to the waste
                            bottle through the hole in the panel to the left of the injector.
                         3. Connect the lines back into the injector. Tighten the PEEK fittings
                            finger-tight and then a 1/4-turn using a wrench.
                         4. Slide the sensors as close as possible to the peak fittings and softly
                            tighten the pre-tee fittings only enough to hold the sensors in place.
                         5. Replace the pick-up line and flask if it is dirty.
                         6. Replace the injector rotor seal if there is evidence of wear.
                        Sample loop full errors with partial injection
                        Dry = 1019                Threshold =1530               Average Wet =3212
                        Dry             Wet       Dry          Wet              Dry          Wet
                        430             1530      10           0                0            0
                        0               0         0            0                0            0
                        0               0         0            0                0            0


                        Liquid had already passed the sample loop full sensor when the injection
                        took place.
                         1. Reduce the “Concentrate sample” step by 5 seconds.
                         2. Ensure that two complete loads of S4 were delivered to the flask. To do
                            this, it will be necessary to analyze the Flask load loop sensor data file
                            which is generated when the “Always report sensor data” box is checked
                            in the Preferences menu.




January 1997            2 Troubleshooting                                                           2-25
PE Applied Biosystems




                         3. If flask is dirty the contents will foam during the Flask Dry function,
                            expelling some of the contents to waste. Clean or replace the flask vial
                            to prevent this from happening.
                        Sample loop full and load errors, no injection
                        An error of this kind will pause the run at the end of the next flask cycle. If,
                        during that next flask cycle, the injection is OK, that cycle’s chromatogram
                        data file will be collected and will appear as the last chromatogram in the
                        610A file. The second-to-last chromatogram will in fact be the data from the
                        flask cycle prior to the cycle in which the error occurred. If a chart recorder
                        is being used to collect data simultaneously, it will include data from the
                        cycle in which the error occurred.
                        The Event log data shows that fluid arrived at the sample loop full sensor
                        (sample loop inlet) in the normal time. However, it never made it through
                        to the sample loop load sensor (sample loop outlet). This can occur when
                        the tail-end of the slug leaving the flask becomes severely fragmented
                        slowing the delivery down to a halt. The problem is exacerbated by a flow
                        restriction, a leaking flask or an incorrectly adjusted pick-up tube.
                         1. Ensure that PEEK fittings are installed at ports #5 & #6 of the injector.
                         2. Remove the orange Teflon lines from ports #5 & #6 of the injector. Cut
                            off 1 inch from each. If this makes the lines too short, they will have to
                            be replaced. Route the line going to the waste bottle through the hole
                            in the panel to the left of the injector.
                         3. Connect the lines back into the injector. Tighten the PEEK fittings
                            finger-tight and then a 1/4-turn using a wrench.
                         4. Slide the sensors as close as possible to the peak fittings and softly
                            tighten the pre-tee fittings only enough to hold the sensors in place.
                         5. Replace the pick-up line and flask if it is dirty. Always replace the pick-
                            up line instead of re-adjusting it to prevent multiple occlusions caused
                            by repositioning the pre-tee tight fitting and ferrule.
                         6. Reset the sequencer:
                              a. Shutdown the Macintosh.
                              b. Power-down the sequencer.
                              c. Unplug the MEL card (firmware)
                              d. Power-up the sequencer.
                              e. Power-down the sequencer.
                              f.   Plug in the MEL card.
                              g. Power-up the sequencer.
                              h. Reboot the Macintosh.


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                        Argon tank pressure too low
                         Argon tank pressure is too low. The sequencer is paused.

                        This message will be generated should the argon supply pressure drop below
                        60psi.
                         1. Check that there is sufficient pressure supplied by the argon tank
                            regulator.
                         2. Ensure that the 1/4 inch tubing is securely connected to the high
                            pressure transducer on the pressure control board (visible from the top
                            of the instrument after removing the top cover).
                        Cannot reach set temperature
                         Unable to reach cartridge, flask or column temperature setpoints.
                         The sequencer is paused.
                        During the Begin step of a cycle, the cartridge, flask and column
                        temperatures are monitored and an error message is generated if either fail
                        to reach setpoint within 20 minutes.
                         1. Ensure that the Method temperatures are in range: ambient to 70 °C.
                         2. Check the thermal fuse on the respective heater PCB.
                        Communication with HPLC lost
                         Communication with the HPLC was lost. Reset the HPLC.

                        The sequencer is no longer able to communicate with the HPLC pump via
                        the RS232 communication link.
                         1. Ensure that the 140 is powered-up
                         2. Ensure that the RS232 cable between the 140 and the sequencer is
                            properly seated.
                         3. Cycle the 140 power.
                        Event buffer overrun
                         Event buffer overrun. Some event messages may have been lost.

                        This message will occur when communication between the Macintosh and
                        sequencer is lost and the sequencer is posting errors in the event buffer.
                        Because this error normally arises as a result of a Sequencer-Macintosh
                        communication failure, this message will not get transferred from the
                        Sequencer to the Macintosh until the communication is re-established.




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                        Injector position error and no corresponding Sample loop sensor errors
                         When the “load injector” function was started, the Rheodyne valve must
                         be in the load position. When starting step (a) of cycle (b), the Rheodyne
                         valve was in the inject position.

                        or
                         When the “Load injector” function is finished, the Rheodyne valve must
                         be in the inject position. When finishing step (a) of cycle (b), the Rheo-
                         dyne valve was in the load position.

                        The injector has not moved to the desired position.
                         1. If a sample loop load error is generated, an injector position error will
                            be generated at the same time by default. Follow the troubleshooting
                            information for the sensor error.
                         2. The flask cycle must include a “Load position” step prior to the “load
                            injector” step
                         3. Check the injector actuator mechanism.
                        Insufficient data collection memory for cycle
                         Insufficient data collection memory for cycle (a). The sequencer is
                         paused.
                        During the Begin step of a cycle, the Procise determines whether there is
                        enough free space in the data buffer to collect that cycle's chromatogram. If
                        there is not, the Procise will wait at the Begin step for up to 4 minutes. If
                        there is still not enough memory available in the data buffer, the above
                        message will result.
                        Because this error normally arises as a result of a Sequencer-Macintosh
                        communication failure, this message will not get transferred from the
                        Sequencer to the Macintosh until the communication is re-established.
                        Invalid sensor Dry reading
                         Sensor (a) does not have a valid dry reading.


                        The dry reading for each fluid sensor is established automatically at the start
                        of each sequencing run during the Init Sensor procedure. These readings
                        are stored in battery-backed memory until overwritten during subsequent
                        Init Sensor procedure execution.
                        If the instrument is reset (by pulling the memory card), these dry readings
                        will be lost. If a function utilizing a sensor is executed after a reset and prior
                        to running the Init Sensor procedure, the above message will result.
                        Run the Init sensor procedure to re-establish Dry readings.




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                        Power failure
                         Power failure occurred on mm/dd/yy, at hh:mm:ssc


                        or during a run
                         A power fail occurred while sequencing. The run will be paused on the
                         End step of the active cycle.

                        If no other instruments in the lab experience the same power failure, check
                        that the power cords are seated correctly in their receptacles.
                        Vacuum assist activated (more than once every 8 hours)
                        There is a leak in the vacuum system. Inspect the valveblock vacuum lines for
                        discoloration. If one of the lines is discolored, the associated valveblock
                        should be replaced. If they look OK, rebuild the vacuum Clippard valve and
                        the vacuum assist assembly which may be partially stuck open.
                        Actual pressure above 5.5psi
                        The Actual pressure in the Pressures & Temperatures view is above 5.5psi
                        (the manual regulator setting).
                         1. Check that the manual regulator gauge is, indeed, reading 5.5psi.
                         2. Reset the sequencer:
                              a. Shutdown the Macintosh.
                              b. Power-down the sequencer.
                              c. Unplug the MEL card (firmware)
                              d. Power-up the sequencer.
                              e. Power-down the sequencer.
                              f.   Plug in the MEL card.
                              g. Power-up the sequencer.
                              h. Reboot the Macintosh.
                         3. Replace the pressure tranducer for the affected position.
                         4. If the fault persists, there is an electronic problem with the pressure
                            control board or the I/O board.
                        All leak tests fail
                         1. Check that the correct grade argon is installed.
                         2. Check that the vent line is free from obstructions.
                         3. Check that the manual regulator is set to 5.5psi.
                        Failing vent test
                         1. Replace the respective checkvalve.


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                         2. Check that the vent line is free from obstructions.
                         3. Check that the vent Angar valve is operational.
                         4. Check for a blockage between the pressure transducer and the waste
                            bottle. See the diagrams in the leak test section.
                        Flask failing vent test
                        If test passes when the X3 bottle is removed, replace the X3 pressure
                        checkvalve.
                        Leak test fails because pressure too high
                        The respective Lee valve on the pressure control board is leaking and needs
                        to be replaced.
                        Cartridge leak test fails
                         1. If all cartridges fail the leak test, the source of the leak is before the
                             cartridge. Use the diagram in the leak testing section to isolate the leak.
                         2. Rebuild the cartridge using a new seal and filter. Ensure that the filter
                            is centrally positioned.
                         3. Ensure that the cartridge ferrule is free from scratches. If it is a multi-
                            cartridge instrument, swap cartridge components until the source is
                            identified.
                         4. If the cartridge itself appears to be the problem, remove the glass
                            blocks and sonicate the cartridge and cap in methanol to clean the
                            threads.
                        Frequent Macintosh lock-ups
                         1. Restart the Macintosh regularly (once a day) to defragment RAM.
                         2. Rebuild the Desktop (once a month). Desktop refers to the application
                            that, among other things, keeps track of where files are located and
                            which, if any, have been marked for deletion. Over time, much of this
                            information becomes redundant and slows down the operation
                            because of the large amount of information that needs to be searched.
                         3. To rebuild the Desktop, restart the Macintosh while holding down the
                            OPTION and keys.
                         4. Ensure that the Memory is correctly allocated. Choose Control panels
                            under the menu and select the Memory control panel. It should be
                            set up as follows:
                         Macintosh type           Quadra 650 (system 7.1)     Power PCs (system 7.5)
                         Disk cache               512K                        512K
                         Modern memory manager    N/A                         ON
                         Virtual memory           As necessary                As necessary
                         RAM Disk                 OFF                         OFF




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                        As a last resort, reformat the hard drive. See Macintosh procedures.
                        Macintosh locks up during data collection
                        If the sequencer is set up to run a considerable number of cycles, set up the
                        610A data analysis software as follows:
                         1. From the Acquisition pull-down menu, select “Configure”.
                         2. In the “Collect Data from:” field, select “Procise Sequencer” but do not
                            select “Leave window open when done”




                        Figure 2-12. 610A configuration menu

                        In this mode, only the chromatogram being “collected” will be displayed.
                        Once collection is complete, the window will close. Therefore, once all
                        cartridges have completed their runs, there will be no sign of a
                        chromatogram on the screen but the data will be stored in the respective file
                        on the hard disk.
                        No communication between Macintosh and sequencer
                        Under normal circumstances, the COMM light behind the front door panel
                        is on to indicate communication between the Macintosh and the sequencer.
                        If the light turns off, there is no communication so sequence data and event
                        information will not be loaded onto the Macintosh hard disk. In order to
                        protect data, this situation will pause a run once the data buffer becomes
                        full.




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                         1. If there is a dialog box on the Macintosh screen which was generated
                            by the Procise application, the COMM light will be off by default. Select
                            one of the prompts in the dialog box to re-establish communication.




                        Figure 2-13. Procise dialog box

                         1. The Macintosh has locked-up. Restart the Macintosh.
                         2. The sequencer has locked up. Reset the sequencer:
                              a. Shutdown the Macintosh.
                              b. Power-down the sequencer.
                              c. Unplug the MEL card (firmware)
                              d. Power-up the sequencer.
                              e. Power-down the sequencer.
                              f.   Plug in the MEL card.
                              g. Power-up the sequencer.
                              h. Reboot the Macintosh.
                         3. The communication cable between the Macintosh and sequencer has
                            become loose. Power-down the two instruments and reseat it.
                        File error every time 610A is launched
                        A corrupted virtual A/D file is causing the problem.
                         1. Restart the Macintosh.
                         2. Move all virtual A/D files from the Procise folder to the Desktop.




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                         3. Launch the 610A application. If it launches successfully, move the files
                            back into the Procise folder, one at a time, until one of them causes the
                            error. Delete the problem file.




                        Figure 2-14. Virtual A/D file location

                        File or disk error every time Procise is launched.
                        Usually [PROCISE error-48]




                        Figure 2-15. Procise file error

                         1. A corrupted virtual A/D file can cause this problem. See item #59
                            above.
                         2. If using system 7.1 operating system, ensure that a printer driver is
                            selected from the Chooser menu (under the menu). This is
                            necessary even if a printer is not physically connected.




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                        File missing error when Procise launched




                        Figure 2-16. Chemistry file missing or incorrectly named

                        The Chemistry file is not in the Procise folder or the name has been
                        changed. For Procise ver. 1.1, the chemistry file name is Chemistry 1.1.
                        Check inside the Procise folder and rename the file if necessary. If it is
                        missing, use the Find file command from the pull-down File menu to
                        locate it.
                        File already exists error generated by the 610A




                        Figure 2-17. 610A file already exists

                        In order to prevent accidental file deletion, the Model 610A Data Analysis
                        application will generate this error message should an attempt be made to
                        use an existing filename.
                         1. A filename entered in the Start Run screen is identical to an existing
                            610A filename. It is good practise to include the date as part of the
                            filename or some other strategy to ensure exclusivity.
                         2. If the 610A application is Quit during data collection, the message will
                            be generated after the application has been relaunched. Try to avoid
                            quitting the 610A application during collection.




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                        “No data has been collected for 12 hours” dialog box message generated




                        Figure 2-18. No new data for 12 hours

                        When a run is completed or stopped by the user, an “End of Run” flag will
                        be set in the virtual A/D file. The 610A constantly monitors these files and
                        transfers any new data to a 610A file of the same name. If the “End of Run”
                        flag is set, the 610A will delete the virtual A/D file because, by this time, a
                        610A version of the complete file will exist.
                        If, for some reason (probably a system crash), the flag does not get set, the
                        610A will generate the above dialog box message.
                        To avoid this message, always check the PROCISE folder (in the Preferences
                        folder) for residual virtual A/D files after a “crash”.
                        610A does not print all cycles
                        Display last chromatogram in chromatogram window before attempting to
                        print.
                        610A Print Quality Poor
                        For best quality, print chromatograms from a report rather than directly
                        from the screen.
                        If the printer is black and white, choose “options” from the “Print” menu
                        and ensure that the print is set to Black and White.




                        Figure 2-19. Print Options dialog box



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                        Macintosh operating system consumes excessive RAM
                        32-bit addressing is turned off.
                         1. Select Control Panels from the Apple menu.
                         2. Select Memory from the list of control panels.
                         3. Turn 32-bit addressing ON.
                         4. Restart the Macintosh.
                        Pump A fails to fill
                        If, when the fill/deliver valve toggles to the fill position, the flag overshoots
                        the fill position optical sensor, a valve error will not be generated because the
                        flag did indeed reach the sensor within the time allowed (about 5 seconds).
                        However, because the flag has gone passed the sensor, the monitoring system
                        will assume that the valve is in the “Midway” position. In this case, due to
                        software logic, only the B cylinder will fill and the A cylinder will not move.
                        To determine whether the above phenomenon is responsible for the failure,
                        press VALVE> from the Main menu and check the status of valve A. If it is
                        reported as being in the “Midway” position, adjust the fill/deliver valve flag
                        and clean the optical sensors carefully with compressed air.
                        Detector (785) beeping
                        The 785A UV detector uses a liquid sensor and beeper to alert the user of a
                        possible flowcell leak.
                         1. The flowcell is leaking. Stop pump, soak up spill and rebuild the
                            flowcell.
                         2. Sensor initialized incorrectly. Cycle the power.
                         3. Sensor is wrongly positions. Ensure that sensor is not touching the drip
                            tray. After repositioning, cycle the power.




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                        Troubleshooting Delivery Problems using Fluid
                        Sensor Data Files
                        A concise delivery troubleshooting guide based on Event log error messages
                        is given in the troubleshooting section of this manual. The following guide
                        is more detailed and uses the fluid sensor data files as a diagnostic.

                        Fluid Sensor Data Files
                        There is the option of generating fluid sensor data files. A separate file is
                        generated for each sensor. The information is similar to that in the Event log
                        but is reported every time a sensor function is executed and not only when
                        an error occurs. This information is temporarily stored in the event buffer
                        but does not result in an Event log error message unless the criteria for a
                        successful delivery has not been met. Instead, the information is saved in
                        individual sensor data files on the hard disk in the Procise folder.




                        Figure 2-20. Location of sensor data files

                        Recording Fluid Sensor data
                        To generate fluid sensor data files, check (X) the “Always report sensor data”
                        box in the Preferences screen. To stop the generation of sensor data, click a
                        second time on the “Always report sensor data” box.

                        Note                If you quit the Procise application, the “Always report sensor data”
                                            box will be deactivated when the application is next launched.




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                        Note                These files can each grow up to 1Mb each(11Mb in total) if the
                                            “Always report sensor data” and “Report valve status” are left
                                            turned on (X'ed). If memory is running out, only use this feature if
                                            delivery problems are evident and purge the files once they have
                                            been resolved.




                        Figure 2-21. Reporting Fluid Sensor Data

                        Valve Status File
                        The valve status file is a report of the “valve on” data for each step in the cycle
                        immediately before it is sent to the valve drivers. This information can be
                        compared with the correct valve list for the respective function in order to
                        expose a fault on the CPU PCB.




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                        To generate the valve status file, check (X) the “Report Valve Status” box in
                        the Preferences screen. See diagram below:




                        Figure 2-22. Reporting Valve status data

                        Opening Fluid Sensor & Valve Status Files
                        These files are stored in the PROCISE folder within the Preferences folder
                        (in the System folder). If they are less than 32Kb, they can be opened with
                        Teachtext or Simpletext which is pre-loaded onto the Macintosh. Otherwise,
                        an alternative text editor such as Word or Excel will need to be used.

                        Note                These files can each grow up to 1Mb each(12Mb in total) if the
                                            “Always report sensor data” and “Report valve status” are left
                                            turned on (X'ed). If memory is short, only use this feature if
                                            delivery problems are evident and purge the files once they have
                                            been resolved.


                        Opening Sensor Data Files using Teachtext
                        (sys. 7.1) or Simpletext (sys 7.5)
                        Providing the sensor data files are below a certain size, they can be opened
                        with Simpletext which is pre-loaded onto the Macintosh.
                         1. Double-click on the Teachtext (sys 7.1) or Simpletext (sys 7.5) icon to
                            launch the application.



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                         2. Select Open from the File menu (in Teachtext it is necessary to close
                            the blank document before you can open a new one).
                         3. Highlight the desired senor data file in the Procise folder and select
                            Open. If the file is too large for Simpletext to open, use an alternative
                            text editor.




                        Figure 2-23. Location of fluid sensor data files

                        The format in which Simpletext displays the sensor information is not ideal.
                        It can be made easier to read by adjusting the size and font of the title bar.
                        Also, reducing the print size and/or orientation will allow each step of
                        information to be reported on the same line.

                        Opening Fluid Sensor Data Files using Excel 5
                        Although Excel is not pre-loaded onto the Macintosh, it is the most suitable
                        application for opening fluid sensor data files.
                         1. Launch the application.
                         2. Select Open from the File menu.
                         3. Highlight the desired sensor data file and select Open.
                         4. Choose the desired formatting.
                              To fit all the horizontal information on the screen
                         5. Highlight the complete document by clicking the box at the top left-
                            hand corner in the window.
                         6. From the Format menu, select Columns and then Autofit Columns
                            from the pop-up menu.
                              To display the column title bar while scrolling vertically


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                         7. Move the cursor over the black box just below the right-hand corner of
                            the window bar. It should change from an arrow into two parallel lines.
                        Click and hold the mouse button and move the cursor downwards until
                        immediately below the column title row. Release the mouse button to split
                        the screen.

                        Interpreting Fluid Sensor Data File Information
                        Date   Time Cycle Step            Air    Fl Th   Fluid   A      F       A       ......etc
                        33108 0.4961 1         6          716    1075    2744    2560   200     0       etc
                               9213
                        Sensor Data Cart Load Large

                                                             Sensor output                    Time



                        Data in the Fluid Sensor files is presented in a similar format to sensor error
                        messages in the Event log. The first four columns are self-explanatory. The
                        other columns are explained below.
                        Air
                        The dry reading established during the Init Sensor procedure.
                        Fl Th
                        Fluid Threshold. The dry reading multiplied by 1.5.
                        Fluid
                        The average of any readings detected above the Fluid Threshold during the
                        delivery function. (The average wet reading [Fluid] is specific for each
                        chemical due to the different refractive indices.) The first dry (A) value
                        represents the time it takes for the respective fluid to reach the sensor. The
                        final wet (F) value will correspond to the number of consecutive wet
                        readings necessary to constitute a successful delivery (see figure below) if
                        the delivery was successful.

                        Note                The Air, Fl Th and Fluid readings represent sensor output level or
                                            voltage.


                        A, F, A, F, A, F...
                        The number of consecutive dry and wet readings (starting with dry)
                        detected from the start of the delivery function. The values in these columns
                        represent time. A reading is taken every 5.2 ms.
                        Sensor ID                                  Necessary consecutive wet readings
                        #1-Cartridge A Outlet                      50
                        #2-Cartridge B Outlet                      50
                        #3-Cartridge C Outlet                      50
                        #4-Cartridge D Outlet                      50



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                        Sensor ID                                Necessary consecutive wet readings
                        #5-Cartridge Load (small loop)           200
                        #6-Cartridge Load (large loop)           200
                        #7-Transfer to Flask                     10
                        #8-Sample Loop Full                      30
                        #9-Sample Loop Load                      30
                        #10-Flask Load (large loop)              200
                        #11-Flask Load (small loop               200


                        Examples of Problem Deliveries
                        The first indication of a delivery problem is normally a fluid sensor error in
                        the Event log. The sequencer may or may not pause as a result depending
                        on which sensor is active. Fluid sensor data files can be used to supplement
                        the Event log messages in order to diagnose the problem. They provide
                        additional information such as answers to the following questions:
                        •     Besides the delivery that generated the error, are any other deliveries
                              affected?
                        •     When, during the run, do the problems tend to occur?
                        •     Is the delivery always “border-line” or is the problem completely
                              random?
                        Examples are given below of sensor data generated under normal and
                        problematic circumstances. Together with the examples are a list of possible
                        causes for the particular problem. Refer to the number appending the
                        problem and see section Causes of Delivery Problems for more information
                        on how to address these problems.

                        Cartridge Load, Cartridge Outlet & Flask Load Sensors
                        Normal Deliveries
                        Listed below are examples of sensor data produced when deliveries are
                        normal. They are to be used as a rough guide only because the sensor output
                        will vary considerably from one sensor to another. There will also be
                        differences in the time it takes a delivery to reach the sensor from
                        instrument to instrument due to vent line back-pressure variation. All of
                        these deliveries are performed using Default pressures.
                        Step   Air      Fl Th    Fluid   A      F     A      F       A       F        A
                        6      716      1075     2744    2560   200   0      0       0       0        0
                        Load R1 Cart (lg loop)
                        Step   Air      Fl Th    Fluid   A      F     A      F       A       F        A
                        39     716      1075     1942    6144   12    6      200     0       0        0
                        Load R3 Cart (lg loop)
                        Step   Air      Fl Th    Fluid   A      F     A      F       A       F        A
                        22     1666 2500         3821    1536   50    0      0       0       0        0



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                        Step    Air      Fl Th Fluid     A      F     A   F      A      F      A
                        Deliver S2, Cart (sensor)
                        Step    Air      Fl Th Fluid     A      F     A   F      A      F      A
                        10      1183 1776 2776           1141   200   0   0      0      0      0
                        Load S4, Flask (lg loop)
                        Step    Air      Fl Th Fluid     A      F     A   F      A      F      A
                        17      1183 1776 2766           1630   200   0   0      0      0      0
                        Load R4, Flask (lg loop)
                        Step    Air      Fl Th Fluid     A      F     A   F      A      F      A
                        14      1183 1776 2822           1621   200   0   0      0      0      0
                        Load R5, Flask (lg loop)


                        In the R3 example above, a few small globules of fluid were detected before
                        the successful delivery criteria was met. This is normal for the more volatile
                        chemicals.
                        Liquid does not reach the sensor
                        Step   Air      Fl Th    Fluid   A      F     A   F      A      F      A
                        6      716      1075     0       3846   0     0   0      0      0      0
                        Load R1 Cart (lg loop)


                        In this case, the Fluid value is “0” because no readings were detected above
                        the Fluid Threshold. There will be no “F” values. An error is generated in
                        the Event log and the run will automatically pause at the end of the current
                        cycle.

                        Possible causes
                        •     Empty bottle (1)
                        •     Manual regulator pressure too low (2)
                        •     Incorrect Set pressure (bottle pressure) (3)
                        •     Bottle pressure leak (4)
                        •     Restricted pressurization path (to bottle) (8)
                        •     Restricted fluid delivery path (11)
                        •     Insufficient vacuum assist (12)
                        •     Restricted waste lines (13)
                        •     Valve failure (15)
                        •     Valveblock port leakage (6)
                        •     Valveblock diaphragm punctured (7)
                        •     Severe leak (22)




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                        Severe outgassing of liquid
                        Step   Air      Fl Th    Fluid   A      F     A   F       A      F       A
                        6      716      1075     2744    2800   180   8   98      78     74      5...
                        Load R1 Cart (lg loop)


                        In this case, the fluid is fragmented as it flows through the sensor. This
                        problem will probably be associated with a longer than normal initial “A”
                        reading because the fragmentation tends to slow down the delivery. Because
                        the sensor never detects a sufficient number of consecutive wet readings, the
                        function will time out and an error will be posted in the Event log.

                        Possible causes
                        •     Fume hood vacuum too high (14)
                        •     Lisk (valveblock) valves are too hot (20)
                        •     Incorrect Set pressure (too high) (3)
                        •     Valveblock port leakage (6)
                        •     Instrument has been idle allowing chemicals to absorb a significant
                              amount of argon (21)

                        Transfer Sensor
                        An error is generated in the Event log if the number of consecutive wet
                        readings detected during a transfer function is less than 10. The run will
                        continue even if an error is generated.
                        Normal delivery
                        Step    Air      Fl Th Fluid     A      F     A   F       A      F       A
                        72      554      832    2008     158    10    0   0       0      0       0
                        Transfer to Flask (gas)


                        As soon as 10 consecutive wet readings are detected the sensor output will
                        no longer be sampled. Therefore, “0”s are reported in subsequent columns
                        even though the function continues to transfer from the cartridge to the
                        flask until the function times out.
                        Step    Air      Fl Th Fluid     A      F     A   F       A      F       A
                        72      554      832    2008     158    7     9   5       4      8       9
                        Transfer to Flask (gas)


                        It is not unknown for the Transfer sensor to report an error even though the
                        instrument is operating correctly. This is due to the speed of transfer and the
                        tendency of the transfer solvent to outgas.




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                        Liquid from cartridge does not reach sensor
                        Step    Air      Fl Th Fluid     A       F       A        F       A       F    A
                        72      554      832    2008     5769    0       0        0       0       0    0
                        Transfer to Flask (gas)


                        The first “A” reading will equate to the function time.

                        Possible Causes
                        •     Incorrect Set pressure (reg #5) (3)
                        •     Restricted fluid delivery path (11)
                        •     Valve failure (15)
                        •     Valveblock port leakage (6)
                        •     Valveblock diaphragm punctured (7)
                        •     Severe leak (22)
                        No liquid delivered to cartridge
                        Step   Air      Fl Th   Fluid    A       F       A        F       A       F    A
                        72     1666 2500        3821     0       50      0        0       0       0    0
                        Del S2 Cart (sensor)


                        Step    Air      Fl Th Fluid     A       F       A        F       A       F    A
                        74      554      832    2008     158     1       5610     0       0       0    0
                        Transfer to Flask (gas)


                        After the initial transfer with S3, the line was not sufficiently dried.
                        Therefore, when the subsequent “Del S2, Cart (sensor)” function took place,
                        the residual liquid in the cartridge line immediately triggered the cartridge
                        output sensor and the delivery valves shut off. Therefore, the volume of S2
                        delivered was not enough to trigger the transfer sensor.
                        Step   Air      Fl Th Fluid A            F      A        F         A       F   A
                        72     1000 1500 1666 0                  50     0        0         0       0   0
                        Del S2 Cart (sensor)-Dry reading established while globule of fluid present


                        Step    Air      Fl Th Fluid     A       F       A        F       A       F    A
                        74      554      832    2008     128     5       5606     0       0       0    0
                        Transfer to Flask (gas)


                        Alternatively, if, during the Init Sensor procedure, a globule of liquid is stuck
                        on the inner surface of the tube in front of the fluid sensor emitter while the
                        Dry reading is being established, an abnormally low “Air” reading will be the
                        result. Consequently a “normal” Air reading will be mistaken as a fluid




January 1997            2 Troubleshooting                                                                  2-45
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                        reading and shut off the valves as soon as a delivery function starts for both
                        the S3 and S2 deliveries. Both the above examples could be caused by the
                        circumstances listed below.

                        Possible Cause
                        •     Cartridge inlet and/or outlet lines restricted (10)

                        Sample Loop Load & Full Sensors
                        A Sample Loop Full sensor error is generated if fluid was not detected when
                        the injection took place. A message will also be generated whenever a
                        Sample Loop Load error occurs. A sample loop full sensor error will not
                        pause the run.
                        A Sample Loop Load sensor error is generated if adequate fluid did not
                        reach the injector sample loop load (outlet) sensor during the “Load
                        Injector” function. A sample loop load error will cause the sequencer to
                        pause at the end of the subsequent Flask cycle.
                        Normal delivery examples
                        Step    Air    Fl Th Fluid     A      F     A     F         A   F      A
                        30      1295 1944 3676         1398   30    0     0         0   0      0
                        Sample loop load sensor


                        Step   Air       Fl Th Fluid   A      F     A     F         A   F      A
                        30     1019 1530 3212          430    997   0     0         0   0      0
                        Sample loop full sensor


                        In the ideal case, the sample loop full sensor will detect the fluid first and
                        continue to detect fluid when the injection takes place. Note that the sum of
                        the “A” and “F” readings will be almost equal for both sensors.
                        Sample over-dried prior to injection
                        Step   Air       Fl Th Fluid   A      F     A     F         A   F      A
                        30     1019 1530 3212          430    987   10    0         0   0      0
                        Sample loop full sensor


                        In this case, the liquid has already passed the sample loop full sensor when
                        the injection takes place.

                        Possible Causes
                        •     “Concentrate Sample” step too long (18)
                        •     Dirty flask (14)




2-46                    2 Troubleshooting                                                   January 1997
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                        Volume of S4 delivered to flask is too small
                        Step   Air      Fl Th Fluid     A      F     A      F      A       F      A
                        24     1183 1776 2776           0      200   0      0      0       0      0
                        Load S4, Flask (lg loop)


                        Step   Air       Fl Th Fluid    A      F     A      F      A       F      A
                        30     1019 1530 3212           430    450   453    0      0       0      0
                        Sample loop full sensor


                        In this case, the first S4 load to the flask was fine but the sensor line was not
                        adequately cleared prior to the second delivery. Therefore, as soon as the
                        second delivery started, 200 consecutive wet readings were detected and the
                        delivery valves shut off. As a result, only half the amount of S4 is delivered to
                        the flask, a proportion of air will be injected and a Sample loop Full error
                        will be generated. Some evidence of air injection may be apparent from the
                        chromatogram.
                        No S4 delivered to flask
                        Step   Air      Fl Th Fluid     A      F     A      F      A       F      A
                        24     600      900      1183   0      200   0      0      0       0      0
                        Load S4, Flask (lg loop)


                        Step   Air       Fl Th Fluid    A      F     A      F      A       F      A
                        30     1019 1530 3212           7692   0     0      0      0       0      0
                        Sample loop full sensor


                        Step   Air     Fl Th Fluid      A      F     A      F      A       F      A
                        30     1295 1944 3676           7692   0     0      0      0       0      0
                        Sample loop load sensor


                        During the Init Sensor procedure, a globule of liquid was trapped between
                        the Load Flask (lg loop) sensor while the Dry reading was being established.
                        Thereafter, a “normal” dry reading is mistaken as a wet reading. No liquid
                        will be delivered during any subsequent Flask Load (lg loop) functions. The
                        flask will be empty prior to the “Load Injector” function so an injection will
                        not take place and Sample loop load and full sensor errors will be reported
                        in the Event log.

                        Possible causes
                        •     Incorrect Set pressure (3)
                        •     Restricted flushing path (9)
                        •     Valve failure (15)
                        •     Valveblock port leakage (6)
                        •     Valveblock diaphragm punctured (7)


January 1997            2 Troubleshooting                                                             2-47
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                        •     3-way valve mechanical failure (16)
                        Premature injection
                        Step   Air       Fl Th Fluid   A       F       A       F       A    F      A
                        30     1019 1530 3212          29      0       0       0       0    0      0
                        Sample loop full sensor


                        Step   Air     Fl Th Fluid     A       F       A       F       A    F      A
                        30     1295 1944 3676          0       29      0       0       0    0      0
                        Sample loop load sensor


                        The orange line into and out of the Rheodyne injector was not flushed
                        adequately after the last injection. Liquid is immediately detected at the
                        Sample loop Load sensor and air is injected. Only the Sample loop Full
                        sensor will report an error.
                        Step   Air       Fl Th Fluid   A       F       A       F       A    F      A
                        30     1019 1530 3212          30      0       0       0       0    0      0
                        Sample loop full sensor


                        Step   Air     Fl Th Fluid A           F        A       F       A   F      A
                        30     1000 1500 1600 0                30       0       0       0   0      0
                        Sample loop load sensor- abnormally low Air reading established


                        In this above example, during the Init Sensor procedure, there was a globule
                        of liquid on the tubing wall in front of the Sample loop Load sensor emitter.
                        As a result, an abnormally low Dry reading was established. However, the
                        Fluid Threshold is larger than a “normal” Dry reading so, in this case, it is
                        not air that is responsible for triggering the injection. Instead, during the
                        “load Injector” step, a small drop of liquid was trapped in the line in front of
                        the Sample loop Load sensor detector. This will generate a low Fluid reading
                        which, under normal circumstances, would not trigger the injection.
                        However, when coincident with a low Dry reading, it can cause intermittent
                        air injections.

                        Possible Causes
                        •     Restricted flushing path (9)
                        •     Valve failure (15)
                        •     Valveblock port leakage (6)
                        •     Valveblock diaphragm punctured (7)
                        •     3-way valve mechanical failure (16)




2-48                    2 Troubleshooting                                                       January 1997
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                        Fluid does not reach Load sensor
                        Step   Air       Fl Th Fluid     A      F      A   F      A      F      A
                        30     1019 1530 3212            430    7232   0   0      0      0      0
                        Sample loop full sensor


                        Step   Air     Fl Th Fluid       A      F      A   F      A      F      A
                        30     1295 1944 3676            7692   0      0   0      0      0      0
                        Sample loop load sensor


                        Possible causes
                        •      Flask pressure leak (5)
                        •      Incorrect Set pressure (3)
                        •      Dirty flask and pick-up line (19)
                        •      3-way valve mechanical failure (16)
                        •      Valve failure (15)
                        •      Valveblock port leakage (6)
                        •      Valveblock diaphragm punctured (7)
                        •      Restricted pressurization path (8)
                        •      Restricted fluid delivery path (11)

                        Causes of Delivery Problems
                            1. Empty bottle
                               As the bottle reaches the empty state, the fluid in the delivery line
                               becomes segmented with argon bubbles. This causes the delivery to
                               slow down until it eventually stops. When installing a full bottle of
                               chemical, ensure that the respective bottle change procedure is run so
                               that the delivery line is backflushed before the line is primed.
                            2. Manual regulator set incorrectly
                               Lower the plumbing plate and check the manual regulator gauge.
                               Ensure that the regulator is set to 5.5psi.
                            3. Incorrect Set pressure
                               If the Set pressure is too low, the delivery will slow down and may stop
                               altogether. If it is too high, the chemical will be subject to increased
                               outgassing.
                               Check the Set and actual pressures in the Pressures & Temperature
                               screen.




January 1997            2 Troubleshooting                                                           2-49
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                        Note                If a set pressure is at “0”, it maybe as a result of aborting a leak
                                            test before it is finished. Always allow a leak test to finish or use
                                            the Next Step radio button to reach the end step prematurely.


                               Click on the Default button to use operating pressures established by
                               Applied Biosystems (if this turns the heaters off, turn them back on).
                               Click the Execute button. The pressure and vent valves will actuate so
                               that the Actual bottle pressures reach the Set pressures (non-bottle
                               Actual pressures may remain higher than the Set pressure until an
                               associated function is activated).

                        Note                In V1.00 firmware, the R3 default operating pressure is 1.2psi. The
                                            R3 liquid-phase delivery may be more reliable at 1.5psi. The R3
                                            gas-phase delivery is optimal at 0.8psi.


                               If the Actual bottle pressure does not follow the Set pressure within ±
                               0.1psi after clicking Execute, there is a problem with the pressure
                               management system. Before replacing parts such as the respective
                               pressure transducer, ensure that the tubing connected to the pressure
                               transducer is unrestricted and free of crimps.
                         4. Bottle pressure leak
                               Examine the bottle seal from the respective bottle and replace if there
                               any cracks. Run the automatic leak test. Make repairs as necessary.
                         5. Flask pressure leak
                               Run the automatic flask leak test. Replace the flask seal or make other
                               repairs as necessary.
                         6. Valveblock port leakage
                               If one or more of the valveblock ports are stuck open, delivery
                               problems of one form or another will result. Chemicals can become
                               cross-contaminated and deliveries can be fragmented with air bubbles.
                               Examine sensor data as necessary to determine whether one of the
                               valveblocks is common to all of the delivery problems. Carry out the
                               Valveblock test procedure. See Testing Valveblocks.
                         7. Valveblock diaphragm punctured
                               If a valveblock diaphragm is punctured, liquid can leak into the vacuum
                               system instead of flowing to its intended destination. The symptoms of
                               a diaphragm leak are frequent recharges of the vacuum and
                               discoloration in the vacuum tube connected to the affected valveblock.
                               See Testing Valveblocks.




2-50                    2 Troubleshooting                                                              January 1997
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                         8. Restricted pressurization path
                              The delivery pressures are monitored at the pressure management
                              PCB. Should there be a flow restriction between the PCB and the
                              respective bottle, the effective bottle pressure will be reduced during a
                              delivery. Run the respective Leak test and ensure that the actual
                              pressure reported in the Event log after the venting portion of the test
                              is no greater than 0.1psi. If it exceeds this value, determine the source
                              of the restriction (the checkvalve is a good place to start).
                         9. Restricted flushing path
                              Before a “Load” function, the plumbing pathway is flushed with high
                              pressure argon to ensure that it is dry. All flushing functions use the
                              high pressure argon supply connected to the respective 3-way valve.
                              The high pressure input to valves #46 & #48 is connected directly to the
                              manual regulator (5.5psi) and for valve #47, it is connected to PMS
                              regulator #. Test for restrictions using the following procedure (you will
                              need a stop-watch):
                              a. Open the plumbing plate to expose the valveblocks and back off
                                 the manual regulator at least ten turns counter-clockwise.
                              b. Scroll to Manual Control and select the respective flush function
                                 (for example Fxn# 137, Flush Input Block). Do not Execute the
                                 function.
                              c. Simultaneously, start the stop-watch and Execute the function. Stop
                                 the timer when the manual regulator needle has dropped to “0” psi.
                              d. The line is not restricted if is has taken less than 13 seconds for the
                                 needle to drop from 5.5 to psi.
                        10. Cartridge inlet and/or outlet lines restricted
                              Check these lines for crimps and over-tightened ferrules. Also check
                              that the line is flush with the end of the cartridge ferrule.
                        11. Restricted fluid delivery path
                              If there is no problem with the bottle pressurization, there could be a
                              blockage in the fluid delivery path. First determine whether any other
                              deliveries are affected by examining sensor data. Concentrate on flow
                              paths that are common to other affected deliveries. If the problem is
                              unique to a single chemical, check the delivery line for crimps or
                              restrictions.
                        12. Insufficient vacuum assist
                              Check the vacuum gauge. The vacuum should be no less than
                              12inchesHg.




January 1997            2 Troubleshooting                                                            2-51
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                        13. Restricted Waste lines
                              Ensure the lines from the waste bottle to the fume hood or duct are free
                              of restrictions such as trapped liquid. Beware of any metallic fittings
                              used in the venting system since they will corrode and become blocked.
                        14. Fume hood vacuum too high
                              If there is too much suction on the vent/waste line, gas will be pulled
                              out of solution during a delivery causing the flow to be severely
                              segmented. Ensure that the fume hood vacuum complies with PE
                              Applied Biosystem's guidelines drawn out in the Pre-installation
                              manual.
                        15. Valve failure
                              Run the Electrical test to check the electrical integrity or all the valves.
                              Should a valve continuity failure be reported, check associated
                              electrical connections. If a valve strike failure is reported, the power
                              supply or I/O PCB may be faulty.
                              Examine the corresponding Valve status data for the respective
                              function. If it is incorrect, there is a problem with the CPU PCB.
                        16. 3-way valve mechanical failure
                              If the 3-way valve fails to switch from the low pressure input to the high
                              pressure (5.5psi) input during a flush function, the effectiveness of the
                              flushing will be compromized. To determine whether the valve is
                              switching correctly, follow the procedure below:
                              a. Scroll to the Pressure & Temperatures menu and set the low
                                 pressure input to the respective 3-way valve to “0”.
                              b. From the Manual Control menu, turn on the 3-way valve.
                              c. Remove the 3-way valve output line from the valveblock and place
                                 the end of the line in a beaker of water. If the valve has switched
                                 correctly, a fast stream of bubbles will flow from the tube.
                              d. From Manual Control, turn the 3-way valve off. The flow of bubbles
                                 should stop.
                              e. Scroll to the Pressures & Temperatures menu and reset the input
                                 pressure to its correct value. A slower stream of bubbles should flow
                                 from the line.
                              f.   Reconnect the line to the valveblock.
                              g. If any of the test fail, replace the 3-way valve.




2-52                    2 Troubleshooting                                                       January 1997
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                        17. Instrument at high altitude. At high altitude there is a greater
                            differential between the set pressure and atmospheric pressure which
                            will encourage the reagent to outgas. To compensate for this, lower the
                            bottle set pressure by 0.2 psi increments.
                        18. Concentrate Sample step too long
                              The “Concentrate Sample” step is included in the Flask cycles so that
                              the injection % can be adjusted. It evaporates the sample immediately
                              prior to injection. If the step time is too long, the sample slug will be
                              too small and will have already passed the Full sensor by the time it
                              reaches the Load sensor.
                        19. Dirty flask and pick-up line
                              If the flask and pick-up line get severely coated, the transfer of sample
                              to the injector will slow down considerably. Also, as the flask empties
                              during the transfer, the liquid has a tendency to segment if the flask is
                              dirty. The segmentation can cause the transfer to stop all together.
                              Replace the flask and pick-up tube. Alternatively, replace the pick-up
                              line only and clean the flask as follows:
                              a. Unscrew the Pre-tee-tite fitting from the top of the flask assembly
                                 and completely remove the pick-up line.
                              b. Unscrew the knurled fitting underneath the flask assembly and
                                 hold the vial as it drops out of the holder (do not lose the small
                                 spring). If the flask does not drop freely, pressurize the vial by
                                 opening valves #24 & #32 and seal the pick-up line port with your
                                 finger. Alternatively, push out the vial using a piece of PEEK tubing
                                 which has been threaded through the vacant pick-up line hole.
                              c. Using a cotton swab soaked in 2M Potassium hydroxide, clean out
                                 the vial. Alternatively, sonicate the vial in this solution for 15
                                 minutes.
                              d. Flush the vial thoroughly with DI water.
                              e. Inspect the flask seal for damage and replace if necessary.
                              f.   Replace the vial, spring and knurled fitting.
                              g. Make up a new pick-up line. Use the indent that the Pre-tee-tite
                                 ferrule made on the old pick-up line to determine its position on
                                 the new line.
                              h. Leak test the flask.




January 1997            2 Troubleshooting                                                           2-53
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                              i.   Run 3 PTH-Standards to season the flask.
                              If the flask becomes very dirty, especially if it is coated with SDS, the
                              contents will foam during the Flask Dry function expelling some of the
                              contents to waste. The reduced volume will cause Sample Loop Full
                              sensor errors.
                        20. Lisk (valveblock) valves too hot
                              If the valve hold current is too high due to a shorted transistor on the
                              I/O board, the valveblocks will heat up and cause the more volatile
                              chemicals to boil during delivery. Since the cartridge and flask
                              valveblocks are powered independently, this phenomenon will tend to
                              affect all cartridge deliveries or all flask deliveries
                        21. Instrument has been sitting idle
                              If the instrument is idle for a long period of time with the default
                              pressure settings, a significant amount of argon is absorbed by the
                              chemicals which will tend to outgas when the chemical is next
                              delivered. To minimize this problem, adjust all of the bottle Set
                              pressures to 1psi before leaving the instrument idle.
                        22. Severe leak. Check all ports for leakage. Check for the smell of
                            chemical as the function is executed.




2-54                    2 Troubleshooting                                                   January 1997
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                        3 Routine Maintenance
                          Procise Planned Maintenance Checklist
                          Instrument location:
                          Serial No.:                                 Service Report No.:
                          Field Service Engineer:                                             Date:

                          Annual planned maintenance for the Procise Protein Sequencer system comprises the
                          following tasks:
                          Place a check next to each completed task.


                                                                      Task
                                Ask customer to turn on “Always Report Sensor Data” from the Preferences menu
                                (have at least 1 run’s worth of sensor data available for analysis).
                          ECO Upgrades
                                Ensure that all ECO upgrades have been completed.
                          Preliminary
                                Run the Delivery Line Backflush procedure.
                                Install clean, empty bottles onto the instrument and ensure that each reaction
                                cartridge is assembled.
                          Replacement Parts
                                Replace the reaction cartridge lines and ferrules (inlet and outlet). (604025 &
                                000160).
                                Replace the conversion flask, flask seal, pick-up line and associated ferrules.
                                Replace the R3 Angar vent valve. (130086).
                                Replace the R3 pressure checkvalve. (130073).
                                Replace the Rheodyne injector rotor seal. (0173-0015).
                                Replace the 0.4mm ID Teflon line into and out of the Rheodyne injector (225108).
                                Install a new Newguard cartridge after the 140 dynamic mixer. (0711-0092).
                                Replace the pump piston and cylinder seals.
                                Replace the switching valve and purge valve rotor seals (100332).
                          Checks, Tests and Procedures
                                Check the Fluid Sensor Data files for abnormal deliveries.
                                Rebuild any damaged bottle caps.
                                Check the vent line for low spots where liquid could get trapped.
                                Rinse out the vent trap bottle (dispose of waste accordingly).
                                Flush any liquid from the bottle vent lines.
                                Remove the top cover. Inspect the moisture filter and replace if necessary.
                                Run the installation Leak Procedures. Repair as necessary to pass all tests.
                                Install a bottle of Methanol onto the X3 position and run the cleanup and backflush
                                procedure.
                                Load chemicals back onto the instrument. Run the Sensor and Delivery test and ad-
                                dress any failures.




November 1996             3 Routine Maintenance                                                                 3-1
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                                                                       Task
                              Run the Flask optimization procedure.
                              Pre-Conversion dry
                              Post Conversion dry
                              Pressure test the Model 140 Pump.
                              Spec 30 psi/min
                              Actual
                              Clean the fan filter at the back of the Model 140.
                              Check the 785A wavelength accuracy (red light at 656nm). Note:- the wavelength
                              range must be set to 190-700nm in the utility menu.
                              Clean the 785A wavelength drive mechanism.
                              Open the Mac and blow out dust from both the inside and the floppy drive.
                              Rebuild the Mac Desktop (hold down OPTION- keys during Restart).
                              Toggle the Procise power and address any power-up error messages in the Event
                              log.
                              Run the “Filter Precycle” method for at least 3 cycles to check the chromatography.
                              Turn off “Always Report Sensor Data”. Otherwise these files will grow to an unac-
                              ceptable size. If possible, print out the sensor data for future reference. Finally, de-
                              lete these files to free up disk space.
                              Ensure that the safety panel is installed (UL requirement).




3-2                     3 Routine Maintenance                                                           November 1996
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                        4 Procedures
                          Pressure System
                          The Power-On Selftest
                          The POST tests the communication to and from the pressure board but does
                          not test the pressure control function of the board.

                          Testing the Control Feedback System
                          To quickly test a regulator, remove the red cap or one of the tubes from the
                          respective output manifold. Put your finger over the port so as to create a
                          slight leak. You should hear a clicking sound as the Lee valve switches on and
                          off. Note that, if you create too large a leak, the Lee valve will remain on so
                          you will not hear it click. Conversely, if the leak is not large enough, the valve
                          will shut off once the set pressure is reached.

                          Testing the 0-5 psi Pressure Transducers
                           1. Scroll to the Pressures & Temperatures view and enter a Set pressure of
                              “0” for each of the regulators. Click on the Execute radio button.
                           2. Create a leak at each regulator output by unplugging either a red cap
                              or a pressure line. Ensure that the Actual pressure falls to “0” ± 0.1 psi.
                              Leave the manifold outlet uncapped.
                           3. Enter a Set pressure of 5.0 psi for each regulator and click on the
                              Execute radio button.
                           4. Reduce the manual regulator pressure to 2.5 psi and reconnect the
                              red plugs or tubing back onto the output manifold. Ensure that the
                              Actual pressure for each regulator reaches 2.5 ± 0.1 psi.
                           5. Return the manual regulator setting to 5.5 psi. The Actual pressure for
                              each regulator should now read 5.0 ± 0.1 psi.
                           6. Click on Default followed by Execute to return to operating pressures.
                              Regulators #5, #9, and #10 will remain at an elevated actual pressure
                              until an associated function is activated.

                          Note            In firmware (MEL card) version 1.0, the P3 regulator should be set
                                          to 1.5 psi for liquid-phase deliveries and 0.8 psi for gas-phase
                                          deliveries.




January 1997              4 Procedures                                                                    4-1
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                        Leak Testing the Pressure Board
                        This procedure is intended to check that the Lee pressure valves are
                        completely closed when turned off. Note that a leak between the sequencer’s
                        manual regulator and the pressure board will also cause the test to fail.
                            1. Power-down the sequencer.
                            2. Create a leak at each regulator output by unplugging either a red cap
                               or a pressure line.
                            3. Back-off the sequencer’s manual regulator.
                            4. Wait 30 seconds and place a piece of sticky tape in line with the gauge
                               needle. If the needle has already fallen to zero, there is a major leak
                               that must be addressed.
                            5. Note the reading on the gauge after five minutes. If the pressure has
                               fallen by more than 0.1 psi, there is a leak at one of the Lee valves or
                               between the sequencer’s manual regulator and the pressure board.
                            6. Power-up the sequencer. Replace the red caps and pressure lines.
                            7. Reset the regulator to 5.5 psi.

                        Replacing a Lee Valve on the Pressure Board
                        Items required:
                        •       5/64-inch hex driver
                        •       Phillips-head screwdriver
                        •       Replacement Lee valve (130091)

                        To replace a Lee Valve on the Pressure Board:
                            1. Turn the main power switch off.
                            2. Disconnect the power cord.
                            3. Shutdown the Mac.
                        Remove the rear cover from the 490
                            4. Unscrew and remove the vent trap bottle.
                            5. Remove the three screws from the top of the rear panel.
                            6. Loosen the three screws at the bottom of the rear panel.
                            7. Lift the panel clear of the lower screws and remove from the
                               instrument.
                        Replacing the Lee valve
                            8. Disconnect the Lee valve cable from the two-pin header.


4-2                     4 Procedures                                                           January 1997
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                            9. Loosen the two hex screws holding the Lee valve in place and remove
                               the valve. Retain the black gasket for later use.
                        10. Install a new Lee valve using the original gasket.
                        11. Power-up the instrument and wait for the Ready light to turn on.
                        12. Restart the Mac.
                        13. Replace the rear panel.

                        Replacing a Pressure Transducer
                        Items required:
                        •      Phillips-head screwdriver
                        •      Replacement pressure transducer (0-5 psi is 291453, 0-100 psi is
                               291454)
                        •      Small tie wrap and side cutters (for 0-100 psi transducer only)

                        Replacing a Pressure Transducer:
                            1. Turn the main power switch off.
                            2. Disconnect the power cord.
                            3. Shutdown the Mac.
                        Remove the rear cover from the 490
                            4. Unscrew and remove the vent trap bottle.
                            5. Remove the three screws from the top of the rear panel.
                            6. Loosen the three screws at the bottom of the rear panel.
                            7. Lift the panel clear of the lower screws and remove from the
                               instrument
                        Replace the pressure transducer
                            8. Pull the pressure board away from the push-on mounts.
                            9. Rotate the pressure board and pull the tubing off the respective
                               pressure transducer.
                        10. Completely remove the two screws holding the transducer onto the
                            board and slide the transducer horizontally towards the centre of the
                            board to remove it from the connector.
                        11. If replacing the 0-100 psi transducer, first shut off the gas pressure at
                            the tank and bleed off residual pressure by opening the argon purge
                            valve. Then use side cutters to remove the tie wrap that secures the 1/
                            4-inch tubing in place.



January 1997            4 Procedures                                                              4-3
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                        12. Install a new transducer and reconnect the tubing. Do not kink the
                            tubing. For the 0-100 transducer, secure the 1/4-inch tube in place with
                            a new tie wrap.

                        Replacing the Pressure Board
                        Items required:
                        •      Phillips-head screwdriver
                        •      Replacement Pressure Board Assembly (603994)

                        Procedure
                            1. Turn the main power switch off.
                            2. Disconnect the power cord.
                            3. Shutdown the Mac.
                        Remove the rear cover from the 490
                            4. Unscrew and remove the vent trap bottle.
                            5. Remove the three screws from the top of the rear panel.
                            6. Loosen the three screws at the bottom of the rear panel.
                            7. Lift the panel clear of the lower screws and remove from the
                               instrument
                        Replace the pressure control board
                            8. Shut off the pressure at the argon tank and open the argon purge valve
                               to bleed off any residual pressure.
                            9. Using tape, bunch together and label the tubes at the output of each
                               regulator. Then disconnect the tubing from the regulator outputs.
                        10. Pull the pressure board away from the push-on mounts.
                        11. Pull the top cover off the instrument. If it cannot be pulled off, it will
                            be necessary to loosen the captive shipping screws from either side. To
                            gain access to these, remove both side panels.
                        12. Leave the 1/4 inch OD tubing attached to the high pressure
                            transducer. Instead, remove it from the injector actuator assembly in
                            the top compartment of the instrument.
                        13. Install a new pressure board. Make sure that you do not trap any cables
                            between the pressure board and the push-on mounts.
                        14. Reconnect the tubing to each manifold.




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                        Delivery Valve System
                        Testing Liquid FlowRates
                        The use of fluid sensors has enabled the instrument to alert the user if a flow
                        or delivery time falls outside the step time window set in the cycle. However,
                        certain flowrate-critical solvent wash steps do not use the fluid sensors. In
                        order to measure the flowrate of the S1, S2 & S3 deliveries, use the
                        procedure below.
                        Items Required:
                        •      10 mL measuring cylinder
                        •      Protective gloves

                        To test the Liquid Flow Rates:

                        WARNING           Wear protective gloves for this procedure.


                            1. Cartridge A must be assembled complete with seal and filter and have
                               passed the cartridge leak test.
                            2. In Manual Control, select cartridge A and activate fxn# 131, Dry Cart
                               Top, for 30 seconds to remove any residual liquid from this flowpath.
                            3. Remove the waste bottle and place it in the fume hood.
                            4. Place a 10 mL measuring cylinder underneath the waste manifold.
                            5. From the manual control view, select cartridge A and activate function,
                               Deliver (solvent) cart (top). Move the measuring cylinder so as to catch
                               the drips from the waste manifold.
                            6. Once 1mL of liquid has been collected, time the delivery for four
                               minutes. See table below for flowrates:-
                            7. Replace waste bottle.
                            8. From Manual Control, activate fxn# 131, Dry cart (top).
                                                                Volume collected in 4 minutes at waste bottle
                        Solvent     Function                    @ 1.7 psi
                        S1          fxn#41, Del S1 cart (top)   4.0 - 5.0
                        S2          fxn#51, Del S2 cart (top)   3.0 - 4.0
                        S3          fxn#61, Del S3 cart (top)   3.5 - 4.5




January 1997            4 Procedures                                                                      4-5
PE Applied Biosystems




                        Testing Gas Flowrates
                        Gas or vapour flowrates are difficult to measure accurately without the aid of
                        a flowmeter. However, the clicking frequency of the pressure control valves
                        is approximately proportional to the flowrate. The table below lists the clicks
                        per second for a given function. The pressures have been altered from the
                        operating values in order to audibly measure the clicking frequency. If an
                        existing function can be used to measure the flowrate, it is given in the table.
                        If not, the group of valves which need to opened are given instead.
                        For convenience, the actual flowrate is listed in the table for default
                        operating pressures (V1.0 firmware). Each were measured using a flow
                        meter connected at the 1/4 inch waste outlet line.
                        The values in brackets are for the actual flush functions which use the
                        manual regulator pressure set at 5.5 psi.
                        Use the following procedure to measure the approximate gas flowrates using
                        the clicking frequency of the Lee pressure valves:

                        WARNING        This procedure allows the instrument to vent into the
                                       laboratory. The waste bottle must be empty and R2 & R3 must
                                       e replaced with empty bottles to measure their respective
                                       flowrates.


                         1. Empty the waste bottle and return it to the instrument.
                         2. Take the vent trap bottle off the instrument to remove any suction due
                            to the fume hood. Ensure that the manual regulator is set to 5.5 psi.
                         3. Flush the respective flowpath with argon for 30 seconds prior to
                            adjusting pressures and taking flow measurements. For example,
                            activate fxn# 131, Dry cart (top) before measuring the flowrate for “Del
                            R2g cart (top).”
                         4. Activate the function (or valves) for at least 30 seconds before taking a
                            measurement.
                         5. Return to operating pressures after the measurements have been
                            taken.




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                        Operating pressures
                                                                                 Actual flowrate for
                                                                                 function at
                                                                                 operating
                        Function               Pressure        Clicks per second pressure (sccm)
                        Del R2g cart (top)
                        Fxn# 11                P2=0.3 psi      2.5±0.3            23@1.0 psi
                        Del R3g cart (top)
                        Fxn# 31                P3=0.5 psi      1.0±0.2            8.0@1,2 psi
                        Dry cart (top)
                        Fxn# 131               P5=0.2 psi      1.9±0.2            81@3.5 psi
                        Flush transfer line
                        Fxn# 141               P5=0.2 psi      1.7±0.2            78@3.5 psi
                        Bubble flask
                        Fxn# 212               P9=0.2 psi      3.4±0.3            75@1.8 psi
                        Empty flask
                        Fxn# 215               P8=0.2 psi      1.8±0.2            74@3.0 psi
                        Flush small loop
                        (flask) Fxn# 217       P8=0.2 psi      2.5±0.3            100@3.0 psi
                        Flush large loop
                        (flask) Fxn# 218       P8=0.2 psi      2.3±0.3            89@3.0 psi
                        Flush flask/injector
                        Fxn# 222 (inject
                        position)              P8=0.2 psi      0.9±0.3            32@3.0 psi
                        Flush flask/injector
                        Fxn# 222 (load
                        position)              P8=0.2 psi      0.8±0.2            30@3.0 psi
                        Flush cart reagent                                        (fxn# 135 yields
                        block Vlv# 1,11,15     P5=0.2 psi      2.3±0.2            150) 100@3.5 psi
                        Flush cart solvent
                        block                                                     (fxn# 136 yields
                        Vlv# 15,16,23          P5=0.2 psi      2.3±0.2            150) 100@3.5 psi
                        Flush input block                                         (fxn# 137 yields
                        Vlv# 7,11,15,16        P5=0.2 psi      2.1±0.2            140) 93@3.5 psi
                        Flush output block                                        (fxn# 138 yields
                        Vlv# 10,15,40          P5=0.2 psi      2.2±0.2            140) 94@3.5 psi
                        Flush small loop
                        (cart)                                                    (fxn# 139 yields
                        Vlv# 7,11,15,22        P5=0.2 psi      1.4±0.2            95)57@3.5 psi
                        Flush large loop
                        (cart)                                                    (fxn# 140 yields 92)
                        Vlv# 7,11,15,21        P5=0.2 psi      1.3±0.2            56@3.5 psi
                        Flush injector
                        Vlv# 42,44 (load                                          (fxn# 221 yields 70)
                        position)              P9=0.2 psi      1.0±0.2            20@1.8 psi


                        Note              At high altitudes, flowrates may exceed the range given in the
                                          table.




January 1997            4 Procedures                                                                       4-7
PE Applied Biosystems




                        Note             Clicking frequency is not an instrument specification.


                        Testing Valveblocks for Leakage to Common Path
                        Use this procedure to determine whether one of the valveblock diaphragms
                        is partially stuck open allowing leakage from that port to the common
                        pathway. This is a general test and is not specific for one particular
                        valveblock.

                        Procedure
                         1. From the Pressures & Temperatures view, set the bottle pressures to “0”,
                            i.e. all regulators except #5, #8, #9 and #10.
                         2. Remove the safety panel.
                         3. Flush the valveblock with argon and backflush the delivery lines of any
                            bottles connected to the valveblock using the appropriate function
                            (usually XX9 for cartridge and XX6 for flask).
                         4. Pressurize the respective block via appropriate port(s). See table below
                            for specific valves.
                         5. If gas flows out of any of the other ports while they are closed, the
                            respective port is stuck open.
                               •   For ports connected to bottle delivery lines, loosen the bottle to
                                   release any pressure. If you see bubbling, the port is stuck open.
                                   Open and close the respective valve. When the valve is open you will
                                   see rapid bubbling. If the bubbling does not stop within a second
                                   after the valve is closed, the port is defective.
                               •   For other ports, Instead of disconnecting a line from its destination,
                                   it may be easier to make up a special piece of line with a 5/16 inch
                                   fitting and ferrule at one end. This line can then be connected to
                                   the port under test while its free and is immersed in a beaker of
                                   water. If you see bubbling, the port is stuck open. Open and close
                                   the respective valve. When the valve is open you will see rapid
                                   bubbling. If the bubbling does not stop within a second after the
                                   valve is closed, the port is defective.
                               •   Do not touch the line with your fingers. As the gas in the line
                                   warms, it will expand and generate bubbles.
                         6. If it was necessary to open a valve on the block under test in order to
                            pressurize it, the valve that was opened has not yet been tested. To test
                            this port, first close this pressurization valve. Next open up a flowpath
                            so that the common port of the valveblock under test is connected to a




4-8                     4 Procedures                                                              January 1997
PE Applied Biosystems




                              bottle delivery line. If there is bubbling in the bottle when the bottle
                              has been loosened, the valveblock has failed. See table below for which
                              specific valves to open.
                                             Open these valves Open these valves to check pressurization
                        Valveblock           to pressurize block valve. Check for bubbling in (bottle #)
                        Cartridge reagent
                        block                46, 15, 11        N/A
                        Cartridge solvent
                        block                46, 15            46, 12 (bottle S2)
                        Cartridge input
                        block                46, 15, 11, 7     N/A
                        Cartridge output
                        block                46, 15, 10        N/A
                        Flask reagent
                        block                24                25 (bottle S4)
                        Flask input block    24                N/A
                        Flask output block   48, 44            48, 41, 32, 25 (bottle S4)


                        If a port is partially stuck open or if there is a blockage in the common
                        flowpath, it may be possible to shift the obstruction by forcing a suitable
                        solvent, such as acetone, through the flowpath using a syringe. Always open
                        the respective valves before applying pressure. Do not operate the valves
                        while pressure is applied.

                        Testing Valveblocks for Ruptured Diaphragms
                        If a diaphragm ruptures chemicals will leak into the vacuum system. There
                        are two methods that can be used to detect such a failure:-
                        Method 1
                        •   Connect a digital manometer to the vacuum manifold purge fitting.
                        •     Pressurize the valveblock.
                        •     Systematically open the valveblock valves to lift the plunger away from
                              the diaphragm.
                        •     Look for an increase in vacuum system pressure that would indicate
                              that a diaphragm has ruptured.
                        Method 1 is most convenient when the vacuum manifold is equipped with a
                        purge cap.
                        Method 2
                        •   Connect a tube to the valveblock vacuum manifold and immerse the
                            other end of the line into a beaker of water.
                        •     Pressurize the valveblock.
                        •     Systematically open the valveblock valves to lift the plunger away from
                              the diaphragm.


January 1997            4 Procedures                                                                   4-9
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                        •       Look for bubbling in the water which indicates that a diaphragm has
                                ruptured.
                        Items required:
                        •       Digital manometer (for method 1)
                        •       3ft length of tubing to connect to valveblock vacuum manifold and a
                                beaker of water (for method 2).
                        •       5/16-inch valveblock blanking plugs.

                        To test Valveblocks for ruptured diaphragms (Method 2):
                            1. Inspect the tubing at the valveblock vacuum manifold. If it is discolored
                               close to the manifold there is a strong possibility that there is a ruptured
                               diaphragm in the valveblock.
                            2. Flush the valveblock with argon and backflush the delivery lines of any
                               bottles connected to the valveblock using the appropriate function
                               (usually XX9 for cartridge and XX6 for flask).
                            3. Substitute empty bottles for any chemicals connected to the valveblock.
                            4. Pressurize the respective block via appropriate port(s). See table above
                               for specific valves.
                            5. Remove the top cover from the instrument and unplug the cable from
                               the vacuum generator assembly.
                            6. Pull the vacuum line off the valveblock under test and replace it with a
                               length of similar tubing or silicon rubber tubing. Immerse the other
                               end of the tube in a beaker of water. Do not touch this line with your
                               fingers. It will warm the gas in the line, cause it to expand and produce
                               slow bubbles.
                            7. If there is a severe rupture, you will already start to see bubbling in the
                               beaker. However, to detect more subtle leaks, it will be necessary to
                               open the valves on the valveblock in order to prevent a plunger from
                               sealing the rupture. As you open the valves, ensure that the associated
                               ports are plugged so that pressure is maintained within the valveblock.
                               For those ports connected to bottle delivery lines, the bottle itself will
                               act as a plug once it has pressurized. However, for those lines connected
                               to waste for example, it will be necessary to plug the port with a
                               valveblock blanking plug.
                            8. If bubbling starts in the beaker as a valve is opened, that particular
                               diaphragm is ruptured.




4-10                    4 Procedures                                                             January 1997
PE Applied Biosystems




                        Testing 3-way Valves
                        If the 3-way valve fails to switch from the low pressure input to the high
                        pressure (5.5 psi) input during a flush function, the effectiveness of the
                        flushing will be compromized. Similarly, if the valve allows high pressure to
                        bleed into the common path, an over-delivery could occur. To determine
                        whether the valve is operating correctly, follow the procedure below.

                        To test 3-way Valves:
                            1. Scroll to the Pressure & Temperatures menu and set the low pressure
                               input to the respective 3-way valve to “0”.
                            2. From the Manual Control menu, turn on the 3-way valve.
                            3. Remove the 3-way valve output line from the valveblock and place the
                               end of the line in a beaker of water. If the valve has switched correctly,
                               a fast stream of bubbles will flow from the tube.
                            4. From Manual Control, turn the 3-way valve off. The flow of bubbles
                               should stop.
                            5. Scroll to the Pressures & Temperatures menu and reset the input
                               pressure to its correct value. A slower stream of bubbles should flow
                               from the line.
                            6. Reconnect the line to the vavlveblock.
                            7. If any of the test fail, replace the 3-way valve.

                        Replacing Valveblocks
                        Items required
                        •       Phillip-head screwdriver

                        To replace Valveblocks:
                            1. Backflush any bottles connected to the valveblock and remove the
                               bottle.
                            2. Flush the valveblock to remove as much residue as possible so that an
                               autopsy can be safely conducted afterwards.
                            3. Power-down the instrument.
                            4. Remove the safety panel.
                            5. Identify the delivery lines connected to the valveblock using tape and
                               then disconnect these lines.
                            6. Loosen the two screws securing the plumbing plate in place and drop
                               it to the horizontal position.



January 1997            4 Procedures                                                                  4-11
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                            7. Pull the vacuum line off the valveblock manifold. If the end is
                               discolored, trim or replace the line. Inspect the vacuum assembly and
                               all other vacuum lines for signs of contamination.
                            8. Unplug the ribbon cable from the valveblock PCB.
                            9. While holding the valveblock, remove the two screws holding the
                               valveblock in place from the front of the panel. Remove the valveblock
                               from the instrument.
                        10. Remove the PCB and bracket from the original valveblock and fit it to
                            the new valveblock.
                        11. Install the new valveblock and remake electrical and plumbing
                            connections.
                        12. Put the chemicals back onto the instrument, power-up the sequencer
                            and prime the delivery lines.

                        Replacing Teflon Lines in the Waste Manifold
                        Items Required:
                        •       Phillips screwdriver
                        •       Razor blade
                        •       Pliers

                        To replace the Teflon Lines in the Manifold:
                            1. Flush the waste line using an appropriate function in Manual Control.
                            2. Remove the waste bottle and store it in a fume hood while carrying out
                               the procedure.
                            3. Loosen, but do not remove, the four Phillips screws accessible from the
                               top of the waste manifold.
                            4. Pull out the old line.
                            5. Shave the end of the new line with a razor blade and thread it through
                               the top of the manifold into the waste bottle.
                            6. Pull all of the shaved section of the line through using a pair of pliers.
                            7. Cut the line flush with the lower manifold.




4-12                    4 Procedures                                                           January 1997
PE Applied Biosystems




                                          Conn, 1/8MP -1/4T
                                          (110044)




                                            Manifold, waste top
                                            (004745)


                                            O-ring, waste (221087)



                                            Manifold, waste
                                            bottom (004746)


                        Figure 4-1. Waste Manifold Assembly




January 1997            4 Procedures                                 4-13
PE Applied Biosystems




                        Vacuum Assist System
                        Adjusting the Vacuum Switch
                        Items required:
                        •       Phillips-head screwdriver
                        •       Flat-blade screwdriver

                        To adjust the Vacuum Switch:
                        Checking the current vacuum range
                            1. Lower the plumbing plate to expose the vacuum and pressure gauges.
                            2. Remove the top cover from the 490. Normally the cover will simply pull
                               off but it may be necessary to first loosen the two captive shipping
                               screws which are accessed by removing both side panels.
                            3. As you look into the top of the unit, the vacuum manifold assembly is
                               located at the rear, right-hand side.
                            4. Newer instruments have a vacuum purge fitting mounted vertically
                               onto the manifold. Loosen this fitting slightly to create a leak in the
                               system. If the manifold does not have a purge fitting, create a leak by
                               carefully pulling a vacuum line off one of the valveblocks.
                            5. Look at the vacuum gauge and listen for when the vacuum assist
                               solenoid valves open to charge the system.
                            6. Note the vacuum gauge pressure when the vacuum assist solenoid
                               valves open (typically 14-15in Hg).
                            7. Reseal the vacuum system fitting you loosened in step 3.
                            8. Wait for the system to charge to the previously set level.
                        To make an adjustment
                            9. Turn the adjustment screw clockwise to increase the vacuum.Turn the
                               adjustment screw counterclockwise to decrease the vacuum. The
                               vacuum should start recharging when the vacuum drops to at least 12
                               in Hg and stop recharging before 20 in Hg is reached.
                        10. Repeat steps #4 through #8 to verify the adjustment.
                        11. Replace the top cover and close the plumbing plate.

                        Setting the Pressure Switch
                        Items required:
                        •       Phillips-head screwdriver


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                        •      Flat-blade screwdriver

                        To Set the Pressure Switch:
                            1. Remove the top cover from the 490. Normally the cover will simply pull
                               off but it may be necessary to first loosen the two captive shipping
                               screws which are accessed by removing both side panels.
                            2. Turn the pressure switch's adjustment screw counterclockwise until the
                               screw protrudes 1/16” from the switch body.
                            3. Open the argon purge valve at the rear of the instrument until a flow
                               of gas can just be heard.
                            4. Note the operating set pressure of the argon cylinder output regulator
                               and then reduce it to 52psi.
                            5. Close the argon purge valve.
                            6. Open the purge fitting on the vacuum manifold to create a leak in the
                               vacuum system.
                            7. Turn the pressure switch's adjustment screw clockwise until the vacuum
                               system starts to recharge.
                            8. Set the argon tank regulator pressure back to the original operating
                               pressure (nominally 65psi) and close the vacuum purge fitting.
                            9. Replace the top cover of the instrument and resume operation.

                        Cleaning the Clippard Valves
                        If the vacuum Clippard valves becomes partially stuck open, it causes the
                        vacuum to leak and, therefore, recharge frequently. A dirty Clippard valve
                        may also delay the recharge.

                        To clean the Clippard Valves:
                            1. Remove the top cover from the instrument. If it does not lift off, it is
                               necessary to loosen the transit screws which are accessible by removing
                               the side panels.
                            2. Unplug the cable from the vacuum assist assembly.
                            3. Turn off the gas and bleed off residual pressure by opening the purge
                               valve.
                            4. Loosen the knurled retaining ring on the vacuum Clippard valve and
                               carefully lift off the solenoid body.
                            5. Clean any dirt from the black plunger and rebuild the valve.
                            6. Carry out the same procedure for the pressure Clippard valve.



January 1997            4 Procedures                                                                4-15
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                        Reaction Cartridge
                        Cleaning the Reaction Cartridge Glass Blocks
                        Use this procedure if high amino acid background indicates that the glass
                        reaction blocks have become contaminated with sample.
                        Items Required:
                        •       Concentrated nitric acid
                        •       Deionized water
                        •       Methanol
                        •       Beaker
                        •       Acid-resistant gloves
                        •       Safety goggles
                        •       Sonicator

                        To clean the Reaction Cartridge Glass Blocks:

                        WARNING             Concentrated nitric acid is extremely corrosive. Wear safety
                                            goggles and gloves.


                            1. Carefully slide the glass blocks out of the cartridge and remove any glass
                               fibre filters.
                            2. Carefully place the blocks into the beaker and cover with nitric acid. Do
                               not place blocks on top of eachother because it can cause them to chip.
                            3. Place the beaker into the sonicator and sonicate for 30 minutes.
                            4. Carefully remove the blocks from the beaker and pour away the nitric
                               acid.
                            5. Rinse the blocks three times with deionized water.
                            6. Give blocks a final rinse with methanol and dry with clean compressed
                               air




4-16                    4 Procedures                                                           January 1997
PE Applied Biosystems




                        Conversion Flask
                        Cleaning the Conversion Flask
                        Use this procedure to remove the white residue from the walls of the
                        conversion flask vial. Cleaning will be more successful if performed
                        routinely before excessive build up of residue occurs. The potassium
                        hydroxide used in the process can etch the glass and accelerate future build
                        up of precipitate. If the cleaning approach is unsuccessful, replace the flask
                        vial and pick-up tube using the flask maintenance kit (P/N 401990). The
                        flask vial and pick-up tube included in the kit are matched which eliminates
                        the need for pick-up tube adjustment.
                        Items Required:
                        •      2M solution of potassium hydroxide (KOH)
                        •      Cotton swab or sonicator
                        •      Deionized water

                        To clean the Conversion Flask:
                        Remove the flask vial from the instrument
                            1. Scroll to the Pressures & Temperatures view, turn off the flask heater
                               and allow the flask assembly to cool until it is comfortable to touch.
                            2. Remove the pick-up tube which should be replaced whenever the flask
                               is cleaned or renewed. Cutting off the coated tip of the tube and
                               repositioning it with respect to the bottom of the vial is not
                               recommended because the pre-tee ferrule forms a slight crimp in the
                               tube every time the fitting is retightened.
                            3. Unscrew the knurled knob underneath the flask vial. As the knob is
                               removed, the constant-force spring, vial and Teflon seal should drop
                               out of the housing. If the vial does not slide out, push it out using a
                               piece of PEEK tubing threaded through the open pick-up line hole. If
                               this is unsuccessful, follow the procedure for Removing a stuck flask
                               vial.
                            4. If the seal does not appear on top of the vial and, instead, remains in
                               the housing, dislodge it by gripping the lip with tweezers.

                        Caution           Do not scratch the Kel-f sealing surface




January 1997            4 Procedures                                                                4-17
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                        Cleaning the flask vial
                            5. Clean the inside of the vial using a cotton swab saturated with the
                               potassium hydroxide solution. Alternatively, sonicate the vial in the
                               potassium hydroxide solution for 15 minutes.
                            6. Thoroughly rinse the vial with deionized water.
                            7. Place a new Teflon seal, lip downward, on top of the vial and install the
                               vial back into the housing.
                            8. Install a new pick-up line. The end of the line should be cut at right-
                               angles to the side of the tube. The gap between the tip of the tube and
                               bottom of the vial should be approximately 1mm.
                            9. From the Test view, select Flow procedures and run the Flask
                               Optimization procedure to optimize the pre- and post-conversion dry-
                               downs.
                        10. Run three PTH-Standard cycles to condition the vial.

                        Removing a stuck Conversion Flask Vial
                        If the vial will not push out with PEEK tubing there has probably been a leak
                        in the past which has caused salt to form and bind the vial in place. Further
                        disassembly is required.
                        Items Required:
                        •      0.035-inch hex wrench
                        •      Tape
                        •      Hammer
                        •      Water
                        •      5/64 inch hex wrench

                        To remove a stuck Conversion Flask Vial:
                            1. Power-down the sequencer.
                            2. Remove the safety panel from the instrument.
                            3. Remove the pick-up tube if you have not already done so.
                            4. Unplug the flask vent valve and the transfer fluid sensor electrical
                               connections from the distribution board.
                            5. Using a 0.035 inch hex wrench, loosen the lens set screw at the bottom
                               of the assembly and carefully remove the lens.
                            6. Loosen the two valve hex screws and the four other hex screws that hold
                               the Kel-f portion of the valveblock onto the remaining flask assembly.



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                         7. The valve will now be slightly proud of the Kel-f due to the force of the
                            valve spring. Use tape to hold the valve in its current position with
                            respect to the Kel-f.
                         8. Remove the lines from ports #32 and #38.
                         9. Lift the Kel-f/valve assembly off the remaining flask assembly and pull
                            off the vacuum line from the valve. Set these parts aside in a clean
                            environment. If the white Teflon seal has stuck to the bottom of the
                            Kel-f, remove it.
                        10. Using a hammer and a 5/16 inch bushing as a punch, tap the vial
                            upwards until it becomes dislodged. Remove the vial completely.
                        11. Clean the vial and the flask holder cavity with water to dissolve any salt.
                        12. Reconnect the vacuum line and screw the valve/Kel-f assembly back
                            onto the main flask assembly.
                        13. Remake tubing and electrical connections.
                        14. Replace the vial, seal, and pick-up line.
                        15. Run the Flask leak test procedure (See Automatic Leak tests).




January 1997            4 Procedures                                                              4-19
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                        Optical Fluid Sensors
                        Reporting Sensor Information
                        When the criteria for a successful delivery is not met, a sensor error message
                        will be generated in the Event Log.
                        There is also the option to report sensor information whenever a sensor
                        function is executed. To generate such files, check the “Always Report
                        Sensor Data” checkbox in the Preferences view. The information is first
                        stored in the event buffer (described in the Diagnostics section), and then
                        saved on the hard disk at 5-second intervals. A separate file is generated in
                        the PROCISE folder for each sensor. See Troubleshooting Delivery
                        Problems using Sensor Data Files.

                        The Sensor & Delivery Test Procedure
                        The Sensor & Delivery Test procedure checks the delivery from each bottle
                        position and the operation of each sensor. The test should be run with
                        normal sequencing chemicals in bottles R1 through S4b (S1 may contain
                        either heptane or methanol). X1 must contain a 50:50 mixture of
                        methanol:water. X2 and X3 must contain100% methanol. Ensure that all
                        delivery lines are primed (use the respective Bottle Change
                        procedure).Default operating pressures must be used.
                        Have a hard copy of the test at hand for reference before running it. If the
                        adequate number of consecutive wet readings for the respective sensor are
                        not detected during a delivery function, an error is generated in the Event
                        log.




                        Figure 4-2. Running the Sensor & Delivery test




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                        To run the Sensor & Delivery Test Procedure:
                         1. Scroll to the Test menu and click on the Flow radio button.
                         2. Select Sensor & Delivery Test from the options.
                         3. Click on the Start Run radio button.
                         4. Using a hard copy of the test as a reference, scrutinize each delivery.
                        When the test has finished, scroll to the Event log and address any failures.




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                        Heaters
                        Testing the Heater Boards
                        All the heaters are tested for open and short circuit condition during the
                        POST. An open circuit condition can be caused by a blown thermal fuse, a
                        closed thermal switch or by a damaged heater element.
                        Items required:
                        •       Multimeter
                        •       Flat-blade screwdriver

                        To test the Heater Boards:
                            1. Power down the instrument.
                            2. Loosen the two screws securing the plumbing plate at the top and lower
                               the plumbing plate to the horizontal position.
                            3. Use the test points shown in the table below to check the element/fuse
                               and thermal switch continuity. The resistance of the element/fuse
                               circuit is given in each case. The thermal switch should be open during
                               normal operation, the resistance of this circuit should be greater than
                               2kW. All test points are on the Distribution board.
                                                                                   Test points for thermal
                                             Test points for                       switch test. (pass if
                        Heater board         fuse/element test   Good resistance   resistance > 2kΩ)
                        Cartridge A          TP9 & TP30          25-30Ω            TP10 & CR6 anode
                        Cartridge B          TP9 & TP31          25-30Ω            TP10 & CR5 anode
                        Cartridge C          TP9 & TP32          25-30Ω            TP10 & CR4 anode
                        Cartridge D          TP9 & TP33          25-30Ω            TP10 & CR3 anode
                        Flask                TP9 & TP34          25-30Ω            TP10 & CR1 anode
                        Column Ovn           TP9 & TP35          20-25Ω            TP10 & CR2 anode


                        Testing for correct Temperature
                        Items required:
                        •       Multimeter and thermocouple probe.

                        To test for correct temperature:
                        Heat cartridge A to 55 °C, the flask to 55 °C and the column oven to 55 °C
                        and wait for front panel LEDs to start blinking. Tape the thermocouple
                        probe to the body of the assembly to be tested. The temperature for each
                        device should be 55 ±3 °C.


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                        Test the remaining cartridge assemblies, one at a time, in the same manner.

                        Note             At higher set temperatures or in very cold laboratories there will be
                                         more heat loss at the surface of the assemblies so the
                                         temperature measured by the thermocouple probe will may be
                                         more than 3 °C lower than the set point.


                        Replacing the Thermal Fuse
                            1. Remove the respective heater board using one of the procedures below.
                               Do not remove the heater element.
                            2. Replace the thermal fuse (S1).
                            3. Dab a spot of thermal compound onto the new fuse and replace the
                               heater board.

                        Replacing a Heater Cartridge Board
                        Items required:
                        •      Flat-blade screwdriver
                        •      Phillips head screwdriver
                        •      Thermal compound (T-5623)
                        •      Replacement cartridge heater PCB (603203)
                        •      Methanol or soldering iron (see below)
                        The cartridge heater assembly has a heater element soldered to the PCB.
                        Either the existing element needs to be removed using methanol or,
                        alternatively, it can be soldered onto the new PCB.

                        To replace a Heater Cartridge Board:
                            1. Power-down the instrument and allow the heaters to cool down.
                            2. Loosen the two screws at the top of the plumbing and lower the
                               plumbing plate into the horizontal position.
                            3. Loosen the two diagonally opposite captive screws behind the
                               respective cartridge assembly.
                            4. Return the plumbing plate to the vertical position.
                            5. Remove the knurled, quarter-turn cap from the respective cartridge
                               assembly.
                            6. Pull the cartridge away from the front of the instrument to disconnect
                               it from its electrical connector.



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                            7. Either, unsolder the heater element wires from the original board and
                               proceed to step #13 or remove the element as described below:
                            8. Remove the panel from the right-hand side of the cartridge assembly.
                            9. Using paper towel, methanol (preferably from a squirt bottle) and
                               gloves, wet the element and allow the glue to dissolve.
                        10. Pull off the old element and dry the surface.
                        11. Peel the backing off the new element and stick it in place.
                        12. Replace the side cover.
                        13. Remove the three screws holding the original heater board in place. Do
                            not lose the black fiber washer that belongs underneath the top screw.
                        14. Take the new board and put some thermal compound on top of the
                            temperature sensor IC (Q1) and on top of the thermal fuse (S1).
                        15. Screw the new board into place. There must be a fiber washer between
                            the upper screw and the PCB.
                        16. Secure the cartridge assembly back onto the plumbing plate and
                            replace the knurled, quarter-turn cap.

                        Replacing the Flask Heater Board
                        Items required:
                        •      Flat-blade screwdriver
                        •      Thermal compound
                        •      5/64 inch hex wrench

                        To replace the Flask Heater Board:
                            1. Power-down the instrument and allow the heaters to cool down.
                            2. Loosen the two screws at the top of the plumbing and lower the
                               plumbing plate into the horizontal position.
                            3. Loosen the two diagonally opposite captive screws behind the flask
                               assembly.
                            4. Return the plumbing plate to the vertical position.
                            5. Pull the flask assembly away from the front of the instrument to
                               disconnect it from its electrical connector.
                            6. Using a 5/64 inch hex wrench to loosen the screws, remove the plate
                               from the rear of the flask.
                            7. Unplug the heater element from the board and remove the board.



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                            8. Take the new board and put some thermal compound on top of the
                               temperature sensor IC (Q1) and on top of the thermal fuse (S1).
                            9. Slot the new board into place and replace the rear plate (the cut-out
                               should face downwards).
                        10. Secure the flask assembly back into place.

                        Replacing the Column Oven Heater board
                        Items required:
                        •      Thermal compound
                        •      7/64 inch hex wrench

                        To replace the Column Oven Heater Board:
                            1. Power-down the instrument and allow the heaters to cool down.
                            2. Remove the top cover from the column oven.
                            3. Remove the knurled outlet column holder end assemblies so that the
                               column and holder can be taken out of the oven assembly and placed
                               elsewhere.
                            4. Using the 7/64 inch hex wrench, loosen the two screws securing the
                               oven heater plate.
                            5. Disconnect the 8-pin plug from the column oven heater board and
                               remove the board and heater plate from the instrument.
                            6. Unplug the heater element, loosen the two screws and remove the
                               board and spacer from the heater plate.
                            7. Take the new board and put some thermal compound on top of the
                               temperature sensor IC (Q1) and on top of the thermal fuse (S1).
                            8. Secure the board and spacer back in place and reassemble the oven.
                            9. Tighten the column end assembly back in place, return the column
                               and holder to the oven and replace the oven lid.




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                        Electronics
                        Digital Controller Board DIP Switch configuration
                        Only DIP switches #1, #2, #3, and #4 have any functionality. They need to be
                        set as follows depending upon how many cartridges the instrument has:
                        Number of Cartridges                Switch #1               Switch #2
                                   1                           off                     off
                                   2                           on                      off
                                   4                           on                      on


                        Instrument Configuration            Switch #3               Switch #4
                        Procise                                off                     off
                        Procise cLC                            on                      off


                        Jumper configuration
                        W3 is the only jumper that should be in place (pins connected together). All
                        other jumper connections must be open.
                        W6, Boot-up jumper. There must not be a jumper in this position. If there is,
                        the instrument will appear to operate normally. However, it will not be
                        possible to erase RAM if corruption is suspected.

                        Replacing the Digital Controller Board
                        Items required:
                        •      Phillips-head screwdriver
                        •      Replacement CPU board (608006)

                        To replace the Digital Controller Board:

                        Caution          Instrument circuit boards and electronics are static-
                                         sensitive. Incorrect handling can cause them to fail through
                                         static discharge. Always follow proper grounding procedures
                                         when working on the instrument and when handling the
                                         circuit boards.

                                         When you remove or ship circuit boards, be sure to enclose
                                         them in ABI-approved antistatic bags.


                            1. Quit the Procise application.
                            2. Turn the 490 main power switch off.



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                         3. Disconnect the power cord.
                         4. Unscrew and remove the vent trap bottle.
                         5. Remove the rear panel from the Procise.
                         6. Remove the MEL card from the socket at the top right-hand corner the
                            digital controller board
                         7. Remove the phillips screws that secure the digital controller board in
                            place.
                         8. Lift the faulty digital controller board upwards to disconnect it from
                            the I/O board and remove it from the 490.
                         9. Ensure that the new board is configured correctly before securing it
                            into place.
                        10. Slide the MEL card into its connector.
                        11. Reconnect the power cord.
                        12. Turn the main power switch on and check that the bank of eight LEDs
                            illuminate in the correct sequence.
                              •   All LEDs turn on.
                              •   All LEDs turn off.
                              •   The LEDs turn on sequentially from the top.
                              •   The Ready LED on the door panel should turn on after 15 seconds.
                        13. Replace the rear panel and vent trap bottle.
                        14. Restart the Mac.

                        Replacing the MEL Card (Firmware)
                        Change the MEL card if it becomes damaged or to replace it with a revised
                        version of the firmware. The MEL card protrudes from the upper, rear left-
                        hand side of the Procise chassis. Its label may or may not face front.
                        Version 1.0                          P/N 603599
                        Version 1.0.1                        Released August 1996


                         1. Turn the main power switch off.
                         2. Press the button adjacent to the MEL card to eject it.
                         3. With the connection holes facing the instrument, gently push the new
                            MEL card into place. If it does not seat, turn it over and try again.
                         4. Turn the main power switch on. If the card is functioning correctly, the
                            Ready LED under the front door visor will light in approximately 15
                            seconds.


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                        Note             If a different version of MEL card has been inserted, the RAM will
                                         be completely erased when the instrument is next powered-up.


                        Replacing the I/O Board
                        Items Requires:
                        •      Medium Phillips-head screwdriver.

                        To replace the I/O Board:

                        Tip              Instrument circuit boards and electronics are static-sensitive.
                                         Incorrect handling can cause them to fail through static discharge.
                                         Always follow proper grounding procedures when working on the
                                         instrument and when handling the circuit boards.

                                         When you remove or ship circuit boards, be sure to enclose them
                                         in ABI-approved antistatic bags.


                            1. Quit the Procise application.
                            2. Turn the main power switch off and disconnect the power cord.
                            3. Unscrew and remove the vent trap bottle and remove the rear cover
                               from the 490.
                            4. Disconnect all cables from the I/O board including the pull-off
                               external event plug and the UV detector cable.
                            5. Remove the five screws attaching the I/O board to the chassis (three
                               across the top and two across the middle.
                            6. Pull the defective board clear of the lower push-on mounts and then
                               pull downwards to disconnect it from the CPU board.
                            7. When installing the new I/O board, be sure not to trap any cables
                               behind the board or underneath the push-on mounts.
                            8. Replace the five attachment screws.
                            9. Reconnect the power cord.
                        10. Turn the main power switch on and wait for the Ready LED, behind the
                            front door visor, to illuminate.
                        11. Launch the Procise application.
                        12. Examine the Event log to ensure that there were no POST failures.
                        13. Replace the rear cover and vent trap bottle.




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                        Replacing the Distribution Board
                        Items required:
                        •      Flat-blade screwdriver
                        •      Replacement distribution board (603235)

                        To replace the Distribution Board:
                            1. Power-down the instrument and quit the procise application.
                            2. Loosen the two screws at the top of the plumbing plate and lower the
                               plate to the horizontal position.
                            3. Use tape to label the cables of those fluid sensors that plug into the
                               component side of the board.
                            4. Remove all cables from the front and back of the board.
                            5. Carefully pull the board clear of the push-on mounts.
                            6. Install a new distribution board. It may be necessary to loosen the
                               cartridge and flask captive screws slightly in order to seat the board
                               properly.

                        Power Supply Configuration
                        The 5 volt adjustment potentiometer is accessible from the right-hand side
                        of the power supply. On later instruments, there is a hole in the chassis to
                        allow the 5 volts to be adjusted in situ. On earlier instruments, however, it
                        will be necessary to remove the power supply in order to make the
                        adjustment. See “Replacing the Power Supply” for removal instructions.
                        To adjust the 5 volts, you will need a volt meter and a plastic, flat blade
                        potentiometer adjustment tool. follow the procedure below:-
                        1. With the instrument powered-up, connect the +ve volt meter probe to
                        TP25 (just below central ribbon cable connector, J7, on the I/O board).
                        Connect the -ve probe to TP40 (top left-hand corner of I/O board).
                        2. Carefully insert the potentiometer adjustment tool into the 5 volt
                        adjustment hole and locate the potentiometer screw. Turn the
                        potentiometer adjustment screw until the voltmeter reads 5.2 volts.




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                                                                                                   V5 +
                                                                                                   V5 -
                                                                                 V3 V4 V5          V4 +
                                                                                 ADJ ADJ ADJ       V4 -
                                                                                                   V3 +
                                                                                           TB2     V3 -
                                                                                                   V2 +
                                                                                                   V2 -
                        LINE                                  PDF                                  V1 -
                                         F1, 10 A NOR BLO           PDV/
                        NEU                                   ADJ          V1                      V1 -
                                          Type 3AG, 250 V           ADJ
                                                                           ADJ                     V1 +
                        GND
                                                                                                   V1 +


                        Figure 4-3. Power supply adjustments

                        Replacing the Power Supply
                        Items required:
                        •      Flat-blade screwdriver
                        •      Phillips-head screwdriver

                        To replace the Power Supply:
                            1. Turn the main power switch off.
                            2. Disconnect the power cord.
                        Remove the rear cover from the 490.
                            3. Unscrew and remove the vent trap bottle.
                            4. Remove the three screws from the top of the rear panel.
                            5. Loosen the three screws at the bottom of the rear panel.
                            6. Lift the panel clear of the lower screws and remove from the
                               instrument.
                        Replace the power supply
                            7. Remove the black plastic cover protecting the line voltage input
                               terminal and disconnect the three wires.
                            8. Disconnect the power supply cables from the I/O board (J17 & J18).
                               Remove these cables from the two “P” clips and thread them through
                               the hole to the right of the power supply.
                            9. Loosen the four captive screws holding the power supply in place and
                               pull the whole assembly firmly from the back of the instrument
                               together with the cables.
                        10. Screw the new power supply into position, connect the three line
                            voltage wires (from the left; line, neutral, ground) and replace the
                            black plastic cover.



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                        Chemistry and Chromatography
                        Optimizing the PTH-Standard Separation
                        To optimize the separation of the amino acids:

                        Table 4-1. Optimization Guidelines

                        Variable                           Major Effect                   Minor Effect
                        Increase initial %B    ←     ←                          ←            ←      ←
                                               DTT    D      PTU;               E DMPTU       H;     S’   R
                        Decrease initial %B    →     →                          ←    →       →            →
                                               DTT    D      PTU;               S    Q;       E    DMPTU H
                        Increase %B at         ←              ←      ←               ←
                        0.4 minutes            E DMPTU         H;     S’   R;   S    Q        T
                        Decrease %B at         →              →
                        0.4 minutes            E DMPTU         H;
                        Increase final %B at         ←
                        18 minutes             I      K        L
                        Decrease final %B at         →
                        18 minutes             I      K        L
                        Increase column        ←     →                          ←    ←       ←
                        temp (2 °C)            M      V                         H    R     PECys
                        Decrease column              →                               →
                        temp (2 °C)            S’     R;                        T    G
                        Increase molarity      ←              ←      ←          ←
                                               H      A;       R     S’;        PECys P
                        Fast Gradients Only:         →               →
                        Decrease flow rate     D     PTU;             S’   R


                        •     If the respective amino acid is greater than 50% resolved, adjust the
                              solvent B composition by 1%. If less than 50% resolved, adjust the
                              solvent B composition by 2%.
                        •     Adjust oven temperature in 2 °C increments.
                        •     Adjust premix buffer concentration in 5ml/liter increments.
                        •     If reducing the initial %B will not provide a satisfactory separation of
                              Asp from DTT, add 100µL of TFA to 1 liter of Solvent A and increase
                              the initial %B to 6-8% to avoid baseline disturbances early in the
                              chromatogram.




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                                                                                                                                             2: Standard 1




                            + 1.00         D N
                                                                 E                                                                           K
                                                     Q
                                                                                                              M              W
                                                                                                                  V
                                                         T                                     Y
                                                                                 A
                                                             G                                                                       F
                                                                             H                            P                                      L
                                                 S
                             0.00                                                                                     DPTU
                                                                                                                                         I
                                                                                                                                 D
                                                                                           R                                     P
                                                                                                   PE-C                          U
                                                                     DMPTU
                            - 1.00




                            - 2.00
                                     3.0                     6.0                     9.0              12.0               15.0                        18.0

                        Figure 4-4. Optimizing the PTH-standard separation




                        Figure 4-5. Default gradient

                        Improving the Shape of the Baseline
                        •       Baseline rises steadily towards end of chromatogram - Solvent B2 has a
                                greater absorbency than solvent A3 at 269nm. Therefore, as the
                                gradient progresses and the concentration of B2 increases, the baseline
                                will rise. To balance out this effect, add acetone to solvent A3. Different
                                systems will produce different baseline profiles but as a general rule:
                                add 1.5mL of 1% acetone in water to 1L of solvent A.
                        •       Negative Sloping Baseline Early in Chromatogram – Some HPLC's
                                exhibit a chronic negative slope from DTT to Glu before flattening out
                                in the latter part of the chromatogram. Add NaH2PO4 or KH2PO4


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                                (monobasic sodium or potassium phosphate, sodium or potassium
                                dihydrogen phosphate) to Solvent A to a final concentration of 100µM
                                (100 micro molar). This will flatten the baseline over several cycles and
                                prevent the reappearance of the slope.

                        Test Pumping System using Run Gradient Method
                        Use this procedure to test the integrity of the pumping system and the
                        efficiency of the mixing. The Run Gradient method automatically starts the
                        pump, equilibrates the column at initial conditions and starts the gradient.
                        No injection takes place.

                        To test the Pumping System:
                            1. Purge the pump.
                            2. Scroll to the Start Run view and select “Run Gradient” as the method.

                                    Run grafient                                              Residue 3




                             0.00




                            -1.00




                            -2.00




                            -3.00




                                                   3.0   6.0   9.0       12.0      15.0      18.0




                        Figure 4-6. Gradient profile with no injection

                        Static Pressure Test
                        Baseline aberrations can often be caused by leaks in the pumping system.
                        Use this test to ensure that the plumbing connections and seals are leak
                        tight. This procedure does not provide a thorough test of the cylinders. Use
                        Dynamic pressure monitoring to check the cylinders.
                        Items Required:
                        •       Rheodyne blanking plug




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                        To test the Static Pressure:
                         1. Purge the pump:
                         2. No. of purges; 1.
                         3. % of syringe; 100.
                         4. Remove the fitting from port #3 of the injector and install a Rheodyne
                            blanking plug into the empty port finger-tight.
                         5. Lodge a paper towel underneath the blanking plug.
                         6. Ensure that the injector is in the “Inject” position.
                         7. Set up the pump to run manually:
                              Flow: 500
                              %B: 50
                              PRESS: 3200 psi
                         8. The pressure should rise to 3200 psi in less than 2 minutes. The flow
                            will stop and the 140 will return to the main menu.
                         9. Take a pressure reading after 5 minutes. If, after this time, it has fallen
                            below 2700 psi, check pump for visible leaks at all fittings, Rheodyne
                            valves and the piston seal leak points.
                        10. Monitor the pressure over an additional 5 minute period. The pressure
                            drop should be less than 75 psi in 5 minutes.

                        Specification
                        The pressure drop should be less than 15psi/min for standard sequencing
                        HPLC solvents (B2/A3 with premix).

                        Troubleshooting the static leak test
                        Should the static pressure test consistently fail and the source of the leak is
                        not obvious, sections of the plumbing can systematically be isolated to help
                        find the leak.
                         1. Perform steps 1. through 8. of the Static leak test.
                         2. From the 140 main menu, select VALVE>. The valve menu will appear
                            but the pressure will still register on the display.
                         3. Select soft key MID A> to move the fill/deliver valve to the midpoint. If
                            the leaking continues, the source may be at or before the fill/deliver
                            valve.




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                            4. If the leaking stops, move valve B (purge valve) to its mid-point and
                               select TOGGLE A to move the fill/deliver valve back to the deliver
                               position. If leaking resumes, the source it at one of the pump’s
                               Rheodyne valves. If it remains leak tight, the source is most probably at
                               the Procise injector.

                        Dynamic Pressure Monitoring
                        Use this procedure to monitor the pressure during a run.
                        Items Required:
                        •       A dual-channel chart recorder is recommended for this procedure.

                        To monitor the Dynamic Pressure:
                        From the 140 Configuration menu, ensure that the D/A channel is set to “A”
                        and the scale is set to “3”.

                        Note              A scale of 3 will ensure that the pressure trace remains on scale
                                          throughout the run (0 to 2040psi). For a more sensitive response,
                                          a scale of less than 3 can be used. In this case, the pressure trace
                                          will autozero at several points during the gradient.




                                                                          785A
                                                                                             COMP
                                                                                     REC



                                                                          140              Event 1
                                                                                        pressure +
                            CHART RECORDER                                              output -
                                                          Chan 1
                                 Paper
                                 feed                     Chan 2


                        Figure 4-7. Pressure monitoring chart recorder hookup

                            1. Connect the red and black input terminals of one of the chart recorder
                               channels to the pressure + & - terminals on the 140 removable
                               connector strip respectively. Set this channel sensitivity to 1V full scale.



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                                    2. Connect the second channel to the REC output on the back of the
                                       785A detector. Set this channel sensitivity to 10mV full scale.
                                    3. Connect the chart recorder external paper feed input to the Event 1
                                       terminals on the 140 connector strip.
                                    4. Use the chart recorder zero controls to position both pens.


           Pen may auto-zero several
           times at the start of the trace




                        pressure                                               pressure
                        Scale = 0                                              Scale = 3




                        Absorbance                                             Absorbance




                                Figure 4-8. Dynamic pressure traces

                                The profile of the pressure trace will be gradient specific but should be
                                consistent from run to run. Negative-going peaks indicate a sudden loss of
                                pressure which could be due to a scratched cylinder. Such a pressure drop
                                would be consistent with a variation in retention time or a sudden step in the
                                absorbency baseline.

                                Calibrating the 140C FlowRate
                                To calibrate the 140C flow rate:
                                    1. Connect a clean, deionized water source to the inlet manifold.


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                         2. Connect the waste lead to the outlet manifold.
                         3. Purge the pump with 2 - 3 full-barrel purges by selecting FILL> to fill
                            the cylinder and then PURGE> . Plug the mixing tee.
                         4. Connect a backpressure column (2.1 x 100 cm) to the dynamic mixer
                            outlet port for the flow test. At a flow of 0.80 mL/min., the
                            backpressure column should generate a backpressure between 2000
                            and 2500 psi. If it does not, replace the backpressure column.
                         5. Follow the procedure outlined in Section 6.1.5. for entering the
                            Dynamic Pressure Test Programs. Then, run program number 2 to
                            calibrate the Syringe A and program number 3 to calibrate Syringe B.
                         6. Allow pressure to stabilize (let the pump run for 2 - 5 minutes).
                         7. During this flow test, measure the pump calibration as follows:
                               a. Using a tared cup, collect the effluent (water) from the
                                  backpressure column for a timed interval (4.0 minutes).
                               a. Weigh the cup containing the 4.0 minute effluent sample.
                               a. Convert the weight of the effluent (water) into milliliters (mL).
                         8. Calculate the pump calibration as follows:
                               Calibration = (Collection Time x Pump Flow Rate Setting x 100) /
                               Weight of Water Collected
                               For example:
                               (4.01 min x 0.80 mL/minute x100)/3.11mL = 102.9

                        Note             Notice that the collection time is not exactly 4 minutes. This
                                         number must be converted from minutes and seconds to minutes
                                         and hundreths of minutes.


                         9. Record the pump calibration on the sticker affixed to the upper front
                            panel of the pump stepper motor so that the pump calibration can be
                            entered into the Model 140C software.
                        The system is now ready for operation.

                        Replacing the Pump Seals (and Cylinders)
                        The pump seals should routinely be changed once every 3-to-6 months.
                        There are two leak points on each pump casting, Figure 4-9. If the piston
                        seals leak, the lower leak point will be wet. If the head seal leaks, the upper
                        leak point will be wet. If you detect any liquid by touching the leak points,
                        the piston seals may need to be replaced if all fittings have been checked for



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                        leaks, but the chromatograms show erratic baselines, broad peaks, or shifts
                        in retention times. Perform the pressure test to confirm that the seals are
                        free of leaks.




                                               GR1030•140C User Manual, Fig. 5.4
                                               Pump Leak Points
                        Figure 4-9. Pump Leak Points

                        Items Required:
                        •     Replacement seals
                        •     Snap-ring pliers (p/n 0392-0003)
                        •     Piston removal/installation tool (p/n 200271)
                        •     Head seal removal tool (p/n 0403-0266)
                        •     1/4-inch open ended wrench (p/n 5400-0010)
                        •     Cylinder head seal insertion tool (p/n 0403-0151)and insertion
                              bushing (p/n 0403-0152)
                        To replace the pump seals, you must remove the cylinder head, cylinder, and
                        piston. The pump seal is made of pure Teflon to give the cylinder the longest
                        lifetime possible. The cylinder should last the life of the pump. However,
                        buffer crystals or other particulates, and lack of careful seal replacement can
                        damage the cylinder, requiring its replacement. During seal replacement,
                        the cylinder can be cleaned first with warm water and then acetonitrile,
                        dried, and inspected internally for damage. The inside diameter of the
                        cylinder is highly polished and uniform to 50 millionths of an inch.
                        Scratches and nicks show up as repetitive patterns on the polished surface.




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                        To replace the Pump Seals:
                         1. Remove the solvent inlet lines from buffers, and cover the solvent inlet
                            filters so that they do not become contaminated.
                         2. Purge the pump.
                         3. Disconnect the stainless steel fittings at the pump heads after purging.
                         4. Fill the pump and then move the knurled retaining collar.
                         5. Remove the cylinder head covers and cylinder heads.

                        Note           Do not interchange parts


                         6. Purge the pump one more time. This will bring the cylinders to the end
                            of the housings with the piston assemblies at the rear-most portion of
                            the cylinders. Carefully remove the cylinders from the piston
                            assemblies.
                         7. Remove the retaining rings from the cylinder heads using snap ring
                            pliers. Discard any rusted or broken snap rings.
                         8. Remove the washers and seals from the cylinder heads being careful
                            not to score the cylinder heads.
                         9. Re-install new seals using the seal installation tool and bushing.

                        Note           When replacing head seals, remember that the spring side always
                                       faces the front of the pumps.


                        10. Using the piston removal/installation tool, loosen the piston seal
                            assemblies.
                        11. Carefully remove the piston seal assemblies making sure that the
                            washers do not fall into the pump housing.
                        12. Replace the two different piston seals for each assembly, and install a
                            new piston retaining clip on each of the assemblies. Loose clips may
                            scratch the inside of the cylinders during normal operation.
                        13. Reconnect the piston seal assemblies, being careful not to overtighten
                            when using the installation tool.
                        14. Inspect the inside of the cylinders: even a small scratch on the inner
                            surface can cause chromatography problems. After inspection, install
                            the cylinders over the front piston seal.
                        15. Fill the pump. This will draw the cylinders into position. Make sure the
                            cylinder have seated themselves properly.



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                        16. Install cylinder heads, covers, and knurled retaining collar, being
                            careful that the inlet ports of cylinder heads are facing down and
                            remain aligned with holes in the cap covers.
                        17. Tighten the 1/4-inch stainless steel tube fittings.
                        18. Put the solvent inlet lines back into the solvent bottles.




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                            19. Purge and fill the pumps. Refer to section 6.1 for performing leak
                                checks.


                                                                              Knurled
                                                                              Retaining
                                                                              Collar


                                                                                               Cylinder
                                                                                               Head Cap

               Retaining Ring                                              Inlet
               Removal Pliers                                              Tubing
                                                                           Fitting




                                                               Stainless
                                                  Cylinder     Steel               Knurled
                                                  Head         Cylinder            Retaining
                           Cylinder                            Head                Collar
                                                  Washer



                                           Cylinder
                                           Seal              Cylinder
                                           Retaining         Head          Black
                                           Clip              Seal          Cap


                                                                                     Front       Retaining
                                                  Rear               Piston          Piston      Clip
                                                  Washer                             Seal




                                                           Rear                                Front
                                                           Piston                              Washer
                                                           Seal
                                                                        Piston Removal/
               Note: Coil side of both piston                           Installation Tool
               seals must face retainer E-clip.
                            Figure 4-10. Pump Seal Replacement




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                          Adjusting the 140 Switching Valve
                          The three small set screws in the active valve stator have been factory set to
                          hold pressure at 5,000 psi (345 bar). If leakage at the valve body is observed,
                          the valve pressure setting should be increased.
                          The end of the valve has three set screws and three larger stator screws,
                          Figure 4-11. Loosen the three set screws 1/20 turn each and tighten the
                          three stator screws by an equal amount. If leakage persists, re-tighten by an
                          additional 1/20 turn. Do not overtighten! This adds nothing to the sealing
                          capacity and increases the wear on the rotor seal.
                          As the valve is adjusted, check to make sure that the motor drive can still turn
                          the valve. Press the VALVE> soft key on the READY Screen to call up the
                          VALVE Screen. Press the TOGGLE> soft key to open and close the valve. If
                          the valve binds or refuses to turn, re-adjust the valve to decrease the torque
                          on the valve slightly. If the valve still leaks, it should be refurbished or
                          replaced.

                                                         Rotor      Stop       Stator
                            Thrust        Spring                    Pins (2)                Set
                            Bearing       Ring           Pin                   Pins (2)
                                                   Rotor     Seal                           Screws (3)
                                                             Pins (4)
                        Body




                                                     Bearing
                                                     Ring             Rotor           Stator
                                         Spring                       Seal
                                         Washers (4)                                         Stator
                                                                       GR0129•140C User's Manual, Fig.
                                                       Isolation           Stator Ring       Screws (3)
                                                       Seal            Valve Adjustment
                          Figure 4-11. Switching Valve

                          Replacing the Pump Rotor Seals
                          The typical lifetime of the valve rotor seal is 6 to 24 months of continuous
                          use. Seal replacement can be accomplished without removing the valve from
                          the pump.
                          Please note that the Rheodyne valve used on the Model 140C, Rheodyne
                          part number 0241-005 (Applied Biosystems part number 0171-0004) is a
                          special item and requires a special rotor seal. The rotor seal is made of
                          Vespel™, a DuPont polyamide material which has exceptionally good wear
                          resistance. The material is, however, susceptible to alkaline attack,




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                        deteriorating rapidly when used with solutions over pH 10. An alternative
                        material is available for alkaline applications. Contact Applied Biosystems
                        for details.
                        Valve Disassembly

                        To disassembly the valve for servicing:
                         1. Remove the three stator screws, Figure 4-11. Do not change the setting
                            of the three set screws in the stator.
                         2. Refer to Figure 4-11 and remove the stator and stator ring from the
                            valve body by pulling axially to disengage the various pins.
                         3. To remove the rotor seal, pry it off of the four seal pins using a
                            screwdriver or a knife blade.
                         4. The isolation seal and bearing ring are usually left in place; they rarely
                            need changing.
                        Valve Reassembly

                        To reassemble the valve with the new rotor seal:
                         1. The rotor pin can be inserted in the rotor from one side only (the side
                            without the peened closure marks around the pin hole). Do not try to
                            force the rotor pin in from the wrong side.
                         2. Be sure that the new rotor seal is correctly oriented as shown in Figure
                            4-12 with the rotor seal slots facing the stator and with the notch in the
                            metal rim of the rotor seal in line as shown (the notch also faces the
                            stator).

                                       Notch              Rotor Pin




                        Figure 4-12. Rotor Seal Orientation (Viewed from Stator)

                         3. In replacing the stator ring, be sure that the two stop pins are replaced
                            in their holes in the stator ring, then push the stator ring squarely onto
                            the rotor assembly so that the stop pins enter the mating holes in the
                            body (with the rotor pin between the two stop pins) and the isolation
                            seal slips inside the stator ring without hanging up.



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                            4. Replace the stator by first pushing it onto the two pins on the stator ring
                               and then adding the three stator screws. Tighten each screw a little at a
                               time to keep the stator surface parallel to the stator ring surface. If the
                               three set screws in the stator were left unchanged, then tighten the
                               three stator screws until all parts are held firmly in place. The three set
                               screws will insure that the gap between stator and stator ring is uniform
                               and exactly as it was before disassembly.
                            5. If the set screws need adjusting because a new rotor seal was installed
                               or because leakage has to be stopped, be sure that each set screw is
                               turned an equal amount so that after the stator screws are tightened,
                               the gap between the stator and the stator ring is uniform all around.

                        Phosphate Wash of Pump and Column
                        Use this procedure to clean the entire pumping system if metal
                        contamination is suspected. This method is preferred over the phosphoric
                        acid method described later because, unlike phosphoric acid, the phosphate
                        can be pumped through the column as well.
                        Items Required:
                        •       Sodium phosphate monobasic or sodium dihydrogen phosphate
                                (NaH2PO4). Potassium phosphate monobasic may be substituted
                                (KH2PO4).
                        •       HPLC grade water.

                        To make up a 0.1M solution of sodium phosphate (approx. pH 5.0):

                        Caution           Ensure that sodium phosphate monobasic is used.
                                          Otherwise the pH may be too high. The pH of the solution
                                          must not exceed 7.0.


                            1. To a 500mL beaker, add 3.45g of sodium phosphate monobasic
                               (NaH2PO4).
                            2. Add 250mL of HPLC-grade water and mix until thoroughly dissolved.
                            3. Flushing procedure
                            4. Remove the lines from solvent A & B and purge the pumps once @
                               100%.
                            5. Place the solvent A and B lines into the phosphate buffer.
                            6. Perform three 100% purges.
                            7. Access Manual Control on the 140 and “free run” at 325µL/min @ 50%
                               B for 45-60 minutes.



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                            8. Place the solvent A and B lines into HPLC-grade water and perform
                               three 100% purges.
                            9. Access Manual Control on the 140 and “free run” at 325µL/min @ 50%
                               B for 15 minutes.
                        10. Place the solvent A and B lines back into the respective solvents and
                            perform a 100% purge.

                        Caution          Sodium phosphate will precipitate in acetonitrile. Do not
                                         allow these two chemicals to mix in the pumping system at
                                         any time.


                        To minimize the possibility of further metal contamination, add sodium
                        phosphate monobasic (NaH2PO4) or potassium phosphate (KH2PO4) to
                        solvent A and mix well until completely dissolved. The final concentration
                        of phosphate should be 100µM. The addition of phosphate may slightly
                        increase the retention time of both aspartic and glutamic acid.

                        Phosphoric Acid Wash of Pump
                        Use this procedure to clean the pumping system if metal contamination is
                        suspected.

                        Caution          Do not run phosphoric acid through the column


                        Items Required:
                        •      Phosphoric Acid
                        •      Acid-resistant gloves
                        •      Safety goggles
                        •      HPLC grade water

                        WARNING          Phosphoric acid is extremely corrosive. Wear safety goggles
                                         and gloves.


                        To clean the pumping system:
                        1. Make up a 5% solution of phosphoric acid by adding 10mL of phosphoric
                        acid to 190mL of HPLC-grade water.
                        2. Place the solvent A and B lines into the phosphoric acid solution.
                        3. Perform five 100% purges.



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                        4. Place the solvent A and B lines into a beaker containing 200mL of HPLC-
                        grade water.
                        5. Perform three 100% purges.
                        6. Place the solvent A and B lines back into the respective solvents and
                        perform a 100% purge.

                        785A Detector Dry Cell Test
                        To run the 785A Detector Dry Cell Test:
                            1. Replace the flowcell with the spare dry cell aperture (normally taped
                               inside the front compartment of the 785A detector). Do not disconnect
                               the flowcell from the plumbing. Set the Wavelength to 238nm, the
                               Risetime to 1.0secs and the Range to 0.001.
                            2. Connect the REC output to a chart recorder set to 10mV full scale and
                               leave the COMP output connected to the Procise. Set the recorder
                               chartspeed to 2mm/min.
                            3. To collect data, scroll to the Start Run view, select Run Gradient as the
                               method and click on the Start Run radio button.
                            4. As soon as the Init Sensor procedure starts, jump to the End step.
                            5. Click on Next step once the Flask cycle starts.
                        6. When the pump starts to run, jump to step #5, Start Gradient.
                        If the 610A is not displaying a “Collecting” window, ensure that 610A is
                        launched and that “Display New Procise Data” is checked under the
                        Acquisition menu.

                        Note              This procedure will start to run the HPLC gradient. If you do not
                                          want the pump to run, press the Stop key on the front of the 140.


                        Specifications
                        •   The noise should be no greater than 2 x 10-5AU.
                        •       20µAU peak-to-peak as read from the 610A.
                                0.2mV peak-to-peak on the chart recorder.
                        •       The drift should be no greater than 1 x 10-4AU/hour after warm-up.
                        •       The baseline should be free of spikes and steps apart from the initial
                                auto-zero.
                        •       If both 610A and chart recorder traces have excessive noise, steps or
                                spikes, suspect the UV lamp, detector electronics of line voltage.




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                        •       If only the 610A data is affected, suspect the COMP output, 785-to-
                                Procise signal cable, Procise I/O PCB or Procise power supply.

                        785A Detector Wavelength Test (Visual)
                        Use this procedure to check that the detector wavelength is correct.

                        Note              This is not an accurate wavelength test but will reveal gross errors
                                          that affect sequencing results.


                        To run the 785A Detector Wavelength Test (Visual):

                        WARNING           Wear UV-protective goggles


                            1. Select the WAVE> soft key on the 785A front panel.
                            2. Press 656 followed by Enter. If the display indicates that the wavelength
                               is driving towards 656nm, proceed to step #8.
                            3. Press the MORE> key.
                            4. When the next menu appears press the UTIL> key to display the utility
                               menu.
                            5. Press the MORE> key.
                            6. Move the cursor to the LIMITS field and press Next to display 190-700.
                            7. Press DONE> and then EXIT> to return to the Main menu.
                            8. Open the detector head door and observe the light from the sample
                               and reference cells. In each case, it should be a bright red color. If it is
                               not, the wavelength is incorrect or the UV lamp is not lit.

                        Testing the Wavelength Drive Mechanism
                        Procedure:
                            1. Remove the detector lid.
                            2. Power-down the detector.
                            3. Touch the limit switch arm gently with your finger. Power-up the
                               detector while pressing the ± and observe the wavelength drive
                               mechanism from the top of the instrument.
                            4. The indexer arm should first rotate counter-clockwise until each of the
                               tabbed washers interlock and force the limit switch arm to activate the
                               lower limit switch. At this point, you should feel the force on your finger




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                               as the limit switch arm moves. If you feel the limit switch arm move
                               before this point, the washers are sticky and should be cleaned with
                               compressed gas.
                            5. The indexer arm should then drive clockwise until the arm cuts the
                               optical sensor (zero order). From this point, the stepper motor moves
                               the number of steps necessary to reach 238nm. Should the limit switch
                               arm snatch at any point during the clockwise rotation and trigger the
                               limit switch, the wavelength displayed will be different from the
                               wavelength entered. Clean the drive with compressed air should this
                               occur.
                            6. Enter a wavelength of 656nm. If you hear a beep alter the LIMIT
                               parameter as follows:
                               •   Select MORE> from the Main menu.
                               •   Select UTIL> to enter the Utility screen.
                               •   Select MORE> and move the cursor to LIMIT.
                               •   Press Next to increase the LIMITs from 190 - 700 nm.
                            7. Once the drive has stopped moving, check that the displayed
                               wavelength is 656nm and check that red light is seen through the
                               reference cell.
                            8. Drive the wavelength between 200nm and 656nm several times to
                               ensure that the mechanism is functioning correctly.
                            9. Power-down the detector.
                        10. Power-up the detector and check that the wavelength drives to the last
                            value entered. If it does not, the battery on the signal electronics board
                            could have failed.

                        Note             The LEDs on the signal electronics board also display information
                                         about the limit switch settings and indexer position.


                        Replacing the 785A Detector UV lamp
                        Items Required:
                        •      New UV lamp (P/N 2900-0484)
                        •      Flat-blade screwdriver

                        To replace the 785A Detector UV lamp:

                        WARNING          Wear UV-protective goggles




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                        WARNING          Allow the lamp to cool down before touching it.


                            1. Power-down the 785A detector and allow the lamp to cool down.
                            2. Rotate the knurled knob at the rear of the detector 1/4-turn
                               counterclockwise to release the back panel catch.
                            3. A spring and catch is used to hold the lamp in place. The catch is
                               located just above the lamp. Using your fingers or a screwdriver, push
                               the catch forward and slightly upwards in order to unhook it.
                            4. Unplug the lamp and pull it horizontally off the locating pins.
                            5. Push the new lamp over the two locating pins and secure the retaining
                               spring.
                            6. Plug in the electrical connector and replace the back panel.

                        Note             There is an interlock switch in the rear compartment of the
                                         detector which disconnects the power when the back panel is
                                         removed.


                        Replacing the 785A Detector Flowcell Windows
                        Items Required:
                        •      Long flat-blade screwdriver
                        •      Small flat-blade screwdriver
                        •      16 inch-ounce torque screwdriver
                        •      1/4-inch torque wrench
                        •      Two 1/4-inch wrenches
                        •      5-10mL disposable syringe

                        Note             For most disposable syringes, the luer adaptor can be screwed
                                         directly into the flowcell adaptor. If this is not the case, an adaptor
                                         tube will have to be made up.


                        •      Compressed gas for drying
                        •      Methanol or IPA
                        •      HPLC-grade water
                        •      Pair of flowcell windows




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                        To replace the 785A Detector Flowcell Windows:
                        Removing the flowcell
                         1. Open the 785A front door panel and loosen the three plastic knurled
                            screws that secure the 785A lid in place.
                         2. Slide the lid back slightly so that you can gain access to the clamping
                            screw, located to the right of the flowcell, from above the instrument.
                         3. Loosen, but do not remove, the clamping screw.
                         4. Once the screw is loose, use a screwdriver to prize open the clamp and
                            remove the flowcell complete with inlet and outlet tubing.
                         5. Disconnect the flowcell tubing. When loosening or tightening the
                            flowcell bushings, always use a second wrench to hold the flowcell
                            adaptor in place.
                        Disassembling the cell
                         6. While holding the front and rear cell apertures in place with your
                            finger and thumb, loosen and remove the three aperture screws.
                         7. Remove the apertures. If the flowcell windows remain in the flowcell
                            body, pry them out with your fingernail or blow compressed gas into
                            one of the flowcell adaptors.
                         8. If there is material trapped inside the flowcell, remove both flowcell
                            adaptors and allow the adaptors and flowcell body to soak or sonicate
                            in HPLC-grade water.
                         9. Use compressed gas to dry the flowcell components.
                        Rebuilding and testing the flowcell
                        10. Being extremely careful not to touch the face of the window, drop one
                            of the windows into to the rear cell counter-bore and place the rear
                            aperture on top so that the aperture and flowcell body screw holes line
                            up.
                        11. While holding the rear aperture in place, drop the second window into
                            the front counter-bore and position the front aperture on top.
                        12. While holding both apertures in place with your finger and thumb,
                            loosely tighten the three aperture screws.
                        13. Tighten each screw in turn slightly so as to keep the apertures parallel
                            with the flowcell body. Tighten each screw to a final torque of 16 inch-
                            ounces.
                        14. If the flowcell adaptors were removed, screw them back into place. Use
                            the 1/4-inch torque wrench.




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                        15. Flush the flowcell with IPA or methanol using the disposable syringe.
                            This will remove any residue from inside the cell minimizing the
                            opportunity for bubbles to become trapped.
                        16. Reconnect the tubing to the flowcell. Always prevent the flowcell
                            adaptor from turning using a wrench while tightening the tubing
                            bushing. Do not over-tighten, the walls of the adaptor are quite thin.
                        17. Freerun the 140 in Manual mode at 90%B with a flowrate of 325µL/
                            min. Monitor the flowcell for any signs of leakage for 5 minutes.
                        Installing the Flowcell
                        18. Push the flowcell back into the clamp. If necessary, lever the clamp
                            open using the blade of a screwdriver. Ensure that the body of the cell
                            is flush with the clamp. The inlet should be to the right-hand side.
                        19. While ensuring that the flowcell is still loose in the clamp, rotate it so
                            that the outlet tubing is about 45° to the left of vertical and then clamp
                            the flowcell in place. This orientation prevents the PEEK from
                            becoming kinked.
                        20. Route both inlet and outlet tubing through the slot in the detector
                            head top plate (if the plate is not slotted, route the tubing so that it sits
                            in the recesses and hold in place with tape).

                        Manual Injection Procedure
                        Use this procedure to manually inject the PTH-Standard if you suspect that
                        the flask chemistry is having a detrimental effect on the results.
                        Items Required:
                        •      Syringe with luer fitting.
                        •      1/4-inch male to luer adaptor (P/N 0382-0007 in Spare parts kit 2)
                        •      1/4-inch female to 5/16-inch female union (P/N 0403-0280 in Spare
                               parts kit 2).

                        To run the Manual Injection Procedure:
                        To make up a 0.1pmol /µL solution of PTH-Standard (8 pmol injected onto column)
                            1. Fill a clean measuring cylinder with approximately 5mL of HPLC grade
                               water.
                            2. Add 10µL of each PTH-Standard stock solution, including PMTC, to
                               the measuring cylinder.
                            3. Make up to a total volume of 10mL using HPLC grade water and mix
                               well.
                        Create a Manual Injection cycle


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                         4. Scroll to the Cycles & Procedures view and select Flask Cycle.
                         5. Select Run Gradient from the list of Flask cycle options.
                         6. Click and hold on File from the top menu bar and select Save Cycle/
                            Procedure as...
                         7. Name it Manual Injection and click OK.
                         8. Highlight the step #1, Begin, and then highlight Fxn# 226, Load
                            Position, from the function list.
                         9. Click Insert. The Global time will be assumed for the inserted step
                            which is OK.
                        10. Highlight, what is now, step #5, Wait, and insert Fxn# 223, Inject
                            Position, in the same way.
                        11. Click and hold on File from the top menu bar and select Save Cycle/
                            Procedure to save the cycle shown below.
                        Table 4-2. Manual Injection Flask Cycle

                             Step      Function Name          Num.        Value          Global
                               1       Begin                   258           0              -
                               2       Load Position           226           0             √
                               3       Wait                    257          30              -
                               4       Prepare Pump            227           0              -
                               5       Wait                    257         480              -
                               6       Inject Position         223           0             √
                               7       Start Gradient          232           0              -
                               8       Wait                    257         690              -
                               9       Wait                    257         690              -
                              10       End                     259           0              -


                        To incorporate the Flask Cycle into a Method:
                         1. Scroll to the Sequence Methods view and select the User Defined
                            Method.
                         2. For the Default line, choose None for Cartridge, Manual Injection for
                            Flask and Fast Normal 1 for the Gradient.
                         3. From the File menu, Save Method as Manual Injection.
                        Performing the Manual Injection
                         4. Purge the pumps.




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                         5. Remove the 5/16 -inch fitting from port #42 and connect it to the white
                            Teflon 5/16-inch to 1/4-inch union.




                                              Luer Apt       Union
                            Syringe                                            Injector inlet line
                                             0382-0007     o403-0280

                         6. Install the 1/4-inch to luer adaptor as shown above.
                         7. From the Start Run view, set up a run using Manual Injection as the
                            Method. Set the number of cycles to “1” and click the Start Run radio
                            button.
                         8. When the Init Sensor procedure starts, jump to the last step of the Init
                            Sensor procedure if you wish.
                         9. If the column has reached the correct temperature (LED is cycling),
                            click Next step if the Flask cycle is “Waiting for Temperatures”.
                        10. As soon as the “Inject Position” LED on the front panel display is OFF,
                            connect the empty syringe to the luer fitting and flush out the sample
                            loop.
                        11. Load the PTH-Standard solution into the syringe and inject it manually
                            into the sample loop.
                        12. After the column has been allowed to equilibrate, the injection will
                            automatically take place.

                        Testing the Injector for blockages
                        To check the HPLC flowpaths for blockages:
                         1. Remove the inlet end cap assembly from the column holder and place
                            it in a beaker.
                         2. On the 140C, select the Manual Control mode and freerun the pumps
                            at 325µL/min @ 50% B. The pressure should not rise above 100psi. If
                            it does so, first replace the Newguard cartridge. If the flowpath is still
                            blocked, determine the source of the blockage by breaking fittings in
                            the flowpath consecutively from the injector to the dynamic mixer.
                         3. From the Manual Control view on the Macintosh, toggle the injector
                            position. The pressure should not vary by more than 5psi.
                         4. Stop the pumps and replace the column end assembly.




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                        Replacing the Injector Rotor Seal
                        The rotor seal used in this 7716 Rheodyne valve is identical to that used in
                        the 120A purge valve (P/N 0173-0015). We recommend that the seal be
                        changed once a year.
                        Items required:
                        •       9/64-inch hex wrench
                        •       1/4-inch wrench
                        •       Rotor seal (0173-0015)

                        To replace the Injector Rotor Seal:
                            1. Remove the safety panel.
                            2. Loosen the pre-tee fittings holding the injector full and load sensors in
                               place. Slide these fluid sensors up the tube out of the way.
                            3. It is unnecessary to disconnect lines from the Rheodyne stator but if
                               you require more room, this can be done.
                            4. Loosen the three hex screws and pull the stator from the assembly. The
                               stator face will either come off with the stator or remain with the valve
                               assembly. There are three dowels in the stator face which allow it to be
                               orientated correctly on the stator.
                            5. The two hex screws holding the retainer ring in place will now be
                               exposed. Loosen and remove these two screws.
                            6. Pull off the retainer ring. The best way to do this is to screw two of the
                               hex screws a little way into two of the retainer ring’s threaded bores.
                               Grasp the screws and pull the ring away from the rest of the assembly.
                            7. With your finger and thumb, pull the rotor seal off the four location
                               pins. If the white isolation seal behind the rotor seals appears worn,
                               replace it (P/N 0173-0014).
                            8. Inspect the rear of the original rotor seal and note the location of the
                               impression made by the shaft bore. Orientate the new rotor seal the
                               same way.
                            9. Inspect the stator face for scratches and replace if necessary.
                        10. Reassemble the injector but do not fully tighten the stator screws.
                        11. Open the plumbing plate to expose the valveblocks and remove the
                            lock ring from the back of the injector assembly.
                        12. Turn the tensioning nut 1/2-turn counter-clockwise.
                        13. Fully tighten the stator screws.



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                        14. Turn the tensioning ring clockwise until the two red marks are aligned.
                        15. Replace the lock ring and close the plumbing plate.




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                        Macintosh Computer
                        Procedures using keystroke commands
                        Task                           Action                         Outcome
                        Rebuild Desktop                Hold down the option and       Remove redundant
                                                       command keys as you reboot information from the desktop
                                                                                      management file
                        Reset the PRAM                 Hold down the P and R keys as
                                                       you reboot
                        Reboot without extensions      Hold down the Shift key as you Prevent extension programs
                        active                         reboot                         running in background


                        Formatting the Hard Drive
                        Power PC 7200/90, System 7.5

                        Caution           Performing a low level format will delete all files from the hard
                                          drive. Ensure that important files are backed-up first.


                        Items required:
                        •       System software

                        To format the hard drive:
                            1. Make a back-up copy of the Procise Chemistry file, 610A data files and
                               any user-specified files.
                            2. Load the system software CD-ROM and restart the Mac while holding
                               down the “C” key. As soon as the selftests have passed (smiley face
                               appears) release the “C” key and hold down the “Shift” key until the
                               message “Extensions off” appears.
                            3. Once the Mac has booted from the CD, open the Utilities folder.
                            4. Double-click on Drive Set-up and highlight the hard disk.0
                            5. Select the Functions menu from the upper menu bar and choose
                               Initialization options.
                            6. Once the dialog box appears, click on (X) both the Low level format
                               and Zero data check boxes and click OK.
                        Install the operating system
                            7. Once the formatting is complete, open the System Software Install
                               folder.
                            8. Double-click the System installer to install the operating system.



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                        Install the Procise /610 software
                         9. Load Procise disk #1 and double-click on Installer.
                        10. Click on OK when the splash screen appears.
                        11. Click Customize and select all but the Chemistry file. Follow the
                            installer instructions.
                        12. Install the Chemistry file that was backed-up earlier into the PROCISE
                            folder (in the Preferences folder).
                        13. Save an alias of the Procise application in the Startup items folder.
                        14. Restart the Macintosh computer.




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                        5 Diagnostics
                          Dialog Boxes
                          A dialog box is a window that the Macintosh displays when a software
                          application requires input from the user. It will normally be in the form of
                          an error alert.
                          A dialog box can be generated by the Macintosh operating system or by any
                          application which is running, such as Procise or 610A. It will usually be
                          apparent from the dialog box text as to which application was responsible
                          for generating it. Most important is that while a dialog box is displayed, the
                          rest of the screen is “grayed out” and the application responsible for
                          generating it effectively freezes until the user clicks on one of the radio
                          buttons within the box.
                          For example, the Procise application responsible for collecting data from
                          the sequencer will no longer communicate with the sequencer while the
                          dialog box is present. If the user does not respond to one of the dialog box
                          prompts, the sequencer will eventually be forced to pause the run because
                          there will not be enough space left in the sequencer’s data buffer to store
                          another cycles-worth of data.

                          Procise Dialog Box Messages
                          During a run, the critical functions of the sequencer are monitored for
                          correct operation. Should a failure be detected, a dialog box describing the
                          error will appear on the computer screen.




                          Figure 5-1. Dialog box generated by the Procise application




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                        It will present two choices: Pause or Continue. If no action is taken, the
                        instrument will pause itself at the end of that cycle to conserve the chemicals
                        and prevent unnecessary chemical interaction with the sample. Once a
                        dialog box has been generated, you must select one of the radio buttons
                        within it in order to access other menus.

                        Caution         While a dialog box is displayed on the screen that has been
                                        generated by the Procise application, there is no
                                        communication between the sequencer and the Macintosh
                                        computer. This may cause the sequencer to pause. One of
                                        the Dialog box prompts must be selected to restore
                                        communication.


                        Detailed information about the error will be reported in the Event log.
                        Always take into account all Event log messages that have been reported
                        since the start of the run.

                        Model 610A Dialog Box Messages

                        Caution         Never set up the 610A to automatically launch when the
                                        Macintosh is booted up.


                        File already exists
                        In order to prevent accidental file deletion, the Model 610A Data Analysis
                        application will generate this error message should an attempt be made to
                        use an existing filename. The following cases will cause this message to be
                        generated:-
                        •     A filename entered in the Start Run screen is identical to an existing
                              610A filename. It is good practise to include the date as part of the
                              filename or some other strategy to ensure exclusivity.
                        •     If the 610A application is Quit during data collection, the message may
                              be generated after the application has been relaunched.




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                        Figure 5-2. File already exists 610A message

                        No data collected for 12 hours
                        When a run is completed or stopped by the user, an “End of Run” flag will
                        be set in the virtual A/D file. The 610A constantly monitors these files and
                        transfers any new data to a 610A file of the same name. If the “End of Run”
                        flag is set, the 610A will delete the virtual A/D file because, by this time, a
                        610A version of the complete file will exist.
                        If, for some reason (probably a system crash), the flag does not get set, the
                        610A will generate the above dialog box message.
                        To avoid this message, always check the PROCISE folder (in the Preferences
                        folder) for residual virtual A/D files after a “crash”.




                        Figure 5-3. No data collected for 12 hours

                        File error
                        When the 610A is launched, it searches for virtual A/D files and attempts to
                        open them. If a file error occurs as soon as the 610A application is launched,
                        it could be caused by a corrupted virtual A/D file. Follow the steps below:-
                         1. Quit the 610A application.
                         2. Move any virtual A/D files out of the PROCISE folder.
                         3. Relaunch the 610A.


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                         4. If the 610A launched successfully, move the virtual A/D files back into
                            the PROCISE folder, one at a time, until the error recurs. Quit the 610A
                            and trash the offending virtual A/D file.
                        As with most other applications, 610A will also generate dialog box messages
                        if illegal entries are attempted.

                        Mac Operating System Dialog Box Messages
                        Errors of this nature can be due to corrupt files or a computer malfunction.
                        Re-booting the Macintosh may be necessary to clear the error condition. It
                        is beyond the scope of this manual to detail and troubleshoot this type of
                        error.




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                        The Event Log
                        The Event Log exists as a file on the Macintosh computer hard disk and
                        serves as the output for the system's many diagnostic routines. Each time an
                        event code is generated, it is stored in the event buffer which is part of the
                        sequencer's battery-backed memory (RAM). At 5-second intervals, the
                        contents of the event buffer will be transferred to the Macintosh computer.
                        The event code will be interpreted and appended to the existing Event log
                        file (PROCISEEventlog) on the hard disk. Immediately after the transfer,
                        the event buffer will be empty. Up to 19 event codes can be stored in the
                        sequencer’s event buffer. If more than 19 messages are generated between
                        transfers, some messages, starting from the oldest, will be overwritten.
                        Besides the event codes that are interpreted as error messages, the event
                        buffer also temporarily stores the fluid sensor data which is generated when
                        the box, Always Report Sensor Data, in the Preferences menu, is checked.
                        Should the hard disk memory used for the Event log file reach 100kB, the
                        operator will be prompted to print out the file and then delete it. The
                        Procise software will not automatically delete the Event log.

                        Opening the Event Log
                         1. Ensure that the PROCISE application is running on the Macintosh
                            computer and that the COMM light is illuminated through the 490A
                            front panel visor.
                         2. Select Event Log from the scrolling menu.




                        Figure 5-4. Procise Event log




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                        Deleting the Event Log
                         1. Open the Event Log.
                         2. Select Delete from the pull-down File menu
                         3. In response to the dialog box, enter the date before which the deletion
                            is to take effect.
                        Printing the Event Log
                         1. Open the Event Log.
                         2. Select Print from the pull-down File menu. Alternatively, press the “P”
                            and “ “keys simultaneously.

                        Note            The PROCISE software does not assign page numbers to the
                                        Event log.


                        Using “WORD” to Archive and Print the Event log
                        If the “Word” application is loaded onto the Macintosh, it is convenient to
                        convert the Event log file into a Word file if it is necessary to print the
                        document in a different format or archive Event logs on the computer once
                        they exceed 100kB.
                         1. Launch the Word application.
                         2. From the pull-down File menu, select Open.
                         3. Select All Files from the List files of type scroll menu on the subsequent
                            dialog box.
                         4. Locate PROCISEEventlog from the Select document scroll menu. It is
                            in the PROCISE folder which, in turn, is in the Preferences folder.
                         5. Double-click on PROCISEEventlog to open this file as a Word
                            document.
                        Save the Event logs in an archive folder
                         6. Select Save from the pull-down File menu.
                         7. Click on Desktop.
                         8. Click on New folder and name it “Event log archive”.
                         9. Name the file “Event log (date)” and click on Save.
                        10. The file, PROCISEEventlog may now be deleted.




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                         Event Log Messages
                        Status Messages
                        The status messages report the activity and run conditions for each
                        cartridge. They are listed below for reference. Where the message is not self-
                        explanatory, more detail is given.
                        Both Cartridge and flask have been paused

                        Temperatures and/or pressures were altered by user

                        Cartridge (ABCD) started. File name is (Filename)

                        Method is (Sequencer Method)

                        Cartridge (ABCD) completed

                        Cartridge (ABCD) terminated. All cartridges waiting to start have been stopped.

                        Hold executed on step (n) of cartridge cycle (n)

                        Hold executed on step (n) of flask cycle (n)

                        Pause executed on step (n) of cartridge cycle (n)

                        Pause executed on step (n) of flask cycle (n)

                        Jump from step (n) to step (m) executed during cartridge cycle (n).

                        Jump from step (n) to step (m) executed during flask cycle (n).

                        (Procedure) started.

                        During cycle xx the “Ready to Transfer to Flask” step was executed before the “Ready to
                        Receive” step.

                        During cycle xx the “Ready to Receive” step was executed more than 10 seconds before
                        the “Ready to Transfer to Flask” step.

                        In cartridge cycle xx the “Transfer Complete” function precedes the “Ready to Transfer to
                        Flask” function. The sequencer is paused.

                        In cartridge cycle xx there is only one synchronization function. For this cycle to
                        synchronize properly, both “the Ready to Transfer to Flask” and the “Transfer Complete”
                        functions must be included. The sequencer is paused.




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                        In flask cycle xx there is a “Ready to Receive” function but in the corresponding cartridge
                        cycle the “Ready to Transfer to Flask” and the “Transfer Complete” functions were omitted.
                        The sequencer is paused.

                        Undercarriage cycle xx there is a “Ready to Transfer to Flask” and a “Transfer Complete”
                        function, but in the corresponding flask cycle the “Ready to Receive” function was omitted.
                        The sequencer is paused.

                        Execution of cold start (all ram has been initialized)


                        On-line Error Messages
                        On-line error messages report failures that occur during routine operation.
                        They are listed below together with an explanation of their meaning.
                        Fluid detection errors
                        During a sensor-controlled function, the respective fluid sensor needs to
                        detect liquid continuously for a predetermined amount of time in order to
                        constitute a successful delivery. Should the function time-out before this
                        criteria is achieved, a fluid detection error message will be generated.
                        Failures of this kind will automatically pause the run at the end of the
                        current cycle for all sensors except the Transfer sensor and Sample loop full
                        sensor.
                        During step (a) of cycle (b), fluid was not detected by the (c) sensor. (The sequencer will
                        pause at the end of this cycle).
                        Dry = (d)                        Threshold = (e)                Average wet = (f)
                        dry,           wet,              dry,           wet,            dry,          wet,
                        (g)            (h)               (I)            (j)             (k)           (l)
                        dry,           wet,              dry,           wet,            dry,          wet,
                        (m)            (n)               (o)            (p)             (q)           (r)
                        dry,           wet,              dry,           wet,            dry,          wet
                        (s)            (t)               (u)            (v)             (w)           (x)


                              Definition of variables
                              a. The step number of the function that is using the fluid sensor.
                              b. The cycle number.
                              c. The fluid sensor designated by the function.
                              d. The “Dry” reading established during the “Init Sensor” procedure.
                              e. The “Threehold” which is 1.5 X the “Dry” reading.
                              f.   The average of all readings detected above the threshold through
                                   the duration of the function. (If no readings were above the
                                   threshold, this value will be “0”.




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                              g. The number of consecutive “Dry” readings detected from the start
                                 of the function. (A reading is taken every 5.2ms).
                              h. The number of consecutive “Wet” readings detected since the first
                                 “Dry” to “Wet” transition.
                              i.   The number of consecutive “Dry” readings since the first “Wet” to
                                   “Dry” transition.
                              j.   through (x)
                                   Numbers will continue to be reported after each transition for the
                                   duration of the function. If there are less than 17 transitions, the
                                   later, redundant readings will be “0”.
                        Argon tank pressure too low
                        During the Begin step of a cycle or procedure, the argon tank pressure is
                        monitored by means of the 0-100psi pressure transducer located on the
                        pressure management PCB. If the pressure drops below 60psi, the sequencer
                        will pause. Expected pressure drops associated with either injector or
                        vacuum generator actuation are ignored.
                        Argon tank pressure is too low. The sequencer is paused.


                        Cartridge, flask or column temperature setpoints
                        During the Begin step of a cycle, the cartridge, flask and column
                        temperatures are monitored and an error message is generated if either fail
                        to reach setpoint within 20 minutes.
                        Unable to reach cartridge, flask or column temperature setpoints. The sequencer is
                        paused.


                        Communication down between 140 and Procise (sequencer)
                        The 490 is no longer able to communicate with the HPLC pump via the
                        RS232 communication link.
                        Communication with the HPLC was lost. Reset the HPLC.


                        Insufficient data collection memory for cycle
                        During the Begin step of a cycle, the Procise determines whether there is
                        enough free space in the data buffer to collect that cycle's chromatogram. If
                        there is not, the Procise will wait at the Begin step for up to 4 minutes. If
                        there is still not enough memory available in the data buffer, the above
                        message will result.




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                        Because this error normally arises as a result of a Sequencer-Macintosh
                        communication failure, this message will not get transferred from the
                        Sequencer to the Macintosh until the communication is re-established.
                        Insufficient data collection memory for cycle (a). The sequencer is paused.


                              Definition of variables
                              a. The current cycle
                        Invalid fluid sensor dry reading
                        The dry reading for each fluid sensor is established automatically at the start
                        of each sequencing run during the Init Sensor procedure. These readings
                        are stored in battery-backed memory until overwritten during subsequent
                        Init Sensor procedure execution.
                        If the instrument is reset (by pulling the memory card), these dry readings
                        will be lost. If a function utilizing a sensor is executed after a reset and prior
                        to running the Init Sensor procedure, the above message will result.
                        Sensor (a) does not have a valid dry reading.


                              Definition of variables
                              a. Fluid sensor identity number (see operator assistance card).
                        A/D converter malfunction
                        Every time the 24-bit A/D converter completes a conversion, a flag is set to
                        “true”. Then, when the contents of the A/D is read, the flag is reset to “false”.
                        If the flag is not “true” at some point within the sampling interval, an error
                        will be reported.
                        The 24-bit A/D converter has malfunctioned. Some data may have been lost. The
                        sequencer will pause at the end of cycle (a).


                              Definition of variables
                              a. Cycle at which error was detected.
                        Power failure (Idle)
                        This error is reported if a power failure occurs while the sequencer is idle.
                        Power failure occurred on mm/dd/yy, at hh:mm:ssc


                        Power failure (Sequencing)
                        This error is reported if a power failure occurs while sequencing.
                        A power fail occurred while sequencing. The run will be paused on the End step of the
                        active cycle.




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                        Rheodyne injector valve in wrong position
                        A mechanical microswitch is used to distinguish between load and inject
                        position. It should be closed open when in the inject position and closed
                        when in the load position. This switch is also responsible for starting the
                        HPLC gradient. If the switch is seen to be open at the start of the “load
                        injector” function or closed at the end, the respective error will be reported.
                        When the “load injector” function was started, the Rheodyne valve must be in the load
                        position. When starting step (a) of cycle (b), the Rheodyne valve was in the inject position.


                        or
                        When the “Load injector” function is finished, the Rheodyne valve must be in the inject
                        position. When finishing step (a) of cycle (b), the Rheodyne valve was in the load position.


                              Definition of variables
                              a. Step # at which failure was detected.
                              b. Cycle # at which failure was detected.
                        Event buffer overrun
                        If more than 19 event messages are generated before the contents of the
                        event buffer can be transferred to the Macintosh computer hard disk, an
                        event code which translates into the above message will be loaded into the
                        last location in the event buffer.
                        This message will most likely occur when a former error generated a dialog
                        box on the screen. The dialog box effectively stops all Mac-490
                        communication until its prompt has been answered. The event codes
                        generated while the dialog box is on the screen may be sufficient to overflow
                        the event buffer before the communication is re-established.
                        If the system is set up to “Always report sensor data”, this data is stored
                        temporarily in the event buffer before being transferred to the respective
                        file on the hard disk. Therefore, if an Event Buffer overrun message is
                        reported there may also be some information missing from the sensor data
                        file.
                        Event buffer overrun. Some event messages may have been lost.


                        Pass/Fail Messages with preset Criteria
                        During execution of the power-up routine (POST), the Electrical selftest
                        procedure or specific functions, Pass or Fail status messages are generated.
                        Below is a list of these messages for reference. The tests that generate these
                        messages are described in detail later in this chapter.
                        (FAIL) The 12 bit A/D has malfunctioned.

                        (FAIL) The upper MUX has malfunctioned (U1).



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                        (FAIL) The lower MUX has malfunctioned (U2).

                        (FAIL) The fluid sensor MUX has malfunctioned (U21).

                        (FAIL) The 24 bit A/D 0 volt test has failed.

                        (FAIL) The 24 bit A/D 124 mV test has failed.

                        (FAIL) The 24 bit A/D 1 volt test has failed.

                        (FAIL) Cart. X heater failure. Open or short to ground.

                        (FAIL) Cart. X heater failure. Overvoltage, thermal shutdown or short to VCC.

                        (FAIL) Column heater failure. Open or short to ground.

                         (FAIL) Column heater failure. Overvoltage, thermal shutdown or short to VCC.

                        (FAIL) Flask heater failure. Open or short to ground.

                        (FAIL) Flask heater failure. Overvoltage, thermal shutdown or short to VCC.

                        (FAIL) Continuity failure on valve xx.

                        (FAIL) Failure detected on Pressure Management Board.

                        (FAIL) First strike pulse failure.

                        (FAIL) Second strike pulse failure.

                        (FAIL) Load Rheodyne valve failure. Open or short to ground.

                        (FAIL) Load Rheodyne valve failure. Overvoltage or short to VCC.

                        (FAIL) Inject Rheodyne valve failure. Open or short to ground.

                        (FAIL) Inject Rheodyne valve failure. Overvoltage or short to VCC.

                        (FAIL) Failure encountered when moving the rheodyne valve to the inject position.

                        (FAIL) Failure encountered when moving the rheodyne valve to the load position.




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                        Pass/Fail Messages with adjustable Criteria
                        The pass/fail threshold for certain functions, such as those used in Leak
                        procedures, is defined by the adjustable parameter of a “Threshold”
                        function within the procedure. The event log messages generated in these
                        situations will be described in the leak testing information.




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                        Door Panel Visor Lights
                        PROCISE™

                        READY

                        COM

                        SEQ

                        ERROR


                        There are 4 diagnostic L.E.Ds (Light Emitting Diodes) and an illuminated
                        PROCISE logo visible through the front panel visor. These can be used to
                        determine the operational status of the system as follows:

                        PROCISE Logo
                        If the PROCISE logo is illuminated, the instrument is powered-up and the
                        main fuses, accessible from the rear of the instrument, are in good order.
                        The cartridge and flask back-lights should also be illuminated in this
                        situation.
                        If the PROCISE and back-lights do not illuminate after the instrument is
                        switched on, first check that power is being supplied to the 490 by plugging
                        the power cord into the pump or detector. If the alternative device powers-
                        up correctly, check the 490's main fuses. There are replacement fuses in the
                        spare parts kit.

                        Caution         Use only fuses of the same current rating as those removed.
                                        4A for an incoming line voltage of between 90 and 180VAC
                                        and 2A for an incoming line voltage of between 190 and
                                        264VAC.


                        Only rarely do fuses blow for no reason. Should the fuses blow again after a
                        short period of time, contact Applied Biosystems Service department for
                        assistance.

                        READY LED
                        When the 490 is powered-up, a program is executed which initializes and
                        tests the various devices on the CPU card. If this “boot-up” routine is
                        successful, the READY LED will illuminate.




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                        If the PROCISE and back lights are illuminated but the READY LED fails to
                        turn on approximately 15 seconds after power-up, power-down the
                        instrument and reseat the Memory card that protrudes from the upper, left-
                        hand side of the 490. Power-up again and observe the READY LED. If it still
                        fails to illuminate, there may be a problem with the CPU PCB EPROMs, U26
                        and U27, or with the CPU circuitry itself.
                        Power-down the instrument and short-out (connect together) the two W6
                        jumper pins. If the instrument now boots up correctly, the EPROMs should
                        be replaced. Otherwise, the CPU itself is most likely the culprit.

                        COM LED
                        The COM LED turns on once the sequencer and Mac establish
                        communication.
                        If the COM LED is off while the sequencer is running and the sequencer is
                        unable to download data to the Mac, the run will continue until the data
                        buffer becomes full. During this time, the Procise Monitor view will not
                        update.
                        A dialog box generated by the Procise software will inhibit communication
                        until one of the prompts is selected.

                        To recover from lost communication:
                         1. Answer the dialog box prompt.
                         2. Reboot the Mac.
                         3. Cycle the sequencer power and then reboot the Mac.
                         4. Reset the sequencer (power-up without MEL card) and reboot the Mac.
                            This is the least desirable option because it will delete any sequence
                            data or Event log messages that is stored in the sequencer’s memory.

                        SEQ LED
                        The SEQ LED is on whenever the sequencer is running.

                        ERROR LED
                        The ERROR LED indicates a CPU malfunction. For more detailed
                        information on the error, check the diagnostic LEDs on the inner panel.




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                        Powering-up the Procise System
                        Power-up sequence
                        To successfully power-up the complete system, it is important that certain
                        individual components are switched on in the following order:
                         1. The 140C solvent delivery system.
                         2. The Procise (490A) protein/peptide sequencer.
                         3. The printer
                         4. The Macintosh computer with the PROCISE application set up to
                            launch at start-up.
                         5. The remaining components.

                        Procise Power-up Sequence of Events
                        When the Procise is powered-up, a Power-on Self Test (POST) is run
                        automatically. If the POST detect s a failure, it will generate an error code in
                        the Event buffer. See Section- Power-on Self Test and Electrical Test for more
                        information.
                         1. The power supply cooling fan begins operating.
                         2. The yellow LEDs behind the conversion flask, reaction cartridges and
                            the PROCISE logo illuminate.
                         3. The vacuum assist system operates if the system's vacuum is low and
                            argon pressure is high enough.
                         4. The ERROR #1 LED on the front panel may illuminate.
                         5. The bank of 8 LEDs on the 68K PCB light in the following sequence:
                              a. All 8 LEDs light.
                              b. All LEDs turn off when the ROM test passes.
                              c. The top (first) LED turns on.
                              d. The second LED turns on to signify that the 68901 multi-purpose
                                 I/O IC test has initialized correctly.
                              e. The third LED turns on.
                              f.   The fourth LED turns on to signify that the RAM test has passed.
                              g. The remaining LEDs light in consecutive order but no further tests
                                 are carried out. If any of these CPU tests fail, the respective
                                 combination of error LEDs will remain illuminated on the front
                                 panel (See section- Power-on Self Test and Electrical Test).


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                         6. A hissing is heard from the Rheodyne actuator valves.
                         7. The ERROR #1 LED turns off.
                         8. The READY LED behind the door panel visor will illuminate (in
                            approximately 15 seconds).
                         9. Any POST errors will be loaded into the Event buffer.

                        Macintosh Computer power-up Sequence
                        An alias of the PROCISE application must be stored in the Start-up Items
                        folder to automatically launch the application at power-up or restart.

                        IMPORTANT       PROCISE should be the only application stored in the Start-up
                                        Items folder.


                         1. The progress of the various steps in the PROCISE launch process is
                            displayed on the screen. During this time, a clock icon will
                            intermittently appear as the different steps are executed. It is normal
                            for the screen to appear frozen for short periods of time. However,
                            should the screen freeze for more than 2 minutes, it is possible that a
                            lock-up has occurred. Refer to section (Resetting the system).
                         2. Once the PROCISE application is launched, the Model screen will be
                            displayed. It should report that the 140C is attached.
                         3. The COMM LED will illuminate behind the 490A visor.
                         4. Any sequencer Power-on Self Test (POST) Event codes generated will
                            be interpreted and displayed as a dialog box and reported as a message
                            in the Event log (if the sequencer was powered-up first).




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                        System Selftest
                        An electrical selftest (POST) is performed every time the 490A is powered-
                        up. The first part of the test is a CPU board test. Any failures detected during
                        this stage will illuminate the respective combination of error LEDs on the
                        front panel. If the CPU is OK, subsequent functional tests are performed.
                        The results of these will be displayed as a dialog box on the computer screen
                        and will also be reported in the Event log.

                        Components Tested
                        The CPU board test
                        •   MEL card.
                        •       RAM.
                        •       68901 IC.
                        •       Basic functionality of 68k CPU PCB.
                        The functional test
                        •   Valve continuity.
                        •       The valve actuation voltages.
                        •       Heaters for open or short circuit.
                        •       Operation of one of the 3 D/A converters on the pressure board.
                        •       12-bit A/D.
                        •       24-bit A/D.
                        •       The 3 multiplexers on the I/O board.
                        •       The Rheodyne injector operation.

                        Performing the Electrical Test
                        The functional section of the Power-on Self Test (POST) can be run as a
                        procedure in its own right as follows:-
                            1. The 490A must be in an Idle state.
                            2. Scroll to the Test screen and click on the Electrical Tests radio button.
                            3. Select Electrical Test Procedure and click on the Start radio button.
                            4. The results of the test are reported in the Event log.




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                        Self-testing Individual Components
                        Because the Electrical Test Procedure is made up of a list of functions which
                        test specific components, it is possible to use any combination of these
                        functions to create an alternative procedure.




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                        Selftest Descriptions
                        CPU Board Tests
                        The low level tests essentially check the operation of the CPU PCB and do
                        not require Macintosh-490 communication. The results of these tests are
                        communicated using LEDs. The only way to run these tests is by cycling the
                        power.
                        ROM test
                        The ROM (Read Only Memory) test checks the integrity of the MEL card
                        memory. When the MEL card was programmed, a checksum was calculated
                        and stored on the card. The test starts by turning on the 8 LEDs on the CPU
                        PCB. Then, the checksum is re-calculated and compared with the stored
                        version. If they agree, the LEDs will turn off and the CPU test will continue.
                        If they disagree, the CPU LEDs will remain on and both the front panel
                        ERROR 3 LED and the ERROR LED behind the door panel visor will turn
                        on to signify that the ROM has become corrupted and that the test has
                        failed.
                        68901 Initialization Test
                        The 68901 is a multi-purpose I/O IC located on the CPU PCB. As the test is
                        started, The top (first) LED of the bank of 8 on the CPU PCB turns on.
                        Initialization information is then sent to the 68091's registers. If the 68091
                        acknowledges receipt of this information, the second LED turns on to signify
                        that the test was successful. Otherwise, the test has failed and the instrument
                        will freeze in this condition.
                        RAM Test
                        The RAM (Random Access Memory) is located on the CPU PCB. As the test
                        is started, the third LED of the bank of 8 on the CPU PCB turns on. Each
                        RAM location will then undergo a write/read test. If the test is successful, the
                        subsequent LEDs will turn on sequentially. If the test fails, both the ERROR
                        4 LED on the front panel and the ERROR LED behind the door panel visor
                        will illuminate.

                        Note            The EPROM ICs U26 and U27 are not tested.


                        Functional Tests
                        The functional tests check beyond the CPU PCB. The results are reported as
                        a detailed message in the Event log.Therefore, it is essential that Macintosh-
                        490 communication is established in order to view the results. When the
                        power is cycled, the functional tests will automatically run as soon as all of
                        the CPU tests have passed. Alternatively, the Electrical Test Procedure can be
                        run independently from the Test menu.



5-20                    5 Diagnostics                                                         January 1997
PE Applied Biosystems




                        Test Valves
                        The Angar, Lisk and 3-way valves are driven by intelligent ICs (TPIC2801)
                        which have both an internal shift register and a valve driver portion. They
                        also have current limiting beyond 1.2A so accidental shorting during
                        troubleshooting should not be a problem. A unique feature of these devices
                        is their ability to test for an open load condition without additional circuitry.
                        To perform this test, 1's (valve OFF) are clocked into the shift registers and
                        latched to the drivers to turn all the valves off. The next high-to-low
                        transition of the SIOE line sets the IC into diagnostic mode. Starting with bit-
                        96, each bit is serially clocked out of the registers. If the bit is a 1, it signifies
                        that the respective valve is open circuit and the fault will be reported.
                        (FAIL) Continuity failure on valve xx.



                                                         24 VS


                                                                           Solenoid Strike
                                                                           Voltage Test

                                                         VSOL



                                                                     MUX        12-bit A/D
                                                             TVSOL
                                                                                             Data bus
                                                                     U1            U3




                                                                           MUX addressing
                        Figure 5-5. Valve voltage test

                        The voltages VSOL1 (First strike voltage) and VSOL2 (Second strike
                        voltage) are used to respectively strike and hold the reaction and flask
                        related valves. The first strike voltage is turned on for 110ms and a portion
                        of the voltage at the centre of this pulse is digitized by the 12-bit A/D
                        convertor. This value is compared with the expected minimum (20.3V). The
                        second strike voltage test is done in the same manner.
                        (FAIL) First strike pulse failure.


                        (FAIL) Second strike pulse failure.




January 1997            5 Diagnostics                                                                    5-21
PE Applied Biosystems




                        Test Heaters



                                                                 Heater 1                   Heater 2
                                         Latch              Driver


                                         U45
                                                                                                         Heater
                                                 Fault                                                   Test
                                               O/C or S/C
                        Data
                        bus

                                         U43


                        Figure 5-6. Heater test

                        The heaters are driven by intelligent devices (TPIC2404) These devices have
                        over-current protection and open/short circuit sensing which is used in the
                        selftest. The IC is a dual driver but only has one short/open status line. By
                        monitoring this status line and each heater return line, it is possible to
                        determine which heater is faulty. The heater return line is monitored while
                        the heater is turned off. If the heater is open circuit or shorted to ground,
                        this line will be at 0 volts and an error message will be generated for that
                        particular heater. The heater is then turned on. If there is a short to the drive
                        voltage (24V), the status line will go low due to an over-current condition
                        and the heater return line will remain high for that particular heater.
                        (FAIL) Cart. x heater failure. Open or short to ground. (FAIL) Cart. x heater failure.
                        Overvoltage or short to VCC.


                        (FAIL) Column heater failure. Open or short to ground. (FAIL) Column heater failure.
                        Overvoltage or short to VCC.


                        (FAIL) Flask heater failure. Open or short to ground. (FAIL) Flask heater failure.
                        Overvoltage or short to VCC.


                        Test Pressure Board
                        The pressure PCB uses a special type of D/A (Digital to Analogue) convertor
                        package. It contains a D/A convertor and four selectable comparators. The
                        “desired” digital pressure is converted to an analogue equivalent and




5-22                    5 Diagnostics                                                                     January 1997
PE Applied Biosystems




                        compared with one of four selectable analogue inputs from the pressure
                        transducers. The output of the comparator will be either high or low, and
                        this signal is used to switch the respective Lee valve.

                                                                  Comparator
                                   A0, A1                         selection
                                                                                      24 V
                                            D/A
                                            Conv.
                          Data Bus                   PX9                                        PSI
                          Set Pressure
                                                    PX10

                                                                                                Test
                                                                                                (TP3)

                          2.5 V
                          Ref                       N/C


                                            U 15 MAX516                     Pressure PCB Test

                        Figure 5-7. Pressure board test

                        Although the Pressure PCB uses three such D/A devices, only one (U15) is
                        used to test the board. One of the analogue inputs is a 5V reference. The first
                        part of the selftest sends the digital equivalent of 5.1V to U15 and looks for
                        a “low” at the test comparator output. Then, digital 4.9V is sent to the chip
                        and a “high” is required at the test comparator output in order for the test
                        to pass. If these criteria are not met, the test fails.
                        (FAIL) Failure detected on pressure management board.


                        Test 12-bit A/D
                        The 12-bit A/D is one of the main components of the I/O PCB. The
                        analogue signals which it converts include fluid sensor output, heater
                        thermocouple output, pressure transducer output and a number of other




January 1997            5 Diagnostics                                                                   5-23
PE Applied Biosystems




                        voltages for test purposes. With so many inputs to deal with, three
                        multiplexers (MUXes) are necessary. These are designated as the upper
                        MUX, lower MUX and fluid MUX.

                         2.5 V          U2
                                        Upper
                                        MUX
                           0V                                  U3
                                                               12-bit
                                                               A/D
                                        U1
                         2.5 V
                                        Lower
                                                                                        Data
                                        MUX
                           0V                                                           Bus



                                        U21
                         2.5 V
                                        Fluid
                                        MUX
                           0V

                                                      MUX Select and input decode


                        Figure 5-8. 12-bit A/D, MUX test

                        Each of these multiplexers has a 0 and 2.5V reference input. Each MUX is
                        tested in turn. The respective MUX is enabled and the 0V input is
                        channelled to the A/D. The output of the A/D is then compared with the
                        digital equivalent of 0V. The 2.5V test is done in a similar manner. The
                        results are then analyzed. First they are checked to determine if the 0V and
                        the 2.5V test fail for all three MUXes. If so, the following message will be
                        reported in the Event log.
                        (FAIL) The 12 bit A/D has malfunctioned.


                        If, however, this check passes, but an individual MUX test fails, one or more
                        of the following messages will result:
                        (FAIL) The upper MUX has malfunctioned (U1).


                        (FAIL) The lower MUX has malfunctioned (U2).


                        (FAIL) The fluid sensor MUX has malfunctioned (U21).




5-24                    5 Diagnostics                                                      January 1997
PE Applied Biosystems




                        Test 24-bit A/D
                        0V, 124mV and 1 volt reference voltages are fed to the 24-bit A/D input via
                        a multiplexer (MUX). Should the respective digital output fall outside the
                        expected range, one or more of the following error messages will be
                        reported in the event log.
                        (FAIL) The 24 bit A/D 0 volt test has failed.


                        (FAIL) The 24 bit A/D 124 mV test has failed.


                        (FAIL) The 24 bit A/D 1 volt test has failed.


                        Self-Test Results
                        The low level stage of the power-up selftest utilizes the front panel and CPU
                        LEDs to display any failures as follows:-
                        CPU LEDS                      FRONT PANEL LEDS        TEST FAILED
                        All LEDs remain on from       ERROR 3 ON              ROM test
                        start of test.
                        LED 1 remains on.                                     68901 test
                        LED 1,2 and 3 remain on.      ERROR 4 ON              RAM test
                                                      Any other combination   Unknown interrupt vector


                        The error LED behind the door panel visor will illuminate in conjunction
                        with any of the above error codes.
                        Results of the high level tests are reported as a detailed message in the Event
                        log. See Section- Self Test Descriptions.




January 1997            5 Diagnostics                                                                    5-25
PE Applied Biosystems




                        Leak Test Procedures
                        The leak test procedures use functions which control the electronic pressure
                        regulators and monitor the pressure transducers. Based on the result of a
                        “decision” function which compares the actual pressure drop to the allowed
                        pressure drop, a Pass or Fail message will be generated.
                        The tests are split into three portions:
                        Pressurization     Check whether volume will pressurize to a set
                                           pressure.
                        Leak rate          Seal volume and check leak rate.
                        Venting            Check venting capacity of volume




5-26                    5 Diagnostics                                                      January 1997
PE Applied Biosystems




                        As an example, an explanation of each step included in the R1 Leak test is
                        given below:
                        Step             Fxn#             Function name    Time/pressure   Comments
                        1                258              Begin            0               Check tank
                                                                                           pressure
                        2                303              Select regulator 1               Select one of the
                                                                                           10 electronic
                                                                                           regulators, in this
                                                                                           case; P1
                        3                7                Vent R1          10              Depressurize
                                                                                           bottle
                        4                9                Backflush R1     20              Clear delivery
                                                                                           line
                        5                304              Save regulator   0               Store current
                                                          setpoint                         operating
                                                                                           pressure


                        Pressurization
                        6                305              Set reg setpoint 38              Start
                                                                                           pressurizing to
                                                                                           3.8psi
                        7                310              Set tolerance    50              ±0.5psi
                                                          (100th psi)
                        8                257              Wait             25              Continue to
                                                                                           pressurize to
                                                                                           3.8psi
                        9                308              Close pressure   1               Stop
                                                          valve                            pressurization
                                                                                           (close P1 Lee
                                                                                           valve)
                        10               257              Wait            15               Equilibrate
                        11               307              Compare         1                Pass
                                                          pressures (10th                  pressurization
                                                          psi)                             portion of test if
                                                                                           current pressure
                                                                                           = 3.8 ± 0.5psi


                        Leak Rate
                        12               317              Save regulator   0               Store current
                                                          pressure                         actual pressure
                        13               310              Set tolerance    5               ± 0.05psi
                                                          (100th psi)
                        14               257              Wait             30              Monitor pressure
                                                                                           drop




January 1997            5 Diagnostics                                                                     5-27
PE Applied Biosystems




                        15              318             Compare saved 0                 Pass leak rate
                                                        pressures                       portion of test if
                                                                                        pressure
                                                                                        change between
                                                                                        step #12 & #14 is
                                                                                        no more than
                                                                                        0.05psi


                        Venting
                        16              7               Vent R1         10              Open R1 vent
                                                                                        valve for 10
                                                                                        seconds
                        17              310             Set tolerance   50              ± 0.01psi
                                                        (100th psi)
                        18              307             Compare         0               Pass venting
                                                        pressures (10th                 portion of test if
                                                        psi)                            current pressure
                                                                                        = 0 ± 0.01psi
                        19              309             Restore reg     0               Restores
                                                        setpoint                        operating
                                                                                        pressure
                        20              8               Flush R1        5               Blanket R1 with
                                                                                        argon
                        21              4               Del R1, waste   10              Prime R1
                                                                                        delivery line
                        22              135             Flush cart      10
                                                        reagent block
                        23              143             Wash cart       5
                                                        reagent block
                        24              135             Flush cart      10
                                                        reagent block
                        25              259             End             0


                        Leak Test Pressurization Paths
                        During bottle leak tests, it is not necessary to open any delivery valves since
                        the bottle pressurization line is connected directly to its controlling
                        regulator. For other Leak tests, however, it is necessary to open delivery
                        valves in order to connect the component under test to the respective
                        regulator. The following diagrams show the plumbing paths that are tested
                        for each Leak test.




5-28                    5 Diagnostics                                                         January 1997
PE Applied Biosystems




               Cartridge Reagent Block                                          Cartridge Solvent Block                                    Flask Reagent Block

      1        2        3        4        5        6        7               8    9   10 11 12 13 14                           24 25 26 27 28 29 30

                                                                                                                                                                        #11
               *
           X1 R2 X3
           gas gas liq
                                    *
                                 * X1
                                   liq
                                               R1
                                               liq
                                                                            * *
                                                                           R3 R3
                                                                           liq gas
                                                                                              S2
                                                                                              liq
                                                                                                    S3 S1
                                                                                                    liq liq
                                                                                                                                   S4 X3 X2
                                                                                                                                   liq liq liq
                                                                                                                                                 * *   R4
                                                                                                                                                       liq
                                                                                                                                                              R5
                                                                                                                                                              liq
      W                                                                                                                                                             W
                                                                                                                          47 Com
                                                                                                             46 Com           NO
                                                                                                                         NC
                                                                                                            NC   NO                                 Flask Input Block
          Cartridge Input Block                                                                                          Argon Argon                   31 32 33
                                                                                                           Argon Argon   High Low
                                                                                                           High Low
 16         18 19 20                      21 22                                                                                                              #10
                                                                                                                                                                    *
                                                                                                                                                                    X2
                                                                                                                                                                    gas
                                     #6                #5
                                                                                                    45                                                 W
                                                                                          W
  W                                       W        W                                                             Flask Output Block
      A        B    C            D
                                                                                                                  41 42 43 44

          #1       #2       #3       #4
                                                                                                                           W
                                               F/C W
                                                                                                                            48 Com
                                              #7                                                                           NC   NO
                                                                                                         Flask
      34 35 36 37                     38 39 40
                                                                                                                           Argon Argon
                                                                                                                           High Low
      Cartridge Output Block




                                                                                                                               Injector

                                                                                                                                   4
                                                                                                                                                 To Column
                                                                                                                               5            3
                                                                                                                         #9                       From Pump
                                                                                                                 W                           2
                                                                                                                               6
                                                                                                                                       1
                                                                                                                                           #8



                                                                Figure 5-9. Cartridge A Leak Test pressurization path




January 1997                                                    5 Diagnostics                                                                                                 5-29
PE Applied Biosystems




                Cartridge Reagent Block                                          Cartridge Solvent Block                                    Flask Reagent Block

       1        2        3        4        5        6        7               8    9   10       12 13 14                        24 25 26 27 28 29 30

                                                                                                                                                                         #11
                *
            X1 R2 X3
            gas gas liq
                                  * X1
                                     *
                                    liq
                                                R1
                                                liq
                                                                             * *
                                                                            R3 R3
                                                                            liq gas
                                                                                               S2
                                                                                               liq
                                                                                                     S3 S1
                                                                                                     liq liq
                                                                                                                                    S4 X3 X2
                                                                                                                                    liq liq liq
                                                                                                                                                  * *   R4
                                                                                                                                                        liq
                                                                                                                                                               R5
                                                                                                                                                               liq
       W                                                                                                                                                             W
                                                                                                                           47 Com
                                                                                                              46 Com           NO
                                                                                                                          NC
                                                                                                             NC   NO                                 Flask Input Block
           Cartridge Input Block                                                                                          Argon Argon                   31 32 33
                                                                                                            Argon Argon   High Low
                                                                                                            High Low
 16 17 18 19 20                            21 22                                                                                                              #10
                                                                                                                                                                     *
                                                                                                                                                                     X2
                                                                                                                                                                     gas
                                      #6                #5
                                                                                                     45                                                 W
                                                                                           W
  W                                        W        W                                                             Flask Output Block
       A        B    C            D
                                                                                                                   41 42 43 44

           #1       #2       #3       #4
                                                                                                                            W
                                                F/C W
                                                                                                                             48 Com
                                               #7                                                                           NC   NO
                                                                                                          Flask
       34 35 36 37                         38 39 40
                                                                                                                            Argon Argon
                                                                                                                            High Low
       Cartridge Output Block




                                                                                                                                Injector

                                                                                                                                    4
                                                                                                                                                  To Column
                                                                                                                                5            3
                                                                                                                          #9                       From Pump
                                                                                                                  W                           2
                                                                                                                                6
                                                                                                                                        1
                                                                                                                                            #8


                                                                 Figure 5-10. Cartridge Reagent Block Test




5-30                                                             5 Diagnostics                                                                                                 January 1997
PE Applied Biosystems




               Cartridge Reagent Block                                          Cartridge Solvent Block                                    Flask Reagent Block

      1        2        3        4        5        6        7               8    9   10 11 12 13 14                           24 25 26 27 28 29 30

                                                                                                                                                                        #11
               *
           X1 R2 X3
           gas gas liq
                                    *
                                 * X1
                                   liq
                                               R1
                                               liq
                                                                            * *
                                                                           R3 R3
                                                                           liq gas
                                                                                              S2
                                                                                              liq
                                                                                                    S3 S1
                                                                                                    liq liq
                                                                                                                                   S4 X3 X2
                                                                                                                                   liq liq liq
                                                                                                                                                 * *   R4
                                                                                                                                                       liq
                                                                                                                                                              R5
                                                                                                                                                              liq
      W                                                                                                                                                             W
                                                                                                                          47 Com
                                                                                                             46 Com           NO
                                                                                                                         NC
                                                                                                            NC   NO                                 Flask Input Block
          Cartridge Input Block                                                                                          Argon Argon                   31 32 33
                                                                                                           Argon Argon   High Low
                                                                                                           High Low
 16 17 18 19 20                           21 22                                                                                                              #10
                                                                                                                                                                    *
                                                                                                                                                                    X2
                                                                                                                                                                    gas
                                     #6                #5
                                                                                                    45                                                 W
                                                                                          W
  W                                       W        W                                                             Flask Output Block
      A        B    C            D
                                                                                                                  41 42 43 44

          #1       #2       #3       #4
                                                                                                                           W
                                               F/C W
                                                                                                                            48 Com
                                              #7                                                                           NC   NO
                                                                                                         Flask
      34 35 36 37                     38 39 40
                                                                                                                           Argon Argon
                                                                                                                           High Low
      Cartridge Output Block




                                                                                                                               Injector

                                                                                                                                   4
                                                                                                                                                 To Column
                                                                                                                               5            3
                                                                                                                         #9                       From Pump
                                                                                                                 W                           2
                                                                                                                               6
                                                                                                                                       1
                                                                                                                                           #8


                                                                Figure 5-11. Cartridge Input Block Test




January 1997                                                    5 Diagnostics                                                                                                 5-31
PE Applied Biosystems




                Cartridge Reagent Block                                      Cartridge Solvent Block                                    Flask Reagent Block

       1        2        3        4        5        6    7               8    9   10 11 12 13 14 15                             25 26 27 28 29 30

                                                                                                                                                                     #11
                *
            X1 R2 X3
            gas gas liq
                                  * X1
                                     *
                                    liq
                                                R1
                                                liq
                                                                         * *
                                                                        R3 R3
                                                                        liq gas
                                                                                           S2
                                                                                           liq
                                                                                                 S3 S1
                                                                                                 liq liq
                                                                                                                                S4 X3 X2
                                                                                                                                liq liq liq
                                                                                                                                              * *   R4
                                                                                                                                                    liq
                                                                                                                                                           R5
                                                                                                                                                           liq
       W                                                                                                                                                         W
                                                                                                                       47 Com
                                                                                                          46 Com           NO
                                                                                                                      NC
                                                                                                         NC   NO                                 Flask Input Block
           Cartridge Input Block                                                                                      Argon Argon                   31 32 33
                                                                                                        Argon Argon   High Low
                                                                                                        High Low
 16 17 18 19 20                            21 22 23                                                                                                       #10
                                                                                                                                                                 *
                                                                                                                                                                 X2
                                                                                                                                                                 gas
                                      #6                #5
                                                                                                 45                                                 W
                                                                                       W
  W                                        W        W                                                         Flask Output Block
       A        B    C            D
                                                                                                               41 42 43 44

           #1       #2       #3       #4
                                                                                                                        W
                                                F/C W
                                                                                                                         48 Com
                                               #7                                                                       NC   NO
                                                                                                      Flask
       34 35 36 37                         38 39 40
                                                                                                                        Argon Argon
                                                                                                                        High Low
       Cartridge Output Block




                                                                                                                            Injector

                                                                                                                                4
                                                                                                                                              To Column
                                                                                                                            5            3
                                                                                                                      #9                       From Pump
                                                                                                              W                           2
                                                                                                                            6
                                                                                                                                    1
                                                                                                                                        #8


                                                             Figure 5-12. Flask Input Test




5-32                                                         5 Diagnostics                                                                                                 January 1997
PE Applied Biosystems




               Cartridge Reagent Block                                      Cartridge Solvent Block                                   Flask Reagent Block

      1        2        3        4        5        6    7               8    9   10 11 12 13 14 15                            25 26 27 28 29 30

                                                                                                                                                                   #11
               *
           X1 R2 X3
           gas gas liq
                                    *
                                 * X1
                                   liq
                                               R1
                                               liq
                                                                        * *
                                                                       R3 R3
                                                                       liq gas
                                                                                          S2
                                                                                          liq
                                                                                                S3 S1
                                                                                                liq liq
                                                                                                                              S4 X3 X2 R4   * *
                                                                                                                              liq liq liq liq
                                                                                                                                                        R5
                                                                                                                                                        liq
      W                                                                                                                                                       W
                                                                                                                     47 Com
                                                                                                       46 Com            NO
                                                                                                                    NC
                                                                                                      NC   NO                                  Flask Input Block
          Cartridge Input Block                                                                                     Argon Argon                   31          33
                                                                                                      Argon Argon   High Low
                                                                                                      High Low
 16 17 18 19 20                           21 22 23
                                                                                                                                                              *
                                                                                                                                                              X2
                                                                                                                                                              gas
                                     #6                #5

  W                                       W        W                                                       Flask Output Block
      A        B    C            D
                                                                                                            41 42 43 44

          #1       #2       #3       #4
                                                                                                                      W
                                               F/C W
                                                                                                                       48 Com
                                              #7                                                                      NC   NO
                                                                                                 Flask
      34 35 36 37                         38 39 40
                                                                                                                      Argon Argon
                                                                                                                      High Low
      Cartridge Output Block




                                                                                                                          Injector

                                                                                                                              4
                                                                                                                                            To Column
                                                                                                                          5            3
                                                                                                                    #9                       From Pump
                                                                                                           W                            2
                                                                                                                          6
                                                                                                                                  1
                                                                                                                                      #8


                                                            Figure 5-13. Flask Leak Test




January 1997                                                5 Diagnostics                                                                                                5-33
   850 Lincoln Centre Drive
   Foster City, CA 94404-1128




P/N 904566, Rev. A

				
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