HPLC LCMS IC Lab

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HPLC LCMS IC Lab Powered By Docstoc
					                                                                                          Kiersten Briggs


HPLC, LC-MS, IC Lab
**Note: the procedure for the HPLC is documented, but was not used due to the instrument being
broken for the remainder of the week.**

Introduction:

        HPLC, or high-performance liquid chromatography, is a method used to separate a mixture
while identifying, quantifying, and purifying the individual components.

Objectives:

       To learn to use the HPLC, LC-MS, and IC instruments through running a series of known samples
and then running unknowns and analyzing them.

Methods:

       LC-MS

       1. Ensure all sections of the instrument are on
       2. Turn on the Nitrogen tank (about 40 psi)
       3. Make sure the flasks are full sitting to the right of the instrument. Prepare more substance if
          needed.
          Software
       1. On the desktop, select ‘Analyst Software’ icon
       2. In vertical menu, click on the hardware Configuration, then LC-MS, then activate profile
               a. There should be 3 icons in the program window to the bottom right that have 3
                   yellow colors to them. If red ask for assistance.
       3. Click Build Acquisition Batch, in window fill in set name.
       4. Select method “Caffeine 2011” or “caffeine 10”
       5. Add set; add sample (at the bottom of the window)
       6. Under vial position, enter numbers where vials reside (tray in the top portion of the
          instrument labeled for you)
       7. Submit tab—submit—view, select “Sample Queue”—ready—start sample
       8. Check pump to ensure it has pressure
          Results
       1. Left panel, click Open Data File, select your name created in Step 3 of software portion
       2. After graph appears, right click on graph and click on List Data
       3. In new window click peak list tab. This is where you record needed data
          Shutdown
       1. In left verticle menu, click Hardware Configuration
        2. Window from startup appears, click on LC-MS again
        3. Click Deactivate profile to shut down instrument
        4. Shut of Nitrogen gas valve at the desk and in gas room

        IC

    1. Check Helium tank in gas room. Pressure should be at 80 psi or above. (Tell Lab assistants or
       instructor know if too low or too high)
    2. A pressure gauge on the instrument should be receiving helium and reading 9 psi. Turn knob
       near the gauge to correct if needed.
    3. Regenerant and Eluent bottles on the instrument should be full. If less than half full they need
       to be refilled.
           a. Gas must be turned off to refill either bottle.
           b. Regenerant is made by diluting 1.7mL of (concentrated) sulfuric acid to 2L (volumetric
               flask) in distilled water.
           c. Eluent is prepared by diluting 10mL of the eluent (in a bottle near the 7 ion standard) to
               1L (volumetric flask) in distilled water.

Equilibration:

    1. Open Chromeleon program from desktop.
    2. From the open program, Click: File→ Open.
           a. In the new menu change the file type to control panel. Click the MyPannels folder and
               click to open the ICS-90 system.
    3. Turn the pump off (in the program window)
    4. Turn the small black knob on the pump (NOT the DO NOT TOUCH KNOB) counter clockwise for
       30 seconds. This purges the system; see if eluent is draining out into a waste jug below.
    5. Turn the pump back on in the program window. Continue to purge for 30 more seconds.
    6. Turn knob on pump clockwise. Make sure not to over tighten.
    7. Stop and let the system equilibrate itself for 20 to 30 minutes. (Perfect time for HPLC or LC-MS
       work)
    8. After the allotted time, check the hoses below. If eluent collects 1mL over a full minute,
       proceed with procedures. If not, wait an additional 10 minutes. Still nothing, summon
       assistance.

Software:

    1. A new sequence is started by clicking, File→ New→ Sequence.
    2. Just click next in the box that appears.
    3. Injection parameters:
           a. Select number of vials to be used
           b. State the number of injections of each vial
           c. Start position is 2
           d. Injection volume is 10μL
          e. Click, Apply→ Next
   4. Do not change any methods or reporting.
   5. Name your sequence with initials and click Done.

Sequence Running:

   1. Click Batch Menu→ Edit→ add. There should be a list of sequences, find yours and click start.
          a. A window will appear telling you to inject the sample, do so now (In the injector port) till
              a small amount drips out into a waste beaker (small hose to the right with beaker).
   2. Click OK on the window. Each run will take 16 minutes

Naming Peaks:

   1. To open the sequence, click: File→ open→ select sequence→ your sequence name (with initials)
   2. Double click on the 7 anion standard in a window that has the names of your injections listed.
   3. In this new window click on the peak analysis tab
           a. Double click on the first positive peak
           b. After a box appears, pull the blue sheet from the drawer below and name the peaks
               from the sheet.

Shutdown:

   1.   Ensure the instrument has stopped running by opening the batch menu and clicking stop.
   2.   Leave Chromeleon program running (it shuts itself off).
   3.   Check eluent and regenerant levels (do they need to be refilled?)
   4.   You may close the program by clicking the close button at the top right of the program window.

   HPLC

Sample Prep:

        For this instrument, its best to have samples prepared and on hand as they are injected
via syringe (found in drawer near the instrument). This should be flushed clean with distilled
water during each separate sampling.

Preparation:

   1. You must ensure:
         a. There is enough degassed methanol and water. (Ask for assistance if more is
            needed)
         b. The lines coming from the degassed species have no air bubbles (labeled A and
            B). If detected, prime the lines by turning the pump knobs. This drains them into
            a waste beaker.
         c. Ensure the computer is still on.
         d. Injector knob should be placed to the left on load.
   2. Turn the detector on. (will be top most box labeled Detector SPD-10 AV)

Software:

   1. On the desktop click the HPLC icon.
   2. Select Shimadzu.
   3. In the program window click on the Methods icon at the top. (There will be a horizontal
      row of icons at the top).
          a. In the methods window select the method TSW.met
   4. Next click the Parameters icon at the top. Set the following:
          a. Flow rate A: .500 mL/min
          b. Flow rate B: .500 mL/min
          c. Flow rate C: 1.000 mL/min
          d. Max. pressure: 6000 psi
          e. Min. pressure: 1 psi
          f. Wavelength: 256 nm
   5. Click Download→ OK

Running Samples:

   1. Prepare sample for injection by drawing it into syringe. Insert into injection port. DO
      NOT inject and DO NOT turn the knob to inject in this step.
   2. Click on the Run Single icon at top of program window.
   3. In the new window assign the file name with initials and click start.
   4. At the very bottom of the program window the program should be listing things in
      preparation. When it says “waiting for trigger” You may inject your sample. Inject until
      3 to 5 drops fall from the waste hose into the beaker below the knob. Switch the knob
      to inject and LEAVE THE SYRINGE IN.
   5. Program will run itself from here. Let run for 5 minutes, the full 10 is unnecessary.

Results:

   1. After 5 minutes Click the red stop sign icon at the top. (same spot as where Run Single
      is)
   2. Click the Analyze icon at the top. This should label peaks on the chromatogram.
   3. Click the Report icon at the top and then click Area% in the dropdown menu.
   4. Record any necessary data. Printing the chromatogram with the chart is easier. Click the
      Printer icon at the top. In the dropdown menu click custom and the printer should
      automatically print both on one sheet of paper.
Shutdown:

   1. Click the Method Icon and this time select the LOWFLOW.met
   2. Click the Parameters Icon and ensure these values are assigned:
          a. Flow rate A: .010 mL/min
          b. Flow rate B: .010 mL/min
          c. Flow rate C: 1.000 mL/min
   3. Once those values are assigned click download. Then exit out the program.
   4. Ensure the screens for A and B (labeled on the boxes) is at 0.010 mL/min. If they are,
      turn the detector off.
   5. Ensure levels of degassed liquids are enough for the next person.

Results

          IC

                                       Anion Standard
                                Retention
                   Peak                         Peak Name         Height (uS)
                                time (min)
                     1             2.92           Fluoride          4.951
                     2             3.49           Acetate           5.547
                     3             3.82           Chloride          7.780
                     4             4.30            Nitrite          5.615
                     5             4.61           Bromide           6.595
                     6             5.03            Nitrate          7.965
                     7             5.54          Phosphate          14.520
                     8             6.25            Sulfate          1.156


                        Unknown Water Samples (Lab Sink Water)
                             Retention
                   Peak                    Peak Name       Height (uS)
                             time (min)
                    1           0.21           N.a.            0.023
                    2           2.25           N.a.            0.172
                    3           2.92           N.a.            1.433
                    4           3.49           N.a.            1.589
                    5           3.82           N.a.            2.106
                    6           4.31        Chloride           1.431
                    7           4.63        Bromide            1.700
                    8           5.03           N.a.            2.352
                    9           5.54           N.a.            4.074
                    10          8.45           N.a.            0.069
                         Unknown Water Samples (Water Fountain)
                              Retention
                    Peak                    Peak Name       Height (uS)
                              time (min)
                     1            2.92          N.a.            5.333
                     2            3.49          N.a.            5.645
                     3            3.82          N.a.            9.121
                     4            4.31        Chloride          6.309
                     5            4.61        Bromide           7.288
                     6            5.03          N.a.            6.909
                     7            5.54          N.a.          15.028
                     8           10.79          N.a.            0.125


                         Unknown Water Samples (Men’s Bathroom)
                               Retention
                    Peak                     Peak Name       Height (uS)
                              time (min)
                     1            2.25           N.a.          0.489
                     2            2.93           N.a.          0.527
                     3            3.49           N.a.          7.253
                     4            3.80           N.a.          0.154
                     5            4.32         Chloride        0.233
                     6            4.63         Bromide         3.101
                     7            5.02           N.a.          0.095
                     8            5.54           N.a.          3.474
                     9           13.22           N.a.          0.025


       LC-MS

               There were a multitude of peaks for each run, therefore are recorded on page 42
       of the lab notebook. No graph could be generated due to the large amount of peaks.

       HPLC

               Was broken during the time period we were given to work on it, therefore no
       results were found.

Calculations:

       Calculations were performed for our standard concentrations for the LC-MS:

       1ppm: M1V1=M2V2

                100ppm(V)= 10ppm(100mL)
                      V= 1mL

Conclusions:

        In my opinion, this lab was not overly successful. The LC-MS peaks were very difficult to
decipher, and there were entirely too many to figure out what it was we were supposed to be
looking at to begin with. And the first lab concerning the IC we could not detect the reason, so
an entire lab was detected to attempting to solve the problem. After we found it was the
regenerate, and it was changed, the IC worked just fine. However, the peaks between the
known anion sample labels and those of our unknowns did not match up as expected. In our
known sample, the chloride and bromide peaks were 3.82 and 4.61 respectively. In our
unknowns, the bromide peaks stayed around the same range, however our chloride peaks
became around 4.31. If more time would have been allowed, we would have rerun the known
sample to double check for errors.

       For the LC-MS however, too low of concentrations were used to detect useable peaks.
To see the graphs and the large multitude of peaks shown for the LC-MS, you can see page 42
of my lab notebook.

				
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