User Guide Varian 240 Atomic Absorption Spectrometer

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					    Oxford University MChem, PTCL Practical Course                                  Varian 240 Flame AA instructions

                                            User Guide
               Varian 240 Atomic Absorption

The Varian 240 is a versatile and powerful research‐grade instrument. Please read through the relevant sections of
the instructions before starting work, and ask for help from Dr Cartwright if at any stage you are in doubt about how
to proceed.

1        Background

Atomic absorption (AA) spectrometers are used to analyze metals at very low concentrations, typically in the parts
per million (ppm) or parts per billion (ppb) range. A liquid sample containing dissolved material whose concentration
is to be measured is aspirated into a thin, wide flame, or is introduced into a small carbon furnace which is heated to
a high temperature; in this experiment you will be using a flame AA. The flame is typically 10 cm wide, but only
about 1 mm thick. At the high temperature of the flame, which may be either oxy‐acetylene as used here, or nitrous
oxide/acetylene, the sample is broken down into atoms and it is the concentration of these atoms that is measured.

Light from a lamp specific to the element being measured is shone along the 10 cm width of the flame so that the
maximum number of atoms can be detected.

It is crucial that the light of a suitable wavelength is used. The spectrum of every element is both complex and
unique, so by selecting light of a wavelength that is absorbed by no other type of atom, any metallic element in the
flame can be monitored, even if other species are present. Light of the chosen wavelength is obtained from a hollow
cathode lamp, which contains a small amount of the metal to be measured. The lamp is heated electrically to a
temperature high enough to vaporize and excite the metal atoms so that they emit light. This is of the precise
wavelength that atoms of the same element in the flame can absorb, so only those atoms absorb the light and are
detected. This makes AA highly element‐specific. The method has the disadvantage that a custom lamp is required
for every element and lamps typically cost several hundred pounds. However, costs can be reduced by including
several different metals within a single lamp.

In the Varian 240 an area behind a hinged door at the front right hand side provides space for several hollow
cathode lamps. With a suitable choice of lamps about twenty elements can be measured in a single experiment.
      Oxford University MChem, PTCL Practical Course                                 Varian 240 Flame AA instructions

At the front of the instrument is a sample feeder. Three streams of liquid can be combined and fed into the flame,
which is behind the panel in the centre of the instrument, directly below the exhaust ducting. On the left of the
feeder tray, samples and standards are introduced into the flame via a peristaltic pump. There is a second pump on
the right, which can be used to feed in chemical modifiers that may enhance the sensitivity of the instrument to a
specific element. The container at the center of the feeder station holds pure solvent (demineralized water) which
can be fed in to keep the overall flow rate constant, while the intake speed of the sample can be varied. This is also
used to dilute the sample to create standards, or to dilute concentrated samples.

Conventionally, the concentration units in AA are Parts Per Million (ppm), rather than moles. The correct definition
of ppm is mg Kg‐1, which is a mass/mass relationship. Because the volume of 1Kg of water is approximately 1000 ml,
this definition has been extended to mg (1000ml)‐1, or mg L‐1 (a mass/volume relationship). Bear in mind that as the
instrument tests for metal atoms, ppm refers to the mass of only the metal in the solution. When preparing
solutions, ensure you use the correct mass for the metal you are analyzing, rather than the whole salt.

2          Preliminaries

2.1        Software

Turn on the computer and log on as user aa; ask the technician for the current password for this account. This is a
local user, specific to the PC that runs the instrument, so do not try to log on to the network. Double click the
SpectraAA icon to start the software.
2.2        Instrument

Turn on the instrument (the power button is at the front bottom left, by the green light) and allow it to warm up.

Open the right hand door of the instrument (the large door with the black glass panel) and check the correct lamps
are fitted; make a note of their positions and the ‘Recommended current’ shown on the white label at the base of
the lamp. If a lamp needs changing ask a technician to do this.

2.3          Gas supplies

Do not turn on the gas supplies until you are ready to light the flame. Do not leave the flame lit when you do not
need it, since acetylene is expensive and you may consume a significant quantity. Instead, when you get to the end
of a set of samples, turn off the flame by pressing the button as the front left of the instrument (if the software does
not automatically do this, which will happen if you have reached the end of a set of samples).

2.4          Samples
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You can start setting up the software to run an experiment before you prepare the samples, but you should not start
the instrument itself until you are ready to take measurements. Acetylene is fairly expensive and you can waste a lot
of gas if you leave the flame running before your samples are ready.

Prepare a full set of standards and samples, using demineralized water and ensure they are properly labelled.

3        Create the instructions to run an experiment

An experiment is run by specifying the operating parameters in a worksheet. When the run starts, the instrument
communicates with you through the Analysis page, providing messages to indicate how the experiment is
progressing, and requests, such as that you should present a fresh sample to the instrument. The first step is
therefore to create a worksheet.

Depending upon how the software was shut down when it was last used, it may either open up with a sand‐coloured
header page or it may go directly to an analysis page. If you have not used the AA before, take time to become
familiar with the software before trying to use the instrument. You do not need to have the instrument running to
do this, so do not ignite the flame yet.

3.1 Creating the worksheet

First you need to load, or create, a worksheet. If the program opened on the header page select the large Worksheet
button; if it was already in a worksheet select the Filing tab and click on the Load Worksheet button.

Generally you will prepare a new Worksheet from scratch. To do this, click New…, type in a name for your worksheet
and press OK. Choose Add Methods and check that Cookbook is selected and only the Flame box ticked, then
select the element of interest and click on OK. The cookbook option will select suitable parameters for most of the
parameters, such as the wavelength to be monitored.

To create a new worksheet by modifying an old one, click on New from… and select a worksheet as a template. If the
worksheet you need is already complete, select Open…

3.2 Menu bars

          At this point the normal Windows menu bar will be visible, together with some tabs. The function of these
tabs is as follows:

Filing tab.                Use to load or save a worksheet.

Develop tab.              Use to add and edit elemental analyses methods when constructing a new worksheet
from scratch, or from a template, or to edit an existing worksheet.
    Oxford University MChem, PTCL Practical Course                                           Varian 240 Flame AA instructions

Labels tab.                Use to enter details about the samples to be measured, such as the name of the sample or
its concentration. This tab, and the Analysis tab beside it will not be “live” until a worksheet has been selected and
at least one element chosen for analysis.

Analysis tab.            Provides information about the experiment as it is run. Messages about progress of an
experiment appear here, generally at the lower right.

3.3 Editing the worksheet

The choice of Cookbook selects for you a set of conditions for the element that are likely to be near optimum; these
can be viewed using the Review button on the Develop tab. However, even though the parameters chosen
automatically should give good results, there will still be some parameters in the worksheet whose values will need
to be specified, and this is the next stage in setting up the experiment.

Click on the Edit Methods… button. Choose the following parameters for an initial run:

Type/Mode tab: Check that the appropriate element has been selected. Choose Manual sampling mode. If the SIPS
        unit is fitted (this is an automatic sample handling system) place a tick in the SIPS box.

Select the Absorbance or Emission Instrument mode as required. The flame type and gas flows will be set by

          applying the cookbook recipe and should not be altered.

Measurement tab: The default parameters are likely to be appropriate. Use PROMT measurement and
        Concentration Calibration mode. The Read Delay (the delay once a sample is being fed into the flame
        before a reading is taken) need not usually be as long as 10 seconds, so can be reduced to 5.

Optical tab: The cookbook assumes single element lamps are being used, however we use multiple element lamps,
          so select the lamp position and current as you noted when starting the instrument. If you are yusing
          emission mode you do not need to specify any lamp details.

SIPS tab: Controls the automatic feeder pumps; this section cane be ignored if the SIPS unit is not fitted, or will not
           be used. Use the default values of 5.0ml min‐1, None under Right pump and Auto Set Standard
           Concentrations under Calibration Mode unless specified differently in the instructions for the experiment.

Standards tab: In order to generate a suitable calibration curve, standards of known concentration are prepared and
         fed in sequentially. Enter the concentrations of your standards in the appropriate boxes. You can change
         the units if desired. The “Upper valid concentration” is the highest concentration for which the instrument
         should be allowed to calculate a reading1.

Calibration tab: Once the instrument has measured your set of standards, a calibration plot of light absorbed or
          emitted by the sample as a function of concentration will be plotted. The calibration tab is used to define

 Any reading above that given by your strongest standard will require the software to extrapolate beyond the end of the
calibration plot. The longer this extrapolation, the larger the uncertainty in the value to be reported, so your standards should
cover the full range of concentration you expect to find in your samples. The Upper valid concentration should be set to a value
about 10% higher than your strongest standard.
       Oxford University MChem, PTCL Practical Course                                     Varian 240 Flame AA instructions

              the parameters that will be used to fit a least squares line through these data. These parameters can be
              modified at this stage or later, when the standards have been run.

Sampler tab: ignore – no sampler is fitted.

Notes tab: This can be use to add notes to the method if required.

Cookbook tab: The cookbook contains the outline of the method for each element selected.

QCP tab: used for quality control purposes; ignore.

Close Edit Methods… and switch to the Labels tab. Set the Total Rows… value to the number of samples you intend
to run, plus one. Label the samples as appropriate for your experiment, by double clicking on the sample name box.
Label the final sample as “Rinse”. When you reach this sample in the process, run distilled water through to check
the instrument zero, and to clean out the left pump.

4             Position the solutions on the SIPS unit

Ignore this section if the SIPS unit is not fitted. If fitted, this unit is attached to the front of the instrument, just below
the window that gives access to the flame. If it has been removed, continue with section 5.

You have now set up the instrument to control the run and must prepare the SIPS unit and the instrument itself.
There should be a rectangular cross‐section plastic bottle in the middle of the SIPS unit; this always contains
demineralized water. If the bottle is not nearly full, fill it now. Check the waste container under the bench to the left
of the instrument is not too full and if necessary empty it.

Place the container holding your sample on the left hand side of the SIPS tray, along with a beaker of distilled water.
If your experimental instructions specify use of a modifier, this is fed into the flame via the right hand pump. This is
controlled in the SIPS tab of the Edit Methods… window. The pump speed is given as a percentage of the maximum
speed of the left hand pump; whatever you set is deducted from the speed of the left hand pump in order to keep
the flow rate into the instrument constant.

5           Light the flame

Turn on the gas supplies.

Air:          Open the main valve on the air cylinder using the black wrench. The valve needs to be turned
              anticlockwise as you face the opening in which the wrench fits, in order to open it. Check that the outlet
              pressure, shown on the gauge closer to the black outlet pipe, is close to 3.5 Bar. The central winged
              handle on the regulator should not need adjustment at any time, either now or at the end of the
              experiment, so do not touch it unless the pressure is not around 3.5 Bar.

Acetylene: Open the acetylene cylinder by turning anticlockwise the handle at the top centre of the regulator. The
         outlet pressure, shown on the gauge directly above the red outlet pipe, should be close to 11 psi; if the
         pressure exceeds 14 psi, do not use the instrument, but consult Dr Cartwright. (Note: If the acetylene
    Oxford University MChem, PTCL Practical Course                                  Varian 240 Flame AA instructions

           pressure in the cylinder, as opposed to the outlet pressure, falls below 100 psi, the instrument may be
           damaged through the introduction of acetone which is present in the acetylene cylinder as a solvent. If the
           pressure is close to 100 psi, or below, do not use the instrument, but consult Dr Cartwright.)

Lower the blind over the window; the instrument has an automatic cut‐off which prevents ignition if there is too
much light sensed by the instrument, since it will assume that the flame has already been lit.

Press and hold the ignition button (the square black button on the front bottom left, to the left of the green light);
gas begins to flow and a small flame from the back left will light the main flame strip. Once this is lit, release the
ignition button. You can open the panel in front of the flame to see the small blue/green flame which should be
burning steadily across the entire width of the burner. Switch on the extractor fan after igniting the flame and set to
Low (the 6 o’clock position on the control dial ‐ the beige round selector on the bench to the left of the instrument.)

6        Running samples

Go to the Analysis page. Select the green traffic light and follow the instructions on screen. A running commentary
will appear in the text box on the left hand side and in a small window near the bottom right. You will be asked, via
pop‐up boxes, to present samples, standards and distilled water for rinsing. It is important that you flush the sample
line with demineralized water whenever the software tells you to do so.

To add samples after starting the experiment, go to the Labels tab and click Ins/Del Rows…, then select Append and
enter the number of rows to insert. Once this has been done, go to the Instrument menu of the Windows menu bar,
click Start At… and select Solution.
7        Correcting calibrations

Most experiments will lead to a suitable calibration curve with no need for manipulation. However, on occasion the
calibration curve may not be suitable, containing one or more points that give an upward slope to the curve, or
points whose scatter is judged by the software to be too large. The instrument will then report that the calibration is
invalid. The most common indication that this has happened is that when you run your samples, instead of a
concentration being reported, the software displays UNCAL.

This is most likely when working in high concentration ranges (≈>50ppm). To rectify this, allow the experiment to
finish with all values noted as “UNCAL”. Now go to the calibration data, select the point which breaks the series,
right‐click on it using the mouse and choose Edit Replicates…. In this menu click Mask then Apply. This removes the
wayward calibration point and should make a calibration curve appear. If it does not, more points need masking.
Continue this process until the software determines that your data are suitable. Sample values should change from
“UNCAL” to calculated values.

8        Printing data
     Oxford University MChem, PTCL Practical Course                                   Varian 240 Flame AA instructions

Results are output by creating and then printing a report. Go to the Window entry in the Windows menu and select
Reports… Choose the items that you wish to have in your report, then select Print Preview to check that you have all
you need, and no more. If you are happy with what is shown, select Print.

9         Fast Sequential Experiments

Experiments can be run in which the instrument analyzes more than one element. Add each individual element as
outlined above; once they have all been included click the Fast Sequential Wizard… button. The wizard can be
followed using all its default values, so keep clicking Next until reaching the final wizard screen, at which point click
Finish. The experiment is now carried out as normal. Follow all the instructions appearing on screen and the
instrument will analyze every element you have specified.

10        Closing down

Before turning off the flame, replace the final sample by demineralized water and allow this to feed into the flame
for a couple of minutes to flush out traces of metal. Press the red button at the front of the instrument to turn off
the flame. Wait a few seconds to check that the sound of the gas has died away, then lift the flap at the front of the
instrument to check that the flame is out. Turn off the air and acetylene supplies at the main valve of each cylinder
but do not touch the regulators on either cylinder.

Remove any standards and samples from the SIPS tray and leave the tubes in demineralized water; leave the central
rectanuglar plastic container of demineralized water in place. Wash out all glassware thoroughly with demineralized
water and leave ready for the next user in the trays for the experiment.

Save and close any worksheets using the Filing tab and close down the software on the PC by selecting the large red
Exit button and log off. Turn off the fan on the exhaust line. Turn off the instrument using the toggle switch at the
lower left hand side. Turn off the printer. Turn off the computer monitor, but leave the computer running.

11        Using the SIPS unit

When using the SIPS unit the normal procedure is to prepare a single standard of known concentration; this can be
used by the instrument to generate a complete set of standards automatically. Within the worksheet ensure that the
SIPS unit box is ticked. Enter the concentration of your standard (in units of ppm) in the Bulk Std Conc box. Set the
number in the Standard Count box to the number of standard solutions to be used (these standards will be created
automatically by the SIPS unit; you should use in the region of 4‐8 standards). Set the Lower valid concentration to
zero and the Upper Valid Concentration to a value about 10% higher than the concentration of your standard
solution. Any samples whose concentration is above the Upper Valid Concentration will automatically be diluted by
the SIPS unit to bring them into range.

12        Optimization of Spectrometer
   Oxford University MChem, PTCL Practical Course                                     Varian 240 Flame AA instructions

Important: only the lamps may need to be adjusted routinely. Do not attempt to adjust the burner or the nebulizer
without consulting Dr Cartwright first.

If the instrument has not been in use for a long period of time, has been set up differently for other experiments, or
if the lamps have been changed, it may be necessary to perform an optimization to ensure correct settings and
optimum results. Start to create the worksheet for your experiment but do not light the burner.

Under the Analysis tab select Optimize. Select the method you wish to optimize and click ok. If more than one
element is on the same lamp it only needs optimizing once for them all. NB. Lamps can only be optimized if your
worksheet method uses absorption rather than emission.

12.1     Alignment of Lamp

Click Optimize Lamps and wait for the signal bar above to display a signal strength. The aim of optimization is to
maximize the signal strength while reducing the gain (shown as a percentage at the bottom of the window).

Open the black glass door on the right of the instrument to access the lamps. The alignment of the lamp is adjusted
with the two black screws on the back of each mount. Adjustment of these screws will affect the signal strength
shown in the bar. Adjust each screw until the signal reaches a peak value, and then continue past it slightly to check
it is a true maximum. Repeat turning in the other direction before returning the screw to the point of the maximum
signal, then repeat the adjustment for the other screw. If the signal goes off the top of the scale, click Rescale. This
will change the gain value of the signal. Repeat the adjustment with the screws. You should aim to get the gain as
low as possible, as this indicates the signal is at its strongest and purest. Once you have finished the adjustment click
Rescale a final time and record the final gain value.                                                                       8
12.2     Alignment of Burner

Remove the guard panel from the gas chamber by unhooking it from the bar it rests on. Ensure the burner is set
parallel to the front panel of the machine. Taking the alignment card, place it with the vertical line in the centre of
the burner. Adjust the height and lateral position of the burner with the black double knob on the front of the
instrument, so the light from the lamp is in the centre of the target area. Once this has been done, move the card to
each end of the burner and check the light is still central. Adjust the angle of the burner if necessary.

12.3     Adjustment of Nebulizer

*NOTE* This is an advanced, difficult and, if it goes wrong, potentially expensive procedure – do not carry it out
unless you are competent in the use of the instrument and need to perform a high‐flow experiment.

Close the Flame Optimization window and light the burner before reopening it. Place the left hand tube in a large
beaker of distilled water.

In the optimization window click Optimize Signal, and click the Start Pump button. Set the nebulizer to the ‘high
solids position’ by gently winding the front black knob surrounding the intake tube clockwise all the way in. Using
your finger block the air vent (the small hole in the lid of the central water bottle) and very quickly disconnect then
reconnect the tubing from the bottle to introduce a small bubble to the tubing. Time the bubble’s travel through the
tubing and wind out the knob until it takes 25‐30 seconds to travel 10cm. When this is achieved click Stop Pump.
    Oxford University MChem, PTCL Practical Course                                         Varian 240 Flame AA instructions

NB. Try to let as small an air bubble into the tubing as possible and allow sufficient time for air to clear before
remeasuring the time. If the nebulizer flow is blocked or inconsistent inform Dr Cartwright.

Problems and solutions

      Observation                                Indicates…                                      Solution

Cannot log onto             Incorrect login domain selected                  Select This computer in drop down menu
Flame fails to light           (i) Instrument not turned on                  Turn the instrument on.
                              (ii) No gas supply                             Check that both gas cylinders are on and that
                                                                             the pressures are set, as explained above.
                              (iii) Ventilation turned on                    Flame cannot light when vent is switched on,
                                                                             turn vent off, light then return vent to ‘Low’
                                                                             setting once lit.
                              (iv) Worksheet incorrectly set                 The flame will not light if parameters that govern
                                                                             its operation are set incorrectly in the
                                                                             worksheet. If the problem cannot be found,
                                                                             reconstruct the worksheet starting from one
                                                                             known to be correct.
Shows 'Flame sensed'        Too much light falling on the sensor in burner   Click ok on error message box. Check the blinds

or ‘Flame control           chamber                                          are shut and if necessary cover the front of the
offline’ and fails to                                                        vent intake while lighting eg. with a folder.
Shows ‘Gas cover open’      Gas cover is open                                Click ok on error message box, ensure central
error message and                                                            cover is hooked correctly and lowered fully then
flame fails to ignite                                                        continue
Has ‘Gas dispersal’         There is a delay while residual gas in the       Wait till the message bottom right shows ‘GCU
error in bottom right       chamber is exhausted before it can attempt to    idle’ then continue
and flame fails to ignite   light again
Cannot change values        There are already results for that worksheet.       (i) Delete the present results, if not needed,
in Edit Methods… tab        Once collected these cannot be overwritten.              by clicking the Delete… button on the
                                                                                     Develop tab and selecting All results &
                                                                                     Signal Graphics, then change the options
                                                                                     before restarting the operation
                                                                               (ii) Load a new worksheet with New from...
                                                                                     edit the method and continue.
Report can be               Default printer incorrectly set (probably to     Go to the Printers and Faxes entry in the start
constructed, but            Microsoft Image writer)                          menu and redefine the default printer.
attempts to print it
always lead to a
request to create a file
Calibration curve is not    Fluid intake from SIPS is not constant, and/or   Inform Dr Cartwright of the problem. Empty,
smooth or values are        dilution is ineffective. This may indicate a     wash out and refill the central water bottle in
not ascending (when         blockage in the instrument.                      the SIPS tray, as this may have been
created using SIPS)                                                          contaminated by backflow.
Program hangs or will       An operation has been performed incorrectly,     Press Ctrl+Alt+Delete, enter task manager and
not close.                  causing the software to cease to respond.        close SpectrAA manually. Reopen the software
                                                                             and continue from where you reached in your
SIPS unit does not          Error in worksheet                               Edit the worksheet. Check that Manual sampling
operate                                                                      mode has been chosen on the Type/mode tab
                                                                             and that a tick has been placed in the SIPS box.
   Oxford University MChem, PTCL Practical Course   Varian 240 Flame AA instructions

February 8, 2010