LAB 4 Separation Science SCBS006H7

Capillary Electrophoresis – The Analysis of Caffeine in Beverages

The traditional method for the determination of caffeine is via extraction with
spectrophotometric quantitation. In this experiment you will compare two different
modes of capillary electrophoresis, capillary zone electrophoresis (CZE) and
micellar electrokinetic chromatography (MEKC), to achieve the same separation.

Experimental Procedure

Preparation of Running Buffers and Standards
Two running buffers are required, these have been prepared for you. The standards
and samples have also been prepared and filtered for you to save time.

CZE Buffer: 0.05M borate buffer, pH=9.0.
(Dissolve sufficient boric acid (61.83 g/mol) in CE grade deionised water to prepare
100mL; add NaOH until pH = 9.0. Filter after preparation.)

MEKC Buffer: 0.05M SDS (sodium dodecyl sulfate) in 0.05M borate buffer, pH =9.0.
(Add sufficient SDS (288.38 g/mol ) to borate CZE buffer to prepare 50 mL of running
buffer, measure pH to ensure pH = 9.0. Adjust pH with either HCl or NaOH if necessary.
Filter after preparation.)

Caffeine Standards
Accurately weigh out 10.0 mg of caffeine. Transfer to a clean 100 mL volumetric flask.
Dilute to the mark with HPLC grade water. Carry out a series of dilutions of the stock
10 mg / 100 mL solution to obtain standards of 1.0 mg /100 mL, 2.5 mg / 100 mL, 5.0
mg / 100 mL, and 7.5 mg / 100 mL. Make 10 mL of each of the dilutions. Use CE grade
water to make the dilutions. Shake the five caffeine solutions to ensure adequate
mixing. Filter after preparation.


Setting up the Instrument
The first experiment you will run is the CZE separation. All the steps are the same for
both methods.
The instrument used in this experiment is an HP (now Agilent) 3D-HPCE. The software
used to control the instrument and collect the data is the HP Chemstation.
You need to start in "Instrument View." If any other view is up, click on the first icon
under the blue "Run Method" at the upper right.
Instrument View shows a diagram of the complete system, with clickable features to
make many actions easier. The menu bar could also be used, but takes more steps per
action.

The instrument must first be initialized before use (if the instrument is switched on
and showing a “Ready” icon you can ignore this step). Click the menu bar item
"Instrument" and select System INIT from the menu. Wait until the system goes to
Ready. This may take some time if the temperature controlled zones need to stabilize.
Click on the Tray icon and select Tray Control. When the window opens, click Unload
under "Unload Lifts." You should then hear the instrument lowering the vials in
positions 1 and 2. "Get Vial" should be set to "1" - click "Get." The tray will rotate vial 1
to the front of the instrument. Open the tray door and check what vials are present.

The instrument should be loaded with vials as follows for these two experiments:
Position 1 NaOH (for capillary cleaning)
Position 2 Water (for capillary washing)
Position 3 Borate buffer (0.05M, pH=9.0) (CZE running buffer inlet)
Position 4 Borate buffer (0.05M, pH=9.0) (CZE running buffer outlet)
Position 30 Borate/SDS buffer (0.05M, pH=9.0) (MEKC running buffer inlet)
Position 31 Borate/SDS buffer (0.05M, pH=9.0) (MEKC running buffer outlet)

Vials should be approx. 2/3 full

Close the tray door and click "Done." The tray will rotate back to its operating position.
Click on the blue Pressure icon and click "Flush Capillary" on the menu. Enter a time of
5 minutes and click OK. The pressure should increase to about 930 mBar and stay there
for the flush time. This will wash out any residue from the prior runs and equilibrate
the capillary with the running buffer.

The instrument can store separation methods and sequences.
A method defines the analysis conditions and includes parameters such as separation
voltage, run time, detection wavelength etc.
A sequence tells the instrument which vial positions to use for the method and whether
to repeat the method for different vial positions to run, for example, standards or
samples.

CZE Separation
Set the method and sequence as follows:

The method and sequence names can be selected directly under the main menu bar.
Use method FB1.M found in C:\HPCHEM\1\METHODS\CE\FB1.M
Use sequence FB1.S found in C:\HPCHEM\1\SEQUENCE\FB1.S.

Click on the tray icon and choose "Tray Control." Set the Get Vial number to 7, and click
Get. You can now open the tray door and put all your samples in.
For sequence FB1.S the vial positions are as follows:

Position 7 Coke Sample
Position 9 100ppm standard
Position 10 75ppm standard
Position 11 50ppm Standard
Position 12 25ppm Standard
Position 21 Dr Pepper Sample

Close the tray door.
On the menu bar, click Sequence and select Sequence Parameters. Enter an operator
name, make sure "Prefix/Counter" is checked, and enter a prefix for your datafile
names. Click OK.
On the menu bar, click Run Control and select Run Sequence. Click "Yes" if satisfied that
you set up your filenames correctly. The instrument will begin running the standards
and samples in sequence.
After the sequence has started, click on the third icon to the right under "Run Method"
to observe the progress of the runs. You should see a peak for your first standard about
5-6 minutes into the run. The electrical current, which can be checked under "Instrument
View," should be typically 15-20 microamperes, values that are very different suggest a
broken or damaged capilliary.
When the entire sequence has completed, find the pulldown menu that currently
displays "Method and Run Control," and change it to Data Analysis. Click on "File – Load
Signal" or click on the Load Signal icon and select one of your data files. Click OK. On the
menu bar, click Report and select Print Report. Repeat for all your other data files.

IMPORTANT: Make sure that you get a peak for each standard. You can always abort
the run and restart it if there is a problem.

MEKC Separation

The MEKC separation is very similar to the CZE separation except it uses an SDS
containing buffer and the run time is longer.

Set the method as follows:

Use method FRANKM.M found in C:\HPCHEM\CE\FRANKM.M
The sequence is the same as the CZE method: FB1.S found in
C:\HPCHEM\1\SEQUENCE\FB1.S
(The method and sequence names can be selected directly under the main menu bar as
before.)

Since this method uses a different buffer you should flush the capillary with the buffer
for 5 minutes before the first run.

Repeat the separation as before except using the new method.

When all reports have been printed, use the peak areas of the standards to plot a
calibration curve.

Report
You should write a standard laboratory report for this assignment with a short
introduction, objectives and a results section that contains calibration figures for the
standard caffeine samples by both CZE and MEKC along with calculations for the
caffeine content of the samples by both methods.

In the discussion section you should cover the following points:
1. Explain the difference in retention time for the two different experiments.
2. What would you expect to happen to the retention time of the caffeine peak if you
decreased the run voltage for the first experiment to 10 kV?
2. Did you get the same answer for the two different CE experiments? Explain.

References
1. Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, Fifth
Edition; Harcourt Brace: Philadelphia, 1998; 591-621.
2. Copper, C. L. Capillary Electrophoresis Part I. Theoretical and Experimental
Background. J. Chem. Ed. 1998, 75, 343-347. pdf
3. Copper, C. L.; Whitaker, K. W. Capillary Electrophoresis Part II. Applications. J. Chem
Ed. 1998, 75, 347-351. pdf
4. McDevitt, V. L.; Rodriguez, A.; Williams, K. R. Analysis of Soft Drinks: UV
Spectrophotometry, Liquid Chromatography, and Capillary Electrophoresis. J. Chem. Ed.
1998, 75, 625-629. pdf