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Lab-10-hplc-2012 chem 3420

Qualitative and Quantitative Analysis of Mixtures Experiment: A computer controlled high performance liquid chromatography system is used in this experiment to gain familiarity with the HPLC instrumentation and the experimental
variables effecting chromatographic separations. Using a C18 RP-HPLC column and
1 mM solutions of theobromine (TB), theophylline (TP), and caffeine (CA), the basic
qualitative and quantitative analysis techniques are demonstrated. In addition, an
external reference calibration curve is generated for caffeine and used to analyze the
caffeine content of popular soft drinks.

Standards and unknown:
Samples of theobromine, theophylline, and caffeine are
provided at a concentration of 5 mM in 1:1 methanol:water (v/v). An unknown mixture
and samples of soft drinks are also provided.
HPLC Operating Instructions

Turning on the instrument: Switch on the Flexar LC pump, Flexar UV-vis detector, and
Flexar Solvent manager, HP printer and computer. The pump and detector have large
rocker switched in the rear, the solvent manager plugs into a transformer. Once the
rocker is in the on position, push the silver buttons on the front of the pump and detector
to turn them on.
Mobile phase preparation: HPLC-grade water and methanol are provided which have
been vacuum filtered through a 0.2 µm membrane. Connect the supply line from the
aqueous solvent to pump A: the methanol supplies pump B.
System purging: Open the front of the Flexar LC pump by removing the gray front cover
(held on by magnets). Open the black prime purge valve located on the front, left of the
binary LC pump by turning it counterclockwise 1-2 turns. Push the A button to pump 6
mL/min of solvent A thru the system to remove any air bubbles. After 30 sec, push button
B to purge the lines. After 30 sec close the drain valve, the pumps will stop as the high
pressure limit is reached and the red error light begins to flash.
Open the Chromera software: Click on the ‘Chem3420 HPLC’ icon on the right side of
computer desktop. When the program loads, go to Run Time > Control Mode > Manual
Control. In the Manual Control Window (lower, center) set the flow rate to 0.8 mL / min
by typing in the value and hitting the apply button. On the status panel, note the Pump
Flow and the Pump Pressure. Using the Manual Control window, set the solvent to be 50% solvent A and 50% solvent B. You will use this solvent mixture for the first six runs. Allow the column to equilibrate with solvent to 8 min. before injecting the first sample. There is no need to equilibrate in between runs. Single Run: Switch from Manual Control to Single Run. Using File > Open Method, select the Method Group Chem 3420 > Isocratic 5050 method and open it. The window should change back to the Run Time window. In the Method (Not Active) window, click the apply button if the Method Name is Isocratic 5050. Another window will open and give the status of the pump and detector. The pump is programmed to equilibrate for 30 sec, after which the pump/detector window will close and another will open on the lower right and state that the HPLC is waiting for your manual injection. Loading the sample loop: Fill the HPLC syringe with >60 µM of the theobromine sample. Turn the injector arm counterclockwise to the LOAD loop position (down). Insert the needle into the port, you will feel a little tightness during the last 2-3 mm of travel as the needle passes through the needle seal and stops against the stator face. Squeeze the syringe plunger to load the loop. Leave the syringe in the injector until after the sample has been injected onto the column. Note: the attached loop only holds 20µL of sample, so any excess goes directly to the waste. Injecting the Sample: Inject the sample onto the column by quickly turning the injection arm clockwise to the inject position. This starts the HPLC run and the status window will update with the chromatogram. After a minute or so, remove the HPLC syringe from the injector and return the injection arm back to the load sample position to get it ready for the next run. Each run lasts 8 min. At the end of that run, the computer will automatically save the chromatogram and clear the data collection window. Once the run is complete, click the Post Run button and use File > Open Data to open the chromatogram. The display will show the column pressure vs. time as the default to show your chromatogram, click on FXUVDet-2 in the Post Run window on the left. The software will report the retention time and peak height/area values. Print the report using the Select Report Template > Sample-1 injection-Single channel. Performing the sequence of HPLC runs: Using the same process, run the following eight samples: Caffeine (2.5 mM, dilute 5.0 mM stock with HPLC grade water) Caffeine (1.0 mM, dilute 5.0 mM stock with HPLC grade water) 5 hr. Energy (dilute 100-fold with HPLC grade water)
Qualitative analysis (Runs 1-5): After printing your initial chromatogram, return to the
Run Time window, apply the Isocratic 5050 method and wait for the system to
equilibrate. After the 30 s equilibration inject the next sample as before. Using this
process, run each of your known compounds alone, in a 1:1:1 v/v/v combination and the
unknown (Runs 1-5). Using the retention times as a guide, identify each component in
the 1:1:1 combination sample and in your unknown mixture.
Quantitative analysis using an external reference: Determine the concentrations of the
components in the unknown mixture using the peak areas of the unknown and standard
solutions. Use detector response factors since the molar absorbtivity of TB, TP, and CA
are not identical at 250 nm.
Quantitative Determination of Caffiene in Soft Drinks: Run two dilutions of the caffeine
standard (2.5 mM and 1.0 mM) and use the peak areas of the three concentrations (5.0
mM, 2.5 mM and 1.0 mM) to generate a calibration curve for caffeine. Analyze the Coca
Cola and the 5-hour energy samples to determine their caffeine concentrations.

Follow the outline of a regular laboratory report as provided on the course web site. Make sure to provide a block diagram of the HPLC with all the components and their purposes. Explain the techniques and methods used (RP-HPLC, internal/external references). Report the resolution for the 1:1:1 TB:TP:CA sample and peak retention times and peak areas for all TB, TP or CA peaks. Use the molecular structure of TB, TP, and CA to interpret your results. Calculate the molar concentrations of caffeine in the soft drinks using your calibration curve. Compare these results to literature values that you find on the web.


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