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Instrumental Anlaysis
CHE 331
Dr. Rahni
EXPERIMENT 7: HPLC Determination of Caffeine in Soda and Coffee
Objectives
The purpose of this lab is to determine the amount of caffeine in various
beverages.
Reference
1. D.T. Sawyer, W.R. Heineman, J.M Beebe, Chemistry Experiments for
Instrumental Methods, John Wiley & Sons, Canada, 1984
Theory
High performance liquid chromatography (HPLC) is a highly versatile method,
which involves the separation of mixtures into their individual components using two
phases, solid and liquid, in contact with one another. It consists of five basic parts;
solvent pump(s), injector, column (stationary phase), detector, and recorder (computer).
Samples are simply injected directly onto the column by means of the injector, and
through this column the mobile phase (solvents) are pumped at high pressure. These
columns are stainless steel tubes packed with the stationary phase. The components will
be separated according to the size and shape of the molecules. The smallest and least
hindered molecules will be eluted first since it is easiest for them to pass through the
finely packed column. As a result, certain components move at different rates depending
on their interaction with the stationary phase.
The components are detected as they leave the column by means of UV
absorbance spectrophotometry by the detector. The detector signals are sent to the
recorder or computer and are seen as "peaks." The area of this peak (in relation to the
area of other peaks) is proportional to the concentration of that particular species in the
sample. Therefore, the area or the peak that is recorded can be used to determine the
concentration of that particular component.

This is done by injecting known

concentrations into the HPLC and recording the corresponding peak area for each

concentration. After multiple concentrations have been injected, a calibration curve can
be made. When an unknown concentration is injected, the peak area can be interpolated
onto the calibration curve to give the unknown concentration. For this experiment the
unknown concentrations will be the concentration of caffeine in various beverages. To
determine the concentration of caffeine in the beverages a calibration curve will need to
be established. Therefore known concentrations of caffeine are prepared and injected
into the HPLC. The known concentration peak areas can then be plotted to give the
calibration curve (peak area vs. concentration of caffeine).
Instrument and Equipment
Instrumentation
This experiment will be conducted using a Varian 5000 Liquid Chromatograph
with a Kipp and Zonen Flatbed recorder.

Procedure
Preparation of soda, tea, and coffee samples
1.

Obtain beverage.

2.

Degas the sample by placing it in a vacuum flask and connecting the flask to a
vacuum pump or water aspirator. Leave it under vacuum until no more bubbles
appear in the beverage. (If no vacuum is available, allow the beverage to stand
open overnight.)

3.

Filter the degassed beverage through 100 micron filter paper.

Preparation of caffeine standards

1.

Prepare a 1000 ppm solution of caffeine.

2.

Prepare standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100
ppm by diluting portions of the 1000 ppm solution with distilled water.

Determination of caffeine content

1.

Obtain the degassed 20% methanol: 80% water solvent for the mobile phase.

2.

Place solvent tube into the solvent and secure. Place the other line into the waste
bottle.

3.

Turn on the HPLC. Prime the pump using one of the 60 L syringes. Make sure
the valve is open before pushing the prime button. If it is not, the column

could be damaged. Bring the flow of solvent up to 2.3 mL/min. Let the HPLC
run approximately 15 minutes before injecting your sample. (Make certain that
waste is collecting in the waste bottle.)
6.

Make sure all bubbles are removed from the tubing and detector.

7.

Clean the syringe by rinsing several times with the solution to be injected.

8.

Draw up sample to the 100 L mark, and expel down to 80 L; make sure that
there is no air in the syringe.

9.

Make sure the injector lever is in the LOAD position, and inject the sample.

10.

Move injector lever to inject position.

11.

Return injector lever to LOAD position.

12.

Print any runs if instructed to do so.

13.

Repeat steps 7 - 13 for each sample (standards, soda, coffee and tea).

Analysis of data
1.

Use standard caffeine samples to identify the caffeine peak and record the
retention time of caffeine. The peak will increase from 20 to 100 ppm and will be
after the small solvent peak.

2.

Use the retention time to determine if caffeine is present in the soda sample.

3.

To quantitatively determine the amount of caffeine in the sample, measure the

caffeine peaks of the standards, and construct a standard curve.

4.

Measure the caffeine peak in the soda sample chromatograph, and use the
concentration to peak area relationship to determine the concentration of caffeine
in the soda sample.

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