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Caffeine Determination by HPLC

High performance liquid chromatography (HPLC) is a relatively new technique when compared
with gas chromatography, paper chromatography or thin layer chromatography. It is a form of liquid
chromatography with the main difference being that much smaller and more uniform stationary phase
particles are used. The spaces between the individual particles are very small and therefore higher
pressures are required to maintain a suitable flow. Another name for this technique is high-pressure
liquid chromatography. In recent years most HPLC analysis is carried out using bonded phase columns.
The solid stationary phase support (usually silica) has the liquid stationary phase chemically attached to
it via covalent bonds. This means the liquid part of the stationary phase cannot be easily washed off by
the mobile phase. This has made it possible to use a large variety of mobile phases and thus has made
many difficult separations possible. Caffeine is a polar organic molecule found in many beverages and
even in pharmaceutical preparations. It is water-soluble and contains an amino group that can be
protonated by the addition of acid. Separation from other components of beverages can be
accomplished on a bonded phase C-18 column using an acidic methanol - water mobile phase.


1) Separation and determination of caffeine in a variety of beverages by HPLC.

2) Explain the differences between normal phase and reverse phase HPLC.

3) Describe the types of detector systems used in HPLC.


HPLC is based on partition chromatography. The components of a mixture partition between

the liquid film, that serves as the stationary phase and the mobile phase passing by the stationary phase.
Components that are more like the stationary phase stay on the column longer than components that are
more like the mobile phase. Size, polarity, and similarity of functional groups affect the degree of
attraction between the individual components in the mixture and the stationary phase.

In normal phase chromatography the column is polar and the mobile phase in non-polar.
Alumina and silica gel are the most common normal phase materials. In our experiment, reverse phase
chromatography is used. The column is non-polar and the mobile phase is more polar. In the early
days of HPLC liquid films similar to those used in GC were coated on an inert support. They worked
fine as long as the mobile phase was very polar (water), but even solvents as polar as methanol tended
to move the stationary phase liquid. Now essentially all reverse phase columns have bonded phases. A
silane material is reacted with small (5 µm), uniform silica beads. A covalent bond is formed between
the silica surface and the organic silane (text page 865). The two most common phases are C-8 and C-
18, which have 8 carbon or 18 carbon long straight chain alkanes attached to the silica. If the phases
were not bonded solvents like acetonitrile or hexane would destroy the column, but with bonded phases
these solvents are commonly used for separations or for cleaning.

The heart of an HPLC system is the pump. The pump forces the mobile phase through the
column at a constant flow rate. Within the pump system are devices to remove pressure pulses caused
by the individual strokes of the pump piston. The pump must be capable of producing high pressure
since the backpressure of the columns can be substantial.

With the GC the sample is injected through a rubber septum and is immediately vaporized into
the gas stream. Injection in HPLC is not as straightforward. The mobile phase is forced through the
column by very high pressures (>1000 PSI) and any injection through a septum would result in a leak.
A special injector valve is used to divert the flow from the pump through a sample loop and then to the
column. The sample in the sample loop is washed onto the column. Since the sample loop is a section
of stainless steel or plastic tubing, with a fixed internal volume, reproducible injections are possible.

Several different detectors are used with HPLC: fixed wavelength UV, variable wavelength
UV-visible, diode array UV-visible, refractive index, fluorescence, IR and several types based on
electrochemical techniques. In our experiment the detector is a variable wavelength UV detector. For
caffeine, the best sensitivity is at 282 nm. The output from the detector goes to the computer, which
serves as a peak integrator. The computer processes the data and gives retention times, peak areas,
and concentrations if the method contains calibration information.


Select two soft drinks, coffee and tea as unknowns. Pour a portion of each soft drink back and
forth between two clean 50 mL beakers to remove the carbonation. You may drink the unused portion
(not in the lab). Pipette 10.00 mL of each soft drink into clean 25 mL volumetric flasks and dilute with
50% methanol. Pipette 2.00 mL of coffee and 5.00 mL of tea in separate 25 mL volumetric flasks.
Again dilute with 50% methanol and mix well. Remember to use glass stoppers, not parafilm. Using the
1000 ppm caffeine standard prepare standards with concentrations of 1, 2, 5, 10, and 20 ppm by
diluting the proper amount of this standard with 50% methanol. Go to the method editor and open
Caff370.met. Do as save as Caff<your initials>.met and use that in the schedule for your part of the

Calibration and Analysis Set up the vials for the auto-sample as shown below.
Vial# 1 2 3 4 5 6 7 8 9 10
Contains blank 1 ppm 2 ppm 5 ppm 10ppm 20ppm Soft Soft Tea coffee
drink drink2
Place the auto-sampler cartridges in to auto-sampler and press the hold/run button on it, once.
The cartridge should move into the sampling position. Start PeakNet and then select schedule. This
will allow you to modify the existing schedule. Be sure to change soft drink labels to reflect what your
unknowns are (i.e. SD1 to Coke). The final schedule should have 11 lines with StopHPLC.met as the
method in the last line. Since we will probably be running several groups of samples all at once the
schedule has to keep straight your samples from the next group. I will help in schedule preparation.
Save the schedule as HPLC370a. Close the schedule window and open the run window. The run
screen will come up with HPLC on top and Anions & Cations under it. Be sure the HPLC screen is on
top. Under file select load schedule and then HPLC370a.sch. This broad range calibration curve has
the correct setting for operating the instrument. This will also make sure the pump and detector are
running correctly. Don’t start the instrument for 20 minutes. This allows time for the instrument and
column come to equilibrium with the eluting solution. After 20 minutes the instrument is ready for you to
inject your samples. Click the run box to start the schedule. After you start the program wait until your
first sample is injected and a line starts on the graph. Your results will be available the next morning. If
your caffeine peaks are not labeled I will have to do a post run reintegration to get your results. If every
thing seems normal, go to the method section of PeakNet and open your method. Click on the
detector block (below the pump block). Near the bottom of the window there are a bunch of boxes.
The 6th box is for calibration, click it and then click details (lower right in window). This should show
you your calibration curve. You can print the calibration graph from this page.

Lab Report

The lab report should contain:

(1) Your chromatograms from the computer

(2) Your calibration graph for caffeine (this can be printed in the method section) .

(3) The concentration of caffeine in each of the beverages (remember to include appropriate
dilution factors), as well as amount of caffeine in a typical serving of each beverage (you decide what a
typical serving is!). Show all calculations.

(4) Answers to the following questions

A) Describe three types of detectors for HPLC.

B) What is the difference between reverse phase and normal phase chromatography?

C) Why does the pH effect the retention time for caffeine?

D) What values did you find on the web for the amounts of caffeine in your samples. Be sure to
give the web address you used.