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In recent years the technology used in chromatographic analysis has greatly improved.
Advances in this area have led to the common use of High Performance Liquid
Chromatography, a laboratory technique that allows for the very efficient separation of small
amounts of the components of a mixture. The technique has essentially the same operational
basis as liquid chromatography, but HPLC allows for far better separation of the components
mixture. The column in an HPLC instrument contains tiny particles of only 5 µm diameter,
ensuring a very large surface area to which molecules may absorb. These particles comprise
the stationary phase of chromatographic system. Because these tiny particles are so tightly
packed, the mobile phase solvent must be forced through the column under very high pressure.
A detector and a computer are connected to the HPLC instrument in order to signal when
eluents are coming off the column and fractions should be collected.

Identification of samples is carried out by comparison of their retention times. A known

amount of a known compound (a standard) is injected, the retention time is recorded and the
area under the peak can be calculated. A sample containing the same analyte is then injected
under the same experimental parameters. If the analyte is the same compound as the standard,
then they should give identical retention times. The ratio of peak areas should give the amounts
of the components present.

Reverse phase partition chromatography in HPLC employs a polar mobile phase and a
non-polar stationary phase. This is the most frequently used form of HPLC. Other modes of
liquid chromatography include normal phase partition (non-polar mobile phase and polar
stationary phase), adsorption, ion exchange and size exclusion.

The column used in this experiment consists of a layer of an alkane C-18 chemically
bonded to the surface of very small silica particles. The alkane stationary phase is non-polar,
the silica is the inert support material with high surface area and the mobile phase is a polar
solvent (water/acetonitrile).

1. To determine the retention time of a standard solution ( caffeine ).

2. To identify the caffeine peak in a soft drink sample.
3. To determine the amount of caffeine in soft drink sample using the respond factor method.


 Beaker
 Burette
 Glass rod
 Volumetric flask 10 mL
 Dropper


 Caffeine standard
 Soft drink ( coke / F&N )
 Distilled water
 Acetonitrile


A. Experimental
1. Caffeine standard solutions.
i. Stock solution, 1.0mg/mL ( solution A )
a) 10 mg caffeine was weighed accurately into a 10 mL volumetric flask.
b) Dissolve in distilled water.
d) Make up to volume with distilled water.

ii. Working solution, (solution B)

a) 1mL of solution A was pipet into a 10 mL volumetric flask.
b) Make up to volume with distilled water.
2. Sample preparation.
i. 10ml of soft drink sample have been poured (coke and F&N) into a small beaker.
ii. Magnetic stirrer has been used to remove any carbocation (sonnicate if necessary).
iii. 2-5 ml aliquot has been filtered through a 0.45 micrometer pore diameter membrane
filter to remove particulate matter.
3. 10 𝜇𝐿 solution B into the HPLC to establish the retention time, tR, of caffeine.
4. Peak area of caffeine has been measured.
5. 10 𝜇𝐿 of the prepared soft drink sample were injected into the HPLC instrument.
6. The peak area was measured corresponding to caffeine by referring to its tR.

B. Operation of the HPLC



Operating Instructions

Starting the instrument (referred on laboratory manual)

Running the software (referred on laboratory manual)

Sample preparation:

1. Use a syringe and a filter to transfer sample (10-20 mL) into a vial.
Type of filter: SRP 15 (0.45 mm).
2. To prepare mobile phase, filter the mobile phase. For methanol, water, buffer and
acetonitrile (ACN), use a membrane filter.
3. Use ultrasonic cleaner (Model : UC -05 ).
4. Sample injection.
Click single run acquisition. Insert syringe and load
Inject sample and turn down
5. Select control > Extent run _____________ min.
6. Select report > View > Method custom report.
7. Print results.
8. For clean up process, click LC setup assistant.
For mobile phase B ,C and D, wash with ACN. Click 100% ACN.
For buffer, wash with water. Click 100 % water.
9. Switch off the instrument.

𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

Response factor, standard =
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑


= 9.741 x 10-3 mg/L

Therefore, concentration of caffeine in coke can be determined

𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒

Concentration of caffeine =
𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑓𝑎𝑐𝑡𝑜𝑟

= mg/L
9.741 𝑥 10−3

= 71.35 ppm


1. State the types of compounds which are suitable for analysis using HPLC.
The compounds that suitable for HPLC are the not easily volatile compound and have high
molecular weight.

2. Differentiate between the HPLC and the GC in terms of:

i. Mobile phase.
HPLC: mobile phase is liquid
GC: mobile phase is gas.

ii. Column.
HPLC: short length column about 20-25 cm
GC: 50 cm long column and the column length can be higher than 50 cm.

In HPLC, it is important to consider are the nature of the stationary phase (or column), the
mobile phase which controls the partitioning or separation of the sample analyte and their rate
and order of elution. The nature of the stationary phase will determine whether the column can
be used fornormal or reverse phased chromatography. Fornormal phase, a polar stationary
phase and non-polar mobile phase so that more polar compounds will be eluted later than non-
polar compounds generally. In reverse phase chromatography, on the other hand, uses a non-
polar stationary phase and polar mobile phase causing polar peaks to generally elute earlier.

The mobile phase for reverse phase is composed of an aqueous buffer and a water
miscible organic solvent that has little to no absorption above 200 nm. Organic solvents are
added to adjust the polarity of the mobile phase and allow the elution of non-polar analytes after
the polar ones. The mobile phase is usually buffered to control the pH, an important parameter
to consider in HPLC.

In the experiment, the stock solution of caffeine was produced with 1000mg/L. Then, the
standard of caffeine with 100mg/L was produced by stock solution. The standard was filtered
about three times and been injected to the instrument. The retention time of the peak
appearance result with 0.910 minutes.

The experiment also covered the determination of caffeine in the soft drink sample. By
applying the same method of preparation of sample, the soft drink sample then was put into the
sonicator to reduce the air bubble in sample. The first injection resulted the retention time with
0.871 minutes. The second and third injection resulted the retention time with 0.877 minutes
and 0.870 minutes respectively. The difference between retention time of sample and standard
show than there is no caffeine in the sample. However, estimation of the presence of derivative
of caffeine is acceptable according to some references.

In conclusion, the retention time of a standard solution is identified which is 0.910

minutes. Even the caffeine peak does not appeared, the derivative of caffeine is believed to be
presented as the appearance of peak with retention time 0.871 minutes, 0.877 minutes and
0.870 minutes for three runs. The determination of the concentration of caffeine in the soft drink
by using the formula and response factor value resulted 71.35 ppm of caffeine in the sample.