TITLE

Determination of Caffeine in Beverages by High Performances Liquid

Chromatography (HPLC)

OBJECTIVE

1. To determine amount of caffeine in sample of beverages by using HPLC.

INTRODUCTION

Caffeine is a bitter, white crystalline purine, a methylxanthine alkaloid, and is

chemically related to the adenine and guanine bases of deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA). It is found in the seeds, nuts, or leaves of a number of
plants native to South America and East Asia and helps to protect them against
predator insects and to prevent germination of nearby seeds. The most well known
source of caffeine is the coffee bean, a misnomer for the seed of Coffea plants.
Beverages containing caffeine are ingested to relieve or prevent drowsiness and to
improve performance. To make these drinks, caffeine is extracted by steeping the
plant product in water, a process called infusion. Caffeine-containing drinks, such as
coffee, tea, and cola, are very popular.

Caffeine can have both positive and negative health effects. It can treat and
prevent the premature infant breathing disorders bronchopulmonary dysplasia of
maturity and apnea of prematurity. Caffeine citrate is on the WHO Model List
of Essential Medicines. It may confer a modest protective effect against some diseases,
including Parkinson's disease. Some people experience insomnia or sleep disruption if
they consume caffeine, especially during the evening hours, but others show little
disturbance. Evidence of a risk during pregnancy is equivocal; some authorities
recommend that pregnant women limit consumption to the equivalent of two cups of
coffee per day or less. Caffeine can produce a mild form of drug dependence –
associated with withdrawal symptoms such as sleepiness, headache, and irritability
– when an individual stops using caffeine after repeated daily intake Tolerance to the
autonomic effects of increased blood pressure and heart rate, and increased urine
output, develops with chronic use.

High-performance liquid chromatography (HPLC) is developed to increase speed and

efficiency in liquid chromatography. Decreasing the size of the solid support material
increased efficiency.

Figure 1 : Structure of caffein

MATERIALS AND PROCEDURES

Chemicals and Materials

Caffeine

Magnesium oxide

Methanol

Beverages sample

Apparatus

HPLC with UV detector and isocratic system

Biker

Volumetric flask

Syringe
Part A : Preparations of standard solutions

1. 100 ml, 1000 ppm of caffeine stock solution prepared.

2. The stock solution diluted to 100 ml of desire concentration of 20 ppm, 40 ppm, 60

ppm, 80 ppm and 100 ppm caffeiene with distilled water.

Part B : Sample preparation

1. 0.5 g beverages sample and 5g MgO mixed in 200 ml water in beaker and stirred
for 20 minutes at 90C in water bath. The solution cooled to room temperature.

2. The solution sample degased by placing it in sonicator for 5 minutes.

3. All the samples filtered through filter papers.

4. The filtered samples diluted 10 times prior for injection.

Part C : HPLC method

1. The sample loop cleaned by flushing 250 µL of mobile phase to clean loading
passages.

2. The syringe refilled, wipe cleaned, reinsert and flushed in another 250 µL, making
sure not to inject any air bubbles.

3. The syringe rinse with several aliquots of the sample to be injected. Before injected
100 µL, the syringe was checked to make sure no air bubbles.

4. The data recorded.

RESULTS AND DISCUSSION

A. Standard Caffeine Sample

Retention time of standard caffeine sample :

 Table 4.1 : Peak area or peak height of standard caffeine sample

Concentration of
Peak Area Peak Height
Caffeine (ppm)

20 2775.79688 179.60068

40 5991.54883 385.86853

60 8834.93457 564.23248

80 1.25160e4 774.64056

100 1.46333e4 895.24420

B. HPLC Result

Retention Concentration % of
Types of Peak
Times of Peak Area of Caffeine Caffeine
Beverages Height
Caffeine Present in Sample

1.030 10.97531 1.68316 100 5.8785

1.218 9.11339 1.51511 100 4.8812

1.318 11.97022 1.76196 100 6.4114

Tea Leaves
1.509 10.85414 1.30932 100 5.8136

2.329 6.96240 1.09867 100 3.7291

6.028 136.82666 8.83598 100 73.2861

HPLC is chromatographic technique used to separate the components in a

mixture, used mainly to identify each component, and to quantify each component.
HPLC is considered as an instrumental technique of analytical chemistry. Separation
is based on the analyte’s relative solubility between two liquid phases. The method
involves a liquid sample being passed over a solid adsorbent material packed into a
column using a flow of liquid solvent. Each analyte in the sample interacts slightly
differently with the adsorbent material, thus retarding the flow of the analyte. If the
interaction is weak, the analyte’s flow off the column in a short amount of time, and if
the interaction is strong, then the elution time is long.

In this experiment, reverse phase HPLC is used. Reversed phase HPLC

(RP-HPLC) used for the separation of molecules based on their hydrophobic
interaction between the solute molecule in the mobile phase and the immobilised
hydrophobic ligand in the stationary phase. For reverse phase HPLC, the mobile
phase is more polar than stationary phase. The process of chromatography starts by
injecting a sample into the instrument. The mobile phase moves it through the column
chromatography which is the stationary phase.

The mobile phase moves particles through the column while the stationary phase
allows particles to remain in the column. On the other hand, the caffeine standards
provide a frame of reference to which the data obtained from the soft drinks can be
compared. A calibration curve for peak area vs. concentration is made. One can
determine the amount of caffeine in the soft drinks by plotting data on this curve.

Lastly, the syringe have to be carefully rinsed before used to remove any
contaminents from the syringe that basically can cause errors occured in the results.
Thus, to make sure a certain a peak in the soda was caffeine and not another substance
with a similar retention time The mobile phase needed to be change and inject the
caffeine and then the sample. Both peaks should be affected similarly if they are
both due to the presence of caffeine.

CONCLUSION

In conclusion, this experiment have determined the average retention time of

standard solution of caffeine which is 2.239 minutes. Then, the caffeine average peak
area in the tea leaves has identified which is 31.11702 of peak area while the average
peak height is 2.70070. Next, the average percentage of caffeine in tea leaves sample
is 16.6667%.
QUESTIONS

1. Briefly explain how HPLC is used as a separation technique.

 HPLC is chromatographic technique used to separate the components in a

mixture, used mainly to identify each component, and to quantify each
component. HPLC is considered as an instrumental technique of analytical
chemistry. Separation is based on the analyte’s relative solubility between two
liquid phases.

 The method involves a liquid sample being passed over a solid adsorbent material
packed into a column using a flow of liquid solvent. Each analyte in the sample
interacts slightly differently with the adsorbent material, thus retarding the flow
of the analyte. If the interaction is weak, the analyte’s flow off the column in a
short amount of time, and if the interaction is strong, then the elution time is
long.

2. What is the purpose of the mobile phase? Of the stationary phase?

 The mobile phase moves particles through the column while the stationary phase
allows particles to remain in the column.

3. What is the purpose of the caffeine standards?

 The standards provide a frame of reference to which the data obtained from the
soft drinks can be compared. A calibration curve for peak area vs. concentration
is made. One can determine the amount of caffeine in the soft drinks by plotting
data on this curve.

4. Why does the syringe have to be carefully rinsed before each use?
 Contaminents must be removed from the syringe.

5. How could you be certain a peak in the soda was caffeine and not another
substance with a similar retention time?

 Change the mobile phase and inject the caffeine and then the sample. Both peaks
should be affected similarly if they are both due to the presence of caffeine.

REFERENCES

Clark, J. (2016). Paper Chromatography. Retrieved from

https://www.chemguide.co.uk/analysis/chromatography/paper.html

HPLC. (n.d). Retrieved from

https://www.omicsonline.org/hplc-scholarly-open-access-journals.php