FST306

FOOD ANALYSIS

LABORATORY REPORT

EXPERIMENT 10:
DETERMINATION OF CAFFEINE CONCENTRATION
BY USING HPLC

DATE OF EXPERIMENT: 2nd DECEMBER 2024

DATE OF SUBMISSION: 14th JANUARY 2025

GROUP: AS1163A3

SUBMISSION TO: DR. SAFIYYAH BINTI SHAHIMI

PREPARED BY:

Name Student ID IC Number

1. Nur Iman Athirah binti Ahmad Asri 2023614652 051217-14-0718
2. Siti Aisyah Faqihah binti Norazman 2023644712 050715-14-1006
3. Najwa Hani binti Zamri 2023623056 050621-10-0932
4. Nurul Izzah binti Mohd ‘Asri 2023614808 051220-06-0364
INTRODUCTION
Caffeine is classified as a stimulant medicine because it speeds up the messages that are sent
from the brain to the body. It is an organic stimulant that can be found in many different types
of drinks, such as energy drinks, coffee, tea, and soft drinks. Since more and more people drink
beverages with caffeine, accurately measuring the amount of caffeine in these goods is crucial
for both legal and medical reasons. Nowadays, High-Performance Liquid Chromatography
(HPLC) has widely been used, it is a general method in analytical chemistry that is used to
separate substances in a mixture. These separations make use of a mobile phase flowing
through a stationary phase-packed column under pressure. The longer time spent engaging with
the stationary phase, the longer time spent on the column, and the longer the retention time (R f)
for that component, the greater the interaction with the stationary phase relative to the mobile
phase. Therefore, HPLC can efficiently identify and measure the amount of caffeine present in
liquid samples by examining the retention time of recognized compounds.

This experiment's three main goals were to find the caffeine standard's retention
duration, locate the caffeine peak in samples of soft drinks, and measure the amount of caffeine
in these drinks. It is possible to identify the caffeine peak in the soft drink samples by using the
retention time of the caffeine standard as a reference. Calculating the caffeine concentration in
the soft drinks by comparing the caffeine peaks in the samples with the standard yield's
important information about the caffeine content and guarantees that the product fulfils
labelling regulations.

OBJECTIVES

1. To determine the retention time of caffeine standard.

2. To identify the caffeine peak in soft drinks.
3. To determine quantity the concentration of caffeine present in the soft drinks.

STANDARD
Caffeine
SAMPLE
Coca-cola
100 Plus
Pepsi

APPARATUS
Beaker
Volumetric flask
Dropper
Syringe filter
Vial

METHODS
HPLC setting
HPLC model : 1200 Agilent
Column : C18
Mobile phase : Acetonitrile: water (70:30)
Flowrate : 1.0mL/ min
Detector : UV at 254nm
Injection volume : 20µL

1. Caffeine standard solution

a) 100 mg caffeine was accurately weighed into a 100 mL volumetric flask.
b) It was dissolved in Dl water and made up to volume.
c) A 5 mL aliquot was filtered through a 0.45 µm pored a diameter membrane filter to
remove particulate matter into a vial.

2. Sample preparation
a) Approximately 10 mL of soft drinks sample was poured into a small beaker.
b) 5 mL of aliquot was filtered through a 0.45 µm pored a diameter membrane filter to
remove particulate matter into a vial.
c) The solutions (standard and sample) were sonicated.
3. 10 µL of the caffeine standard solution was injected into the HPLC to determine the
retention time.
4. 10 µL of the prepared soft drink sample was injected into the HPLC.
5. The peak area of caffeine was measured by referring to the retention time.

RESULTS AND DATA SHEETS

Table 1: Retention Time and Peak Area of Caffeine for Standard and Sample
Standard/ Sample Retention time, TR Peak area of caffeine
Caffeine 0.830 56.617
Coca-cola 1.307 5358.417
Pepsi 1.085 3318.106

Table 2: Peak Area Data for Caffeine

Standard Peak area Response factor, Rf
Caffeine 56.617 0.56617

Table 3: Peak Area Sample for Sample

Sample Peak area Amount
1. Coca-cola 5358.417 9467.99
2. Pepsi 3318.106 5861.09

Graph 1: Peak Area for First Result of Each Sample

CALCULATION
Response factor, R = Peak Area of Standard
Concentration of Standard

= 56.617
100
= 0.56617 (area/ppm)
Amount of caffeine = Peak Area of Sample
Response factor

Coca-Cola = 5358.417/0.56617
= 9467.99 ppm
100 Plus = 3318.106/0.56617
= 5861.09 ppm
DISCUSSION
Based on experiment, the chromatograms obtained show caffeine peaks in both Coca-Cola and
100 Plus drinks and the retention time and peak area of the shove image the identification the
caffeine content in both drinks. The caffeine standard yielded a retention time of 0.830 minutes
and was used to determine the presence of caffeine in the samples. Coca-Cola had 1.307
minutes retention time and 5358.417 peak area which proved it was relatively higher caffeine
content than to 100 Plus which had 1.085 min retention time and 3318.106 peak area.

These values of retention times also correlate with the standard and thus supports the
work’s assumption that the chosen method is reliable. Resting upon the bar graph showing the
difference between the peak areas of the two carbonated and the sweet sodas, it is equally
evident that the caffeine level is significantly high in Coca-Cola, which is intended to serve as
energy drink, while 100 Plus isotonic drink contains surprisingly lesser amount of caffeine.
Since response factors were not determined because concentration data were not available, the
analysis provides a clear indication of the efficiency of the method in differentiating as well as
determining the concentration of caffeine in the various beverages. In general, the presented
results concord with predicted expressions and demonstrate the effectiveness of the HPLC
method for the determination of caffeine concentration.

The observed data align with the theoretical principles of HPLC, where analytes with
higher affinity for the stationary phase exhibit longer retention times, and peak areas correlate
with analyte concentrations. Instrumental sensitivity, column efficiency, or sample preparation
may be the cause of discrepancies or variances in the results. This analysis emphasizes the
reliability of HPLC method in quantifying caffeine levels, which providing a clear
differentiation between caffeinated and non-caffeinated beverages.

To obtain accurate results in an HPLC method experiment, several precautionary steps

must be followed. Samples must be carefully prepared with precise amounts and filtration to
prevent contamination or column blockage. Using hygienic and appropriate glassware or
containers is crucial to avoiding contamination or sample deterioration. During the experiment,
the flow rate and injection volume must be accurately set and monitored to ensure consistency
across runs. Besides, the data must be analyzed with care, which ensuring peak integration is
performed correctly and any irregularities in the chromatograms are investigated thoroughly.
By adhering to these precautions, the accuracy and reproducibility of the HPLC results can be
significantly improved.

CONCLUSION
In conclusion, the objectives of this experiment successfully achieved where we managed to
determine the retention time of caffeine standard, we managed to identify the caffeine peak in
soft drinks as well as we managed to determine the quantity of concentration of caffeine present
in the soft drinks. The amount of caffeine in soft drink sample was determined using response
factor method. It shows that the retention time and peak area of caffeine is lowest than Coca-
cola and 100 Plus sample, which means that caffeine has lowest sugar composition than Coca-
cola and 100 Plus while Coca-cola has the highest sugar composition between caffeine and 100
Plus. As expected, 100 Plus is a non-caffeinated beverage, the results showed that Coca-Cola
had the highest caffeine concentration (9467.99 ppm), followed by Pepsi (5861.09 ppm).
Caffeine had the shortest retention period (0.830 minutes), which was indicative of its less
interaction with the stationary phase than the more complex beverage matrices. The retention
times varied among the samples. The response factor of 0.56617 derived from the caffeine
standard was validated by the peak areas, which were directly proportional to the caffeine
concentrations. Overall, the experiment demonstrated the reliability and precision of HPLC
method in quantifying caffeine content and distinguishing between caffeinated and non-
caffeinated beverages, making it a valuable tool for quality control and analytical studies in the
food and beverage industry.
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