UNIVERSITI KUALA LUMPUR

MALAYSIAN INSTITUTE OF CHEMICAL & BIOENGINEERING

TECHNOLOGY
LABORATORY TECHNICAL REPORT
SUBMISSION FORM

To: KHAIRUL FAIZAL BIN PA’EE Code Subject: CFD30003

From: Student ID. No.:
NUR ASIAH BT AHMAD MUSA 55104316002
NUR HADIRAH BT HASHIM 55104316025
NUR AISYAHTON BT WAN SABRI 55104316047

No. of Group: Date of Experiment: 27 MARCH 2018

Title of Experiment: DETERMINATION OF CAFFEINE IN SOFT DRINK BY HPLC

Received by: Date of Submission: 10 APRIL 2018

Note: Late submission will not be accepted.

*To be filled by the marker*

VERY VERY
POOR GOOD EXCELLENT
CRITERIA POOR GOOD
2 3 5
1 4
1.0 ABSTRACT & OBJECTIVES (HALF PAGE 2 4 6 8 10
ONLY) (TOTAL: 10%)
1. State the summary to the experiment
conducted.
2. State the objectives of the experiment (point
form)

2.0 PROCEDURES (TOTAL: 5%) 1 2 3 4 5

1.Methodology is presented in suitable and
understandable flowchart.
3.0 RESULTS (TOTAL: 10%) 2 4 6 8 10
1.Data are presented as deemed suitable with
complete label and units in tables and/or graphs.
4.0 DISCUSSIONS (MAXIMUM 1 PAGE) (TOTAL: 3 6 9 12 15
15%)
1. Explanations of the referred tables and/or
graphs are presented after it.
2. Discuss on the findings and relations to the
theory and objective of experiment.
5.0 CONCLUSIONS (TOTAL: 5%) 1 2 3 4 5
1. Summary of the results to relate the findings or
results with the theory applicable to the
experiment.
6.0 REFERENCES (TOTAL: 5%) 1 2 3 4 5
1. Minimum of 4 references.

TOTAL MARKS
 Lab Technical Report
Course code/name

Abstract & Objective(s):

 To study the type of detector system used in HPLC.

 To identify the present of caffeine in soft drink.
 To determine the amount of caffeine in a sample of beverages.
 To determine the concentration of caffeine in soft drink.
 To apply the principles of reverse phase high performance liquid chromatography.

This experiment provides an introduction to the application of High Performance Liquid

Chromatography (HPLC) to the solution of complex analytical problems. Cola type drinks are
complex chemical systems that contain varying amounts of caffeine. The amount of caffeine present
in these soda drink can be determined by HPLC. In this experiment, sample was prepared with
standard caffeine samples of 5ppm, 15ppm, 25ppm, 50ppm, and 100ppm by diluting with distilled
water. HPLC is technique for the separation of the components of a sample by means of a high
pressure pump forcing the eluent through a column. Polar, dispersive or ionic forces working on the
samples allow its components to either be retained in the stationary phase or eluted out by the mobile
phase thereby producing responses with different retention times. The area under each response
(peak) can be used to obtain its concentration. In this experiment, reverse phase HPLC was used to
determine the caffeine content of cola products. External calibration was done to generate the
standard curve where the respective concentrations of caffeine in the samples were intrapolated from.
In this experiment, serial dilution also will be prepared to be as standard caffeine and to determine if
caffeine is present in the soda sample by use retention time. The increase the concentration, the bigger
the getting area. This shows that the higher concentration of caffeine will make a bigger effect.

Methodology:

Using a suitable flowchart, state the steps involve in this lab work.

1. calculate for prepare 2. prepare serial dilution for

caffeine standard caffeine standard 5ppm,
1000ppm from the 15ppm, 25ppm, 50ppm and
caffeine standard 100ppm and mark up with
deionized water.

4. degas the sample by 3. obtain the sample and soft

placing it in a vacuum drink.
flask pump or water
aspirator.

5. leave it under
vacuum until no 6.filter the degas soda
more bubbles appears through 45L filter syringe.
in the soda sample.
Result/ Discussion:

High Performance Liquid Chromatography [HPLC] is a form of liquid chromatography used to

separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column, and a detector. Compounds are separated by
injecting a sample mixture onto the column. The different component in the mixture pass through
the column at differentiates due to differences in their partition behavior between the mobile phase
and the stationary phase. In this experiment, reversed phase is used. Reversed phase chromatography
includes any chromatographic method that uses a stationary phase. In this column the packing
material is relatively non-polar and the solvent is polar with respect to the sample. Caffeine is a polar
molecule. Retention is the result of the interaction of the non-polar components of the solutes and
the nonpolar stationary phase.

Based on these experiment, the level of concentration of caffeine in beverages was determined using
HPLC. A chromatogram was obtained for each analysis for each concentration. The response is
displayed on a graph where the x-axis is the retention time and the y-axis is a measure of the intensity
of the response. The size of the peak is proportional to the concentration of the analyte was obtained
after calibration and necessary computations have been made. There are different concentration area
for volumetric flask. Based on table 2, the results obtain for 5ppm is 63326, 15ppm is 168125, 25ppm
is 47770, 50ppm is 526737 and 100ppm is 1046985.

Before chromatographic analysis of the sample, calibration is done to establish a relationship

between signals or the response detected and the concentration of the analyte. External calibration,
which was the one used in the experiment, involves the construction of a standard curve by injecting
different concentrations of a known standard and intrapolating from there the concentration of
subsequent runs. The samples must have no variation in injection volume all throughout because the
calibration parameters will be specific to the chromatographic system established

The Beer-Lambert law states that the quantity of light absorbed by a substance dissolved in a fully
transmitting solvent is directly proportional to the concentration of the substance and the path length
of the light through the solution. Reversed phase chromatography employs a polar (aqueous)
mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the
hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the
column and are eluted first. Using the law, the concentration of the chemical to the amount of UV
absorption can be relate.

Based on graph 1: Peak area vs Concentration, it shows that the results obtained was not accurate
since all the point are not met with the straight line where it does affect the value of R 2 where the
value is 0.94493. The obtained was not statifying because the value was not even near to 1 where it
shows there are wrong steps during preparation of solution. Peaks that are tall, sharp, and relatively
narrow indicate that separation method efficiently removed a component from a mixture; high
efficiency. Efficiency is very dependent upon the HPLC column and the HPLC method used.
However, the peak hasn't shown a good peak separation, the wider the peaks, the poorer the
separation. Efficiency factor is synonymous with plate number, and the 'number of theoretical plates'.
Based on table, at retention time of 1.541 at concentration of 7.097 mg/L it shows the presence of
caffeine in sample (carbonate drink).

Some errors might occur during the experiment. The inefficiency could also come from the column
such as ineffective capping of silanol residues, packing of silica particles, uniformity of C18 coating.
In conclusion, during experiment was conducted there might have some errors occur since the value
of R2 is too far from value of 1.0000, which is in the inefficiency of calibration, measurement of
chemicals in the preparation of standards or in the injection volume which is very vital in external
calibration.
Result:

Table 1: Preparing Serial Dilutions

Stock Diluted

Concentration of Volume of Concentration of Volumetric

Copper (ppm) Caffeine (ml) Copper (ppm) flask (ml)
M1 V1 M2 V2

1 1000 0.5 100 5

2 1000 1.5 100 15

3 1000 2.5 100 25

4 1000 5.0 100 50

5 1000 10 100 100

Table 2: Sample (caffeine )

Concentration Peak Area
5 63326
15 168125
25 477705
50 526737
100 1046985
 Peak area vs Concentration
1200000
1046985

1000000
y = 9841.3x + 72765
R² = 0.9443
800000
AREA

600000 526737
477705

400000

168125
200000
63326

0
0 20 40 60 80 100 120
CONCENTRATION

Graph 1: Peak area vs Concentration

Table 3: Peak
Peak Retention time Concentration
1 1.541 7.097
2 1.980 0.000

Conclusion:

High performance liquid chromatography is a powerful tool for separation, purification and
analysis of analytes in the sample. It is relatively simple to use and the necessary computations
needed for analysis are fairly basic. The main objectives of operating the High Performance Liquid
Chromatography (HPLC) is to determine caffeine content in coke. Caffeine standard with
concentration of 5ppm, 15ppm, 25ppm, 50ppm and 100ppm were prepared. HPLC is a form of liquid
chromatography used to separate compounds that are dissolved in solution. The type of column used
in this experiment is reverse phase. In this column the packing material is relatively non-polar and
the solvent is polar with respect to the sample. Caffeine is a polar molecule. The result obtained
shows that r2 is quite far from 1.000 and the peak hasn't shown a good peak separation. It is therefore
recommended that the procedure be re-evaluated and optimized for the determination of caffeine in
the beverage samples. Other than that, the column should be washed and equilibrated thoroughly as
well as assessed for its efficiency prior to analysis.

References :

1) Reference book- e book:

 Food Analysis Laboratory Manual Second Edition
 Food Analysis Fourth Edition

2) Internet
 https://www.sciencedirect.com/science/article/pii/S016792449780011X/pdf?md5=19826bfeda
7b807d70899ff247f3c9dc&pid=1-s2.0-S016792449780011X-main.pdf&_valck=1
[accessed on 10.35 pm, 9 April 2018]
 https://laboratoryinfo.com/hplc/
[accessed on 8:12 pm, 8 April 2018]

3) Lecture note (HPLC)

Appendices :

Equation:

M1V1 = M2V2

 Calculation: Volume of Caffeine , V1 (ml)

1. 5 ppm concentration
M1V1 = M2V2
(1000)V1 = (100)(5)
V1 = 0.5ml

2. 15 ppm concentration
M1V1 = M2V2
(1000)V1 = (100)(15)
V1 = 1.5ml

3. 25 ppm concentration
M1V1 = M2V2
(1000)V1 = (100)(25)
V1 = 2.5ml

4. 50 ppm concentration
M1V1 = M2V2
(1000)V1 = (100)(50)
V1 = 5.0ml

5. 100 ppm concentration

M1V1 = M2V2
(1000)V1 = (100)(100)
V1 = 10ml
 Information from machine (printed)