Experiment 3 Determination of Caffein in Soft Drink

UNIVERSITI KUALA LUMPUR
MALAYSIAN INSTITUTE OF CHEMICAL BIOENGINEERING

TECHNOLOGY
LABORATORY REPORT SUBMISSION FORM
July Semester 2020
SECTION A – Filled up by Student

Subject & Code ANALYTICAL AND ORGANIC CHEMISTRY CLB 10803
DETERMINATION OF CAFFEIN IN
Title of Experiment SOFT DRINKS
Date of Experiment Section L01-P2

Marks Peer Evaluation Total
Group Members ID No. (90%) (100%)
(10%)
TM1) Leader: : NORYUSHAINA ADLINA 55217119075
BINTI YUSMAINI
TM2) HARITH SAIFULLAH BIN JOFFERRY 55217119091
TM3) MAISARAH BINTI OTHMAN 55217119116
TM4) NUR ANNISA FARAHIN BINTI 55216119077
MOHD FAUZI
TM5) SHAIDATUL NAJWA BINTI MOHAMAD 55217119083
TM6) MUHD SYAHMI BIN YUSOP 55213117108
Name of Lecturer Ms Norhayati Binti Mohd Idrus
SECTION B – Filled up by Lecturer

NOT VERY VERY
CRITERIA Included POOR POOR GOOD GOOD EXCELLENT
1.0 JOTTER NOTE (5%) – Prepare a summary on the methodology 0 1 2 3 4 5

of the experiment (flowchart, diagram etc)
2.0 SUMMARY / ABSTRACT (10%) - Briefly review on the
experiment as a whole including the results and conclusion. 0 2 4 6 8 10
3.0 INTRODUCTION (10%) – State the background or theory of the

experiment. 0 2 4 6 8 10
4.0 MATERIALS & PROCEDURE (5%) – Include the list of chemicals

and equipment used and the experimental procedure. 0 1 2 3 4 5
5.0 RESULTS & DISCUSSION (30%) – Results are presented a 0 6 12 18 24 30

deemed suitable with complete label & units. Discuss on the
findings and the relations to the theory and objectives of the
experiment.
6.0 CONCLUSION & RECOMMENDATIONS (15%) – The conclusion 3 6 9

should begin with a restatement of the results. The results should
be compared with literature values/theory whenever possible. 0 12 15
Any possible sources of error should be identified. Suggest your
recommendation (s) for the improvement of the experiment.
7.0 TUTORIAL (10%) 0 2 4 6 8 10

8.0 REFERENCES (5%) 0 1 2 3 4 5
9.0 PEER EVALUATION (10%) - Team work, submit the peer
evaluation form along with respective lab report. 0 2 4 6 8 10
TOTAL PERCENTAGE (100%)
Acknowledgement of Received
Received Date : __________________________
-------------------- --------------------------------------------------------
-----------
-----------------------------------------------
Student’s Copy
Lecturer Chop / Sign
Submitted by : ________________________________
Experiment No. : _______________________________
Subject : Physical Chemistry CLB 10703
1.0 SUMMARY
High performance liquid chromatography (HPLC) technique is used to separate a mixture of

compounds in analytical chemistry with the purpose of identifying and quantifying the
individual components of the mixture. HPLC is also considered an instrumentation technique
of analytical chemistry. Benzoic acid and caffeine standard were prepared with different
concentration. Then, both sample and standard were analyzed by using HPLC. The mixture
of compound in the soft drink sample that was separated were caffeine and benzoic acid.
In this experiment, the presence of caffeine and benzoic acid in soft drink sample was
acknowledged and the amount of caffeine in soft drink sample was determined. Compound
separately retained in the stationary phase each gradually reacted to produce a set of peaks
along the timeline. Each component of the mixture reaches the detector at a different time and
produce a signal at a specified time called the retention time. The area under a peak is related
to the amount of the component present the mixture. In this experiment, serial dilution also
will be prepared to be as standard caffeine and to determine if caffeine was present in the
soda sample by use retention time.
Other than that, by using the concentration to peak area relationship, the concentration of
caffeine in the soda sample can be determined. The peak of caffeine appeared after second
and by measuring caffeine peaks of the standards, the amount of caffeine in a sample can be
determined. The area under the caffeine graph increase when the concentration was
increased. This proved that the mixture contained a higher quantity of caffeine.
2.0 OBJECTIVES
1. To Identify the present of Benzoic acid/ Caffeine in soft drink sample

2. To determine amount of caffeine in soft drink sample.
3.0 INTRODUCTION
The analytical technique used for separating mixtures into their components due to their
molecular composition and structure is called chromatography. In recent years, the
technology used in chromatography analysis has greatly improved. The improvement in
this area have led to the common use High Performance Liquid Chromatography that
allows for the very efficient separation of small amounts of the components of a mixture.
It is essentially the same as basic liquid chromatography but HPLC allows for far better
separation of the components of a mixture. It contained tiny particle of only 5 μ m diameter
in the column of the HPLC ensuring a very large surface area to which molecules may
absorb. These particles comprise the stationary phase of the chromatographic system. The
mobile phase solvent must be forced through the column under very high pressure because
these tiny particles are so tightly packed. The HPLC are connected to a detector and a
computer to give signal when eluents are coming off the column and fractions should be
collected.
Below is an image of the pathway used in HPLC. The mobile phase we used contained
Methanol. The high pressure provided by the pump solvent manager as the sample is
injected. It then passes with the mobile phase down through the HPLC column where the
interaction between both phases separates the solution. The component then passes out
from the column and are detected with the results being passed through to a computer data
station with the results being shown as peaks on a chromatogram.
There are some factors that are being considered when using HPLC and other forms of
chromatography include the retention time, resolution between peaks and column
efficiency. Retention time or Rt can be defined as the time taken for the components to
flow through the column. Long retention times may lead to higher costs and wasted time
while analysing solutions. Resolution can be explained as how well two elution peaks can
be differentiated on a chromatogram. It can be calculated by using the difference in
retention times between two peaks, divided by the combined widths of the elution peaks.
By altering the mobile phase, the flow rate, and the wavelength these factors can be
optimised to give the best separation. Mobile phase composition alters results due to
changes in its polarity. For normal phase chromatography the stationary phase generally
used is polar for example Silica.
4.0 MATERIALS AND PROCEDURE
Materials:
Isocratic HPLC system with UV detector, C18 column, Vacuum, Funnel, 0.45 μm filter
paper, 0.45 μm filter syringe,100 μL syringe,60 mL syringe,Volumetric flask
Chemicals:
Caffeine 1000 ppm standard (stock solution), Methanol (HPLC grade), Double distilled
water (filtered with 0.45 μm filter paper), Soft drink sample
Methods:
1. Preparation of caffeine standards
 Standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm
were prepared by diluting portions of the 1000 ppm solution with distilled water.
2. Preparation of soda samples

 A soft drink sample was obtained.
 The sample was degassed by placing it in a vacuum flask and connecting the flask
to a vacuum pump or water aspirator. It was left under vacuum until no more
bubbles appear in the soda sample. (If no vacuum is available, allow the soda to
stand open overnight)
 The degassed soda was filtered through #42 filter paper.
3. After preparing the serial dilution and sample, the instructor briefed on standard
operating procedure of HPLC.
5.0 RESULS AND DISCUSSION
Concentration of Retention Time Area

Type of Sample
Sample (ppm) (min) (µV.s)
20 1.894 454723.31
40 1.872 869653.17
Standard
60 1.881 1378881.14
Caffeine
80 1.872 1788539.99
100 1.874 2234898.49
Coke 45 1.856 1176173.38
Pepsi 70 1.871 1589421.26
Graph of Concentration of Sample (ppm)

against Area (µV.s)
2500000.00
2000000.00
Area (µV.s)
1500000.00
1000000.00
500000.00
0.00
0 20 40 60 80 100 120
Concentration of Sample (ppm)
Based on the objectives of the experiment, this experiment is conducted to identify the
present of Benzoic acid or caffeine in soft drink sample. Not just that, this experiment also
conducted to determine the amount of caffeine in soft drink sample. Samples that have
been using in this experiment are standard caffeine with different concentrations, coke,
and Pepsi. To identify caffeine in stock solutions, High-Performance Liquid
Chromatography or known as HPLC was used. HPLC relies on pumps to pass a
pressurized liquid solvent containing the sample mixture through a column filled with a
solid adsorbent material. Each component in the sample interacts slightly differently with
the adsorbent material, causing different flow rates for the different components and
leading to the separation of the components as they flow out of the column.
After all stock solutions have been scanned with HPLC, we have gained the peak area and
the concentration of the stock solutions of every concentration which are 20ppm, 40ppm,
60ppm, 80ppm, and 100ppm. The retention time that were used is the highest peak of the
graph while for the rest, the nearest retention time was used. In this experiment, the
retention time that were used is around 1.8 minutes. Based on the results, a standard
calibration curve can be conducted with a graph of concentration against the peak area.
standard calibration curve

(concentration vs peak area)
2500000
concentration (ppm)
2000000
1500000
1000000
500000
0
0 20 40 60 80 100 120
peak area (µV. s)
Figure 1 : Graph of concentration against peak area

From the standard calibration curve that has been constructed as above, concentration of
caffeine in coke and soda can be determined. For coke, with same retention time and after
calculating the real peak area with dilution factor, the concentration of caffeine determined
is 45ppm while for Pepsi, the caffeine determined is 70ppm.
Comparing coke to Pepsi, Pepsi has more amount or concentration of caffeine in it.
Caffeine is a central nervous system (CNS) stimulant of the methylxanthine class. Caffeine
is one of the world's most widely consumed psychoactive drug. Different to many other
psychoactive substances, it is legal and unregulated in nearly all parts of the world. There
are several known mechanisms of action to explain the effects of caffeine to our body and
health. The most important is that the caffeine reversibly blocks the action
of adenosine on its receptors and consequently prevents the onset of sleepiness induced by
adenosine. Caffeine also stimulates certain portions of the autonomic nervous system.
For conclusion, we can conclude that there are presence of caffeine or benzoic acid in soft
drinks as in in this experiment, coke and Pepsi has been used. It is just how much different
the amount of caffeine in the soft drinks. Nevertheless, we also can conclude that High-
Performance Liquid Chromatography can be used to determine the amount of caffeine in
the soft drinks as HPLC has a function of separating components from a mixture.
HPLC has been used widely in industries nowadays. HPLC is used in clinical diagnosis and
health industry, in scientific research, in pharmaceutical industry, and many more. The
effectiveness and uses of HPLC applications in recent days are further enhanced due to
coupling with detectors such as Mass Spectrometer, Nuclear Magnetic Resonance
spectrometer, and many more.
6.0 CONCLUSION AND RECOMMENDATION
The objectives of this experiment are to identify the present Caffein in soft drink sample
and to determine the amount of Caffein in that sample by using High Performance Liquid
Chromatography. Based on the calibration curve plotted, the concentration of Caffein in
Pepsi is 70 ppm while Coke is 45 ppm. This shows that Pepsi contains more Caffein than
Coke. During this experiment, a few precautions need to care about. For example, we must
always wear safety goggles and gloves as caffein may cause irritation to some people.
Next, all waste must be poured into the waste container following their own category. To
improve the accuracy of the results, we can repeat the experiment five more times and get
the average value.
7.0 TUTORIALS
Post – Lab Questions:
1. Why does the syringe have to be carefully rinsed before each use?
In general, assume that all syringes are dirty. Each syringe should be rinse first with
the sample you plan to inject two or three times to make sure there is no residue left
from the last sample injected.
2. Retention of caffeine in standards. How could you identify a peak in the soda
was caffeine and not another substance by using retention time?
The pump keeps a precise flow rate so that the positions of the peaks in time can be
used to identify the species in a sample. This is done by comparing the
chromatographs of prepared standards of the species to be determined. The common
peak is an indication of the standard. So, from the graph the highest peak is identified.
8.0 REFERENCES
1. HPLC Determination of Caffeine, Lab report [online] Available from

https://www.studocu.com/en-ie/document/dublin-city-university/analysis-of-
environmental-samples/practical/hplc-determination-of-caffeine-lab-
report/1925536/view [Accessed on: 1 November 2020]
2. HPLC [online] Available from

https://www.academia.edu/11572035/LABORATORY_REPORT_BASIC_INSTRU
MENTAL_ANALYSIS_CHM260_TITLE_HIGH_PERFOMANCE_LIQUID_CHR
OMATOGRAPHY_HIGH_PRESSURE_LIQUID_CHROMATOGRAPHY_HPLC_
EXPERIMENT_6_GROUP_AS120_6A2_AS114_4A1_Objectives_1_Methods_3_R
esults_Data_Calculations_6 [Accessed on: 2 November 2020]
3. High-Performance Liquid Chromatography, 2020 [online] Available from

https://en.wikipedia.org/wiki/High-performance_liquid_chromatography [Accessed on
2nd November 2020]
4. HPLC Applications | In Diagnosis, Research & Industry, 2012 [online] Available from
https://www.studyread.com/application-hplc-pharmaceutical-industry/ [Accessed on
2nd November 2020]
5. Caffeine, 2020 [online] Available from https://en.wikipedia.org/wiki/Caffeine
[Accessed on 2nd November 2020]
9.0 APPENDIX
Figure 1: 20 ppm
Figure
1(a): 20
ppm
Figure
2: 40
ppm
Figure 2(a):40 ppm
Figure 3: 60 ppm
Figure 3(a): 60 ppm
Figure 4: 80 ppm
Figure 4(a): 80 ppm
Figure 5: 100 ppm
Figure 5(a): 100 ppm
Figure 6: Coke Sample
Figure 6(a): Coke Sample
Figure 7: Pepsi Sample
Figure 7(a): Pepsi Sample
10.0 JOTTER NOTES
EXPERIMENT 3: DETERMINATION OF CAFFEINE IN SOFT DRINK
NAME: SHAIDATUL NAJWA BT MOHAMAD

ID NUMBER: 55217119083
OBJECTIVES:
• To Identify the present of Benzoic acid/ Caffeine in soft drink sample
• To determine amount of caffeine in soft drink sample.
MATERIALS:
• Isocratic HPLC system with UV detector
• C18 column, Vacuum
• Funnel
• 0.45 μm filter paper
• 0.45 μm filter syringe
• 100 μL syringe
• 60 mL syringe
• Volumetric flask
CHEMICALS:
• Caffeine 1000 ppm standard (stock solution)
• Methanol (HPLC grade)
• Double distilled water (filtered with 0.45 μm filter paper)
• Soft drink sample
METHODS:
1) Preparation of caffeine standards
-Prepare standard caffeine samples of 20 ppm, 40 ppm,
60 ppm, 80 ppm and 100 ppm by diluting portions of
the 1000 ppm solution with distilled water.
2) Preparation of soda samples

-Obtain a soft drink sample.
-Degas the sample by placing it in a vacuum flask and
connecting the flask to a vacuum pump or water aspirator.
Leave it under vacuum until no more bubbles appear in the soda
sample.
-Filter the degassed soda through 42 filter paper.
3) After preparing the serial dilution and sample, your

instructor will brief on standard operating procedure of
HPLC.
DATA AND RESULTS:
Concentration
Type of Rentation Area
of Sample
Sample Time (min) (µV. s)
(ppm)
20 1.894 454723.31
40 1.872 869653.17
Standard
60 1.881 1378881.14
Caffeine
80 1.872 1788539.99
100 1.874 2234898.49
Coke 45 1.856 1176173.38
Pepsi 70 1.871 1589421.26
PRE-LABORATORY QUESTION
1. Briefly explain how HPLC is used as a separation technique.
-HPLC used as an instrumental technique of analytical chemistry which mainly used
to identify each component and to quantify each component.
2. What is the purpose of the mobile phase and the stationary phase?
-Mobile phase carries the components of the mixture through the medium being used
and the stationary phase acts as constraint on many of the components in a mixture
that slowing them down to move slower than mobile phase.
3. What is the purpose of the caffeine standard?

-Caffeine standard used to determine the level of caffeine in beverages.
EXPERIMENT 3
DETERMINATION OF CAFFEINE IN SOFT DRINK
NAME: NORYUSHAINA ADLINA BINTI YUSMAINI
ID NUMBER: 55217119075
OBJECTIVES
• To identify the present of Benzoic acid/caffeine in soft drink sample.

MATERIALS
Chemicals:
• Caffeine 1000 ppm standard (stock solution).

• Methanol (HPLC grade).
• Double distilled water (filtered with 0.45 μm filter paper).
• Soft drink sample.
Apparatus:
• Isocratic HPLC system with UV detector.

• C18 column.
• Vacuum.
• Funnel.
• 0.45 μm filter paper.
• 0.45 μm filter syringe.
• 100 μL syringe.
• 60 mL syringe.
• Volumetric flask.
METHODS
After preparing the

serial dilution and
sample, your
Preparation of instructor will brief
on standard
soda samples operating procedure
Preparation of of HPLC.
caffeine i. Obtain a soft drink
standards sample.
ii.Degas the sample by

i. Prepare standard placing it in a
caffeine samples of vacuum flask and
20 ppm, 40 ppm, 60 connecting the flask
ppm, 80 ppm and to a vacuum pump or
100 ppm by diluting water aspirator.
portions of the 1000 Leave it under
ppm solution with vacuum until no
distilled water. more bubbles appear
in the soda sample.
iii.Filter the degassed

soda through 42
filter paper.
DATA AND RESULTS
Concentration of Rentation Time Area

Type of Sample
20 1.894 454723.31
40 1.872 869653.17
Standard
60 1.881 1378881.14
Caffeine
80 1.872 1788539.99
100 1.874 2234898.49
Coke 45 1.856 1176173.38
Pepsi 70 1.871 1589421.26
Table 1
PRE-LABORATORY QUESTIONS
- HPLC used as an instrumental technique of analytical chemistry which mainly

used to identify each component and to quantify each component.
5. What is the purpose of the mobile phase and the stationary phase?
- Mobile phase carries the components of the mixture through the medium being
used and the stationary phase acts as constraint on many of the components in a
mixture that slowing them down to move slower than mobile phase.
6. What is the purpose of the caffeine standard?
- Caffeine standard used to determine the level of caffeine in beverages.

NUR ANNISA FARAHIN BINTI MOHD FAUZI 55216119077
OBJECTIVES
• To Identify the present of Benzoic acid/ Caffeine in soft drink sample

MATERIALS AND APPARATUS
MATERIALS
• Caffeine 1000 ppm standard (stock solution)

• Methanol (HPLC grade)
• Double distilled water (filtered with 0.45 μm filter paper)
• Soft drink sample
APPARATUS
• Isocratic HPLC system with UV detector

• C18 column, Vacuum
• Funnel
• 0.45 μm filter paper
• 0.45 μm filter syringe
• 100 μL syringe
• 60 mL syringe
• Volumetric flask
PROCEDURES
PREPARATION OF CAFFEINE STANDARDS

- prepre standard caffeine samples of 20ppm, 40ppm, 60ppm, 80ppm,
and 100ppm by diluting portions of the 1000ppm solution with
distilled water
PREPARATION OF SODA SAMPLES

- Obtain a soft drink sample
- degas the sample by placing it in a vacuum flask and connecting the
flask to a vacuum pump or water aspirator. leave it under vacuum until
no more bubbles appear in the soda sample. (if no vacuum is
available, allow the soda to stand open overnight)
- filter the degassed soda through #42 filter paper
After preparing the serial dilution and sample,

your instructor will brief on standard operating
procedure of HPLC
DATA AND RESULTS
CONCENTRATION OF SAMPLE RETENTION TIME AREA

TYPE OF SAMPLE
(ppm) (min) (µV. s)
20
40
STANDARD
60
CAFFEINE
80
100
COKE
PEPSI
PRE-LABORATORY QUESTIONS
It uses a mobile phase (mobile solvent) to carry a sample through a tube containing a stationary phase
which is a liquid coated on solid particles such as silica. The molecules in the sample interact differently
with the stationary phase, the more they interact with it the more they are slowed down. This tends to
separate them out with those interacting least emerging first from the HPLC tube. The mobile phase
speed through the tube is kept uniform by a pump that can sustain a constant (non varying) pressure
level
2. What are the purposes of the mobile phase and the stationary phase?
The mobile phase refers to the liquid or gas, which flows through a chromatography system, moving
the materials to be separated at different rates over the stationary phase while stationary phase refers
to the solid or liquid phase of a chromatography system on which the materials are to be separated or
selectively adsorbed
3. What is the purpose of the caffeine standards?
The purpose of the caffeine standards is to determine if caffeine is present in the beverage samples
JOTTER NOTES
MAISARAH BINTI OTHMAN (55217119116)
OBJECTIVES

MATERIALS AND APPARATUS
Materials:
Isocratic HPLC system with UV detector, C18 column, Vacuum, Funnel, 0.45 μm filter paper
0.45 μm filter syringe,100 μL syringe,60 mL syringe,Volumetric flask
Chemicals:
Caffeine 1000 ppm standard (stock solution), Methanol (HPLC grade), Double distilled water
(filtered with 0.45 μm filter paper), Soft drink sample
PROCEDURE
Preparation of caffeine standards
Prepare standard caffeine samples of

20 ppm, 40 ppm, 60 ppm, 80 ppm and
100 ppm by diluting portions of the
1000 ppm solution with distilled water.
Preparation of soda samples
Obtain a soft drink sample.
Degas the sample by placing it in a vacuum flask

and connecting the flask to a vacuum pump or
water aspirator. Leave it under vacuum until no
more bubbles appear in the soda sample. (If no
vacuum is available, allow the soda to stand open
overnight)
Filter the degassed soda through #42 filter

paper.
After preparing the serial dilution and sample, your instructor will
brief on standard operating procedure of HPLC.
DATA AND RESULTS
Concentration of Rentation Time

Type of Sample Area (µV.s)
Sample (ppm) (min)
20 1.894 454723.31
40 1.872 869653.17
Standard
60 1.881 1378881.14
Caffeine
80 1.872 1788539.99
100 1.874 2234898.49
Coke 45 1.856 1176173.38
Pepsi 70 1.871 1589421.26
PRE – LAB QUESTIONS

High-performance liquid chromatography is a 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
The stationary phase remains fixed in place while the mobile phase carries the components
of the mixture through the medium being used. The stationary phase acts as a constraint on
many of the components in a mixture, slowing them down to move slower than the mobile
phase.

Caffeine standard used to determine the level of caffeine in beverages.
Jotter Note
Harith Saifullah Bin Jofferry (55217119091)
Experiment 3: Determination of Caffeine in Soft Drink
Objectives
Materials and Apparatus
Materials:
Chemicals:
(filtered with 0.45 μm filter paper), soft drink sample
Procedure
Prepare standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100
ppm by diluting portions of the 1000 ppm solution
Obtain a soft drink sample.
Degas the sample by placing it in a vacuum flask and connecting the flask to a
vacuum pump or water aspirator. Leave it under vacuum until no more bubbles
appear in the soda sample. (If no vacuum is available, allow the soda to stand
open overnight)
Filter the degassed soda through #42 filter paper.
After preparing the serial dilution and sample, your instructor will brief on
standard operating procedure of HPLC.
Data and Results
Concentration of Rentation Time

Type of Sample Area (µV.s)
Sample (ppm) (min)
20 1.894 454723.31
40 1.872 869653.17
Standard
60 1.881 1378881.14
Caffeine
80 1.872 1788539.99
100 1.874 2234898.49
Coke 45 1.856 1176173.38
Pepsi 70 1.871 1589421.26
Pre-Lab Questions

A pump is used to pass a pressurized liquid solvent containing the sample mixture
across a column filled with absorbent material.
Mobile phase: To move the sample via the column and divide the components of the
sample by slowing them down.
Stationary phase: Constraint on many of the components in a mixture, slowing them
down to move slower than the mobile phase
To calibrate HPLC
JOTTER NOTES
NAME: MUHD SYAHMI BIN YUSOP
ID MATRIC: 55213117108
OBJECTIVES:
2. To determine amount of caffeine in soft drink sample
MATERIAL AND APPARATUS:
Materials:
Chemicals:
(filtered with 0.45 μm filter paper), Soft drink sample
PROCEDURE:
1.Preparation of caffeine standards
Standard caffeine samples of 20 ppm, 40 ppm, 60

ppm, 80 ppm and 100 ppm was prepared through
diluting portions of the 1000 ppm solution with
distilled water
2. Preparation of soda samples
The soft drink sample was obtained.

The sample was degassed by placing it in
a vacuum flask and connecting the flask
to a vacuum pump or water aspirator.
Then, it was leave under vacuum until no
more bubbles appear in the soda sample.
Take note that If no vacuum is available,
allow the soda to stand open overnight
The degassed soda was

filtered through #42 filter
paper.
After preparing the serial dilution and sample, your

instructor will brief on standard operating procedure of
HPLC
DATA AND RESULTS.
Concentration of Rentation Time Area

Type of Sample
20 1.894 454723.31
40 1.872 869653.17
Standard
60 1.881 1378881.14
Caffeine
80 1.872 1788539.99
100 1.874 2234898.49
Coke 45 1.856 1176173.38
Pepsi 70 1.871 1589421.26
PRE-LAB QUESTIONS.

High-performance liquid chromatography or high-pressure liquid chromatography
(HPLC) is a chromatographic method that is used to separate a mixture of
compounds in analytical chemistry and biochemistry so as to identify, quantify or
purify the individual components of the mixture. HPLC relies on pumps to pass a
pressurized liquid and a sample mixture through a column filled with adsorbent,
leading to the separation of the sample components.
In very small amounts, the sample mixture to be separated and tested is sent into a
stream of mobile phase percolating via a column. There are different types of
columns available with sorbents of varying particle sizes and surfaces. The
mixture moves through the column at varying velocities and interacts with the
sorbent, also known as the stationary phase. The composition of the mobile phase
is chosen based on the intensity of interactions between several sample
components and the stationary phase. The composition of the mobile phase is
either maintained as a constant or as varied during the chromatographic analysis.
Caffeine standard used to determine the level of caffeine in beverages.

11.0 PEER EVALUATION

MALAYSIAN INSTITUTE OF CHEMICAL
BIOENGINEERING TECHNOLOGY
PEER EVALUATION FORM

Analytical & Organic Chemistry (CLB
10803) July Semester 2020
STUDENT NAME : NORYUSHAINA ADLINA BINTI YUSMAINI
STUDENT ID : 55217119075
TITLE OF EXPERIMENT: DETERMINATION OF CAFFEINE IN SOFT DRINKS

LECTURER : MISS NORHAYATI BINTI MOHD IDRUS
1. Rate your team members on the relative contribution that were made in preparing and submitting your lab report. Please refer to
the rubric provided to assess your peers
2. In rating your peers, use to five point scale.
3. Every single group member is to fill in this form and be honest, do not favour anyone. Form is to be submitting along with
the respective submission
5- Excellent 4- Good 3- Average 2- Below Average 1-Poor
Names: HARITH Names: MAISARAH Names: NUR ANNISA Names: SHAIDATUL Names: MUHD
SAIFULLAH BIN BINTI OTHMAN FARAHIN BINTI NAJWA BINTI SYAHMI BIN YUSOP
JOFFERRY MOHD FAUZI MOHAMAD
ID: 55217119091 ID: 55217119116 ID: 55216119077 ID: 55217119083 ID: 55213117108
(1) Participated in group
5 5 5 5 5
discussions
(2) Contribution of useful ideas 5 5 5 5 5
(3) Focus on the task 5 5 5 5 5
(4) Quality of Work 5 5 5 5 5
(5) Working with others 5 5 5 5 5
TOTAL MARK (25%) 25 25 25 25 25
TOTAL MARK (10%)

STUDENT NAME : SHAIDATUL NAJWA BT MOHAMAD
STUDENT ID : 55217119083
TITLE OF EXPERIMENT: DETERMINATION OF CAFFEINE IN SOFT DRINK

LECTURER : MS NORHAYATI MOHD IDRUS
Names: HARITH Names: Names: Names: MAISARAH Names: NUR

SAIFULLAH BIN MUHAMMAD NORYUSHAINA BT OTHMAN ANNISA FARAHIN BT
JOFFERRY SYAHMI BIN YUSOP ADLINA BT MOHD FAUZI
YUSMAINI
ID: 55217119091 ID: 55213117108 ID: 55217119075 ID: 55217119116 ID: 55216119077
(1) Participated in group discussions 5 5 5 5 5
TOTAL MARK (25%) 25 25 25 25 25
TOTAL MARK (10%) 10 10 10 10 10

STUDENT NAME : MAISARAH BINTI OTHMAN
STUDENT ID : 55217119116

Names: HARITH Names: Names: Names: SHAIDATUL Names: NUR

SAIFULLAH BIN MUHAMMAD NORYUSHAINA NAJWA BT ANNISA FARAHIN BT
JOFFERRY SYAHMI BIN YUSOP ADLINA BT MOHAMAD MOHD FAUZI
YUSMAINI
ID: 55217119091 ID: 55213117108 ID: 55217119075 ID: 55217119083 ID: 55216119077
TOTAL MARK (25%) 25 25 25 25 25
TOTAL MARK (10%) 10 10 10 10 10

STUDENT NAME : NUR ANNISA FARAHIN BINTI MOHD FAUZI
STUDENT ID : 55216119077

Names: HARITH Names: Names: Names: SHAIDATUL Names: MAISARAH

SAIFULLAH BIN MUHAMMAD NORYUSHAINA NAJWA BT BINTI OTHMAN
JOFFERRY SYAHMI BIN YUSOP ADLINA BT MOHAMAD
YUSMAINI
ID: 55217119091 ID: 55213117108 ID: 55217119075 ID: 55217119083 ID: 55217119116
TOTAL MARK (25%) 25 25 25 25 25
TOTAL MARK (10%) 10 10 10 10 10

STUDENT NAME : HARITH SAIFULLAH BIN JOFFERRY
STUDENT ID : 55217119091

Names: NUR Names: Names: Names: SHAIDATUL Names: MAISARAH

ANNISA FARAHIN MUHAMMAD NORYUSHAINA NAJWA BT BINTI OTHMAN
BINTI MOHD FAUZI SYAHMI BIN YUSOP ADLINA BT MOHAMAD
YUSMAINI
ID: 55216119077 ID: 55213117108 ID: 55217119075 ID: 55217119083 ID: 55217119116
TOTAL MARK (25%) 25 25 25 25 25
TOTAL MARK (10%) 10 10 10 10 10

STUDENT NAME : MUHAMMAD SYAHMI BIN YUSOP
STUDENT ID : 55213117108
TITLE OF EXPERIMENT: DETERMINATION OF CAFFEINE IN SOFT DRINK

LECTURER : MISS NORHAYATI BINTI MOHD IDRUS
Names: HARITH Names: MAISARAH Names: NUR ANNISA Names: SHAIDATUL Names:
SAIFULLAH BIN BINTI OTHMAN FARAHIN BINTI NAJWA BINTI NORYUSHAINA
JOFFERRY MOHD FAUZI MOHAMAD ADLINA BT
YUSMAINI
ID: 55217119091 ID: 55217119116 ID: 55216119077 ID: 55217119083 ID: 55217119075
(1) Participated in group
5 5 5 5 5
discussions
TOTAL MARK (25%) 25 25 25 25 25
TOTAL MARK (10%) 10 10 10 10 10

Experiment 3 Determination of Caffein in Soft Drink

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Experiment 3 Determination of Caffein in Soft Drink

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UNIVERSITI KUALA LUMPUR

MALAYSIAN INSTITUTE OF CHEMICAL BIOENGINEERING

SECTION A – Filled up by Student

Date of Experiment Section L01-P2

SECTION B – Filled up by Lecturer

1.0 JOTTER NOTE (5%) – Prepare a summary on the methodology 0 1 2 3 4 5

3.0 INTRODUCTION (10%) – State the background or theory of the

4.0 MATERIALS & PROCEDURE (5%) – Include the list of chemicals

5.0 RESULTS & DISCUSSION (30%) – Results are presented a 0 6 12 18 24 30

6.0 CONCLUSION & RECOMMENDATIONS (15%) – The conclusion 3 6 9

7.0 TUTORIAL (10%) 0 2 4 6 8 10

TOTAL PERCENTAGE (100%)

Received Date : __________________________

High performance liquid chromatography (HPLC) technique is used to separate a mixture of

1. To Identify the present of Benzoic acid/ Caffeine in soft drink sample

4.0 MATERIALS AND PROCEDURE

1. Preparation of caffeine standards

2. Preparation of soda samples

5.0 RESULS AND DISCUSSION

Concentration of Retention Time Area

Graph of Concentration of Sample (ppm)

Concentration of Sample (ppm)

standard calibration curve

peak area (µV. s)

Figure 1 : Graph of concentration against peak area

6.0 CONCLUSION AND RECOMMENDATION

Post – Lab Questions:

1. HPLC Determination of Caffeine, Lab report [online] Available from

2. HPLC [online] Available from

3. High-Performance Liquid Chromatography, 2020 [online] Available from

EXPERIMENT 3: DETERMINATION OF CAFFEINE IN SOFT DRINK

NAME: SHAIDATUL NAJWA BT MOHAMAD

2) Preparation of soda samples

3) After preparing the serial dilution and sample, your

DATA AND RESULTS:

3. What is the purpose of the caffeine standard?

DETERMINATION OF CAFFEINE IN SOFT DRINK

NAME: NORYUSHAINA ADLINA BINTI YUSMAINI

• To identify the present of Benzoic acid/caffeine in soft drink sample.

• Caffeine 1000 ppm standard (stock solution).

• Isocratic HPLC system with UV detector.

After preparing the

ii.Degas the sample by

iii.Filter the degassed

Concentration of Rentation Time Area

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

- HPLC used as an instrumental technique of analytical chemistry which mainly

6. What is the purpose of the caffeine standard?

- Caffeine standard used to determine the level of caffeine in beverages.

EXPERIMENT 3: DETERMINATION OF CAFFEINE IN SOFT DRINK

• To Identify the present of Benzoic acid/ Caffeine in soft drink sample

MATERIALS AND APPARATUS

• Caffeine 1000 ppm standard (stock solution)

• Isocratic HPLC system with UV detector

PREPARATION OF CAFFEINE STANDARDS

PREPARATION OF SODA SAMPLES

After preparing the serial dilution and sample,