EXPERIMENT 3

DETERMINATION OF BENZOIC ACID / CAFFEINE IN SOFT DRINK

1.0 objectives
1) To identify the present of Benzoic acid/ Caffeine in soft drink sample
2) To determine amount of caffeine in soft drink sample.
2.0 Summary
In this experiment, two sample was prepared with standard caffeine and Benzoic acid
samples of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm by diluting with distilled water. In this
experiment, presence of caffeine and benzoic acid in soft drink sample is identified and the
amount of caffeine in soft drink sample was determined. High-performance liquid
chromatography (HPLC) referred to as high-pressure liquid chromatography, is a technique
in analytic chemistry used to separate the components in a mixture, to identify each component,
and to quantify each component. Each component of the mixture reaches the detector at the
different time and produces a signal at the characteristic time called the retention time. The area
under a peak is related to the amount of the component present the mixture. Ideally the
calibration curve of benzoic acid generated by this assay will display in good linearity. The area
of this peak (in relation to the area of other peaks) is proportional to the concentration of that
particular species in the sample. . 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. From the result obtained, the caffeine
retention time was 1.6 minutes. From the result the highest peak area for this experiment is at
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100ppm with 158416.83 v.s it shows that the higher the concentration, the higher the peak area
whereas a higher concentration gives a greater effect towards the area. The graph 1, shows not in
a straight line and the R2 value is 0.9896 while for caffeine retention time is 1.

3.0 Introduction
High-performance liquid chromatography (HPLC) referred to as high-pressure liquid
chromatography, is a technique in analytic chemistry used to separate the components in a
mixture, to identify each component, and to quantify each component. It 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 the column.
Instead of a solvent being allowed to drip through a column under gravity, it is forced
through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows
you to use a very much smaller particle size for the column packing material which gives a much
greater surface area for interactions between the stationary phase and the molecules flowing past
it. This allows a much better separation of the components of the mixture.

The time taken for a particular compound to travel through the column to the detector is
known as its retention time. This time is measured from the time at which the sample is injected
to the point at which the display shows a maximum peak height for that compound. When a
substance has passed through the column the detector will detect using ultra-violet absorption.
Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV
light shining through the stream of liquid coming out of the column, and a UV detector on the
opposite side of the stream, you can get a direct reading of how much of the light is absorbed.
The amount of light absorbed will depend on the amount of a particular compound that is passing
through the beam at the time. The output will be recorded as a series of peaks, each one
representing a compound in the mixture passing through the detector and absorbing UV light.
Benzoic acid is most commonly used as a food preservative, protecting against fungi,
bacteria, and yeast in acidic environments while caffeine frequently ingested pharmacologically
active substances present in a variety of foods, beverages, and medications The least complex
aromatic carboxylic acid, benzoic acid (C6H5 -COOH) is very soluble in most organic solvents.
Once dissolved, benzoic acid and its salts have the capability to react with ascorbic acid to form
benzene. HPLC presents an effective, time conscious method by which concentration of
substances within a densely combined mixture can be determined. The established procedure for
simultaneous determination of benzoic acid and caffeine involves length and wavelength
scanning. Ideally the calibration curve of benzoic acid generated by this assay will display in
good linearity.
The area of this peak (in relation to the area of other peaks) is proportional to the concentration
of that particular species in the sample.

4.0 Methodology

1)

2)
5)

3)

4)

6)

5.0 Result and Discussion

5.1 Result:
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Table 1: Retention Time of Caffeine in Standard

Retention time (min)

Peak area ( v.s)

1.467

39513.43

40

1.393

70510.01

60

1.386

110529.08

80

1.371

131669.78

100

1.628

158416.83

Soda drink

1.599

1245470.16

Concentration (ppm)
20

Concentration vs. Peak Area

180000
y = 29897x + 12438
R = 0.9896

160000

Peak Area (v.s)

140000
120000
100000
80000
60000
40000
20000
0
20

40

60

80

100

Concentration (ppm)
Figure 1: Standard curve for peak area vs. concentration

5.2 Discussion:

The objective of this experiment was to identify the present of benzoic acid or caffeine in
soft drink sample and to determine the amount of caffeine present in soft drink sample. The
sample run in the High Performance Liquid Chromatography (HPLC) was the standard caffeine
samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm was prepared in 50mL volumetric
flask. Based from the result obtained, peak area for 20ppm was 39513.43 v.s with time 1.467
minutes, peak area for 40ppm was 70510.01 v.s with time 1.393 minutes, for 60ppm the peak
area was 110529.08 v.s with time 1.386 minutes, 80ppm was 131669.78 v.s with time 1.371
minutes minutes and 100ppm is 158416.83 v.s with time 1.628 minutes, while the caffeine
retention time was 1.6 minutes and for the soda drink retention times obtained was 1.599
minutes.The retention time used to determine caffeine are present in the Coca-cola sample and
the amount of the caffeine in the sample was determined. The caffeine peak of the standards was
measured in Table 1, and a standard curve was constructed in Figure 1. The peak was increased
from 20ppm to100ppm.
From the result the highest peak area for this experiment is at 100ppm with 158416.83
v.s. This shows that the result obtained obeys the theory, it states that the higher the
concentration, the higher the peak area whereas a higher concentration gives a greater effect
towards the area. The graph 1, shows not in a straight line and the R2 value is 0.9896 while for
caffeine retention time is 1. This shows the result obtained was inaccurate due to parallax error
or wrongly measured the titration solution and also might be some bubbles present in the syringe
injected into HPLC. Although the comparison of the calculated and literature values of the
analyte concentrations, the standard addition plots yielded R2 values was closed to 1, which
means that the method of standard addition was successful. The objectives in this experiment
was achieved as there was presence of caffeine in soft drink sample and the amount of caffeine

in soft drink sample can be determined. By using the equation y = 29897x-12438, the value
concentration of soft drink calculated was 41.24 ppm. This proved that the caffeine/benzoic acid
presence at concentration 41.24 ppm.

6.0 Conclusion and Recommendation

The objectives in this experiment was achieved as there was presence of caffeine in soft
drink sample and the amount of caffeine in soft drink sample can be determined. The sample run
in the High Performance Liquid Chromatography (HPLC), result shows the caffeine retention
time was 1.6 minutes and for the soda drink retention times obtained was 1.599 minutes. . This
shows that the result obtained obeys the theory, it states that the higher the concentration, the
higher the peak area whereas a higher concentration gives a greater effect towards the area and
the R2 value is 0.9896 while for caffeine retention time is 1. The concentration of calculated
value was 41.24 ppm. However, the percentage of error was small because R2 value was near to
1. Highly recommend to make sure that there is no bubbles inside the syringe to HPLC to get
more accurate result and make sure the measured solution was titrate properly with eye level for
the next experiment.

7.0 References
1. Skoog, D.; Holler, F.; Crouch, S. Principles of Instrumental Analysis 2007.
2. Backes, Gabriella. 2006.HPLCHigh Pressure Liquid Chromatography. Quantitative
Determination of Caffeine in Beverages. Accessed April 28, 2013.
http://spot.pcc.edu/~gbackes/ORGANIC/CH%20242/Labs.CH242.W05/HPLC.htm.
3) Leacock, R.E., Stankus, J. J., Davis,J.M.(2011).Journal of Chemical Education.
Simultaneous Determination of Caffeine and Vitamin B6 in Energy Drinks by HighPerformance Liquid Chromatography. Volume 88, pp.2
4) Union of European Soft Drink Association (UNESDA), (2010). Qualitative and
quantitative control of benzoic acid and caffeine in soft drinks. United States: UNESDA
Publications.

8.0 Lab question

8.1 Pre lab question:
1) Chromatography is essentially a physical method of separation in which the components
are separated between two phases one of which is stationary phase and mobile phase
through it in definite direction. So sample is injected into the injector port and will be
carried away by the mobile phase to the column. Once it reaches the column it is
separated based on different boiling point and interaction with column.

2) The stationary phase is attached to a support, a solid inert material. The sample gas,
liquid or solid which may or may not dissolved in solvents is carried across the stationary
phase by a mobile phase. The mobile phase in his separation was the solvent.

3) Caffeine standard usually prepared and ruined throughout the column. When the
unknown sample is inserted the presence of caffeine can be determined based on the
similar retention time with the caffeine standard.

8.2 Post lab Question:

1) High Performance Liquid Chromatography (HPLC) was sensitive device. Any small
contamination will give wrong result which will produced a small peak graph. That why
syringe need to be rinsed every time before it is used and also need to make sure there is
no bubbles in syringe to HPLC.

2) By plot the standard calibration curve of caffeine. A comparison of the caffeine peak area
in the soft drink sample compared to standard curve allows determination the amount of
caffeine. The retention time in standard curve of caffeine was compared with retention
time of soft drink.

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APPENDIX
I.

Sample of calculation:

A) 20 ppm
M1 = 1000 ppm
M2 = 20 ppm
V1=? Ml
V2 = 50 mL

M1V1= M2V2
(1000)(V1) = (20) (50)
V =1 mL

II.

Determining concentration of soft drink

Y = 29897x + 12438
1245470.16 12438 = 29897x
1233032.16 / 29897 = x
X = 41.24 ppm

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