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.

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 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.

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4.0 Methodology

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 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

Concentration (ppm) Retention time (min) Peak area ( v.s)

20 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 vs. Peak Area

180000
y = 29897x + 12438
160000 R = 0.9896

140000
Peak Area (v.s)

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:

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 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

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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.

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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 High-

Performance 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.

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 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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