Name: Wambugu Florence Waithira.

Reg.No: SCP212-0378/2016.

Course: BSc. Industrial Chemistry

Unit Code: SCH 2356

Unit Name: Separation Methods.

Report: Determination of the amount of Caffeine in Coca Cola.

Lecture: Dr. Anam Onditi

Date: 21st March 2019.

DETERMINATION OF THE AMOUNT OF CAFFEINE IN COCA COLA SODA USING

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY.

Objectives.

1. To determine the amount of caffeine in coca cola using high performance liquid

chromatography.

2. To find out whether the amount of caffeine in the coke soda is within the required

standards of Kenya Standards of Bureau.

Literature Review.

High performance liquid chromatography is a type of chromatography which is similar to

liquid chromatography. However, in high performance liquid chromatography, the stationary

phase column is more tightly packed than in other types of chromatography. In this lab, the

column is packed with Carbon-18 particles that are less than 10 micrometers in diameter. The

small diameter of the particles allows for unprecedented resolution and high efficiency. Since the

particles in the column are very small, it is necessary to pump the mobile phase through the

column at very high pressure. The pump keeps a precise flow rate so that the positions of the

peaks in time can be used to identify the species in the sample. This is done by comparing the

chromatograms of prepared standards of the particular species to be determined. The common

peak is an indication of the standard.

A sample is injected into the injector port where the mobile phase moves it through the

column. Each component being different in physical composition will move at a different rate

through the Carbon-18 column. Therefore, the components will be separated according to the

size and the shape of the molecules. The smallest and the least hindered molecules will be eluted
first since it is easy for them to pass through the finely packed column. As each set of molecules

elutes from the column, a detector (most often UV) recognizes it and records a peak. The area of

this peak is proportional to the concentration of that particular species in the sample. The identity

can also be found by comparing the sample peaks to standards. Identical peaks will have

identical retention times. It is important to note than an isochratic high performance liquid

chromatography using a reverse phase is used in this experiment. The reverse phase means that

the stationary phase is non-polar whereas the mobile phase is polar.

Materials and Reagents.

1. Anhydrous caffeine fir preparation of the standards.

2. Acetonitrile for the HPLC mobile phase

3. Deionized Water

4. High Performance Liquid Chromatography Equipment.

5. Coca-Cola

6. UV/Visible Detector

7. Volumetric flask

8. Pipettes

9. Sonicator

Procedure.

25mg of caffeine was weighed and was transferred to a 250ml volumetric flask.

Deionized water was added to get a 250ml bulk standard solution. The standard was further

diluted to obtain six working standard solutions of 10,20,40,60,80 and 100ppm were made.

The standards were then run in the HPLC-UV 278nm at 0.8ml/min flow rate for the mobile
phase. Afterwards, 1ml of coke soda was pipetted and put in a 5ml measuring cylinder. 4ml

of deionized water was then added and the solution was then degassed in a sonicator. An

aliquot of these diluted samples was injected into the HPLC to quantitate the caffeine

concentrations. The data was then recorded.

Calculations.

1. Plot a calibration curve of peak areas against the concentrations and determine the

amount of caffeine in the soda.

Calibration Curve for the Determination of Caffeine in Soda

8000000

7000000

6000000 f(x) = 62712.05 x + 1946.18

R² = 0.68

5000000
Peak Areas

4000000

3000000

2000000

1000000

0
0 20 40 60 80 100 120

Concentrations in ppm

Sample of the diluted soda had a peak area of 315382.

Thus, y = mx + c.

(315382 – 1946.2)/ 62712 = x

X = 4.998ppm

Actual Concentration = (x × dilution factor × volume)/weight

= (4.998 × 20 × 1ml)/1g

= 99.96ppm

For the raw undiluted soda, y = mx + c.

X = (6215453 – 1946.2)/62712

X = 99.08ppm

Actual Concentration = (99.08 × 1 × 1ml)/1g

= 99.08ppm

Discussion.

Caffeine is a substance that is found naturally in coffee beans and tea leaves that provides

stimulant properties but also has flavor properties as well. For this reason, caffeine is added

in soft drinks to complement the sweeteners used in the drinks, partly to counter the

sweetness thus, enhancing the flavor. Caffeine is known to be addictive and hence is

regulated in the drinks. Coffee for example is known to contain about 80ppm of the caffeine

while tea is known to contain about 50ppm. For the cola drinks, the allowed amount of

caffeine varies from 9.7ppm to about 15ppm as per the Australian and New Zealand

standards followed by Coca-Cola company. However, this was not found to be the case in the
 coke soda drink used for the experiment. It was found out to have about 99ppm of the

caffeine. This was a very huge deviation from what the company`s site had given and

therefore meant that the regulations in the country were not being strictly followed. This

could be attributed to the fact that the testing of caffeine levels in the products was not being

done on site. However, it could not be ruled out that the experiment had errors and hence the

large amount obtained. Nevertheless, despite the presence of errors, the deviation was very

large and hence it indicated that most Kenyans were exposed to high caffeine levels in the

soda and hence, were more prone to addiction of the soda.

Conclusion.

The amount of the caffeine in the coke soda was found out to be about 99ppm. This amount

was not within the allowed amounts of caffeine given by the Coca-Cola website that followed

the Australian and New Zealand food regulations. All objectives were met and hence, the

experiment was a success.

References.

1. https://www.coca-colacompany.com/stories/0000014b-079f-da6b-ad6f-

6fdf6be40000#ath

Retrieved on 21/03/2019 at 1200 hours.

2. https://scialert.net/fulltextmobile/?doi=jas.2006.831.834

Retrieved on 21/03/2019 at 1210 hours.

3. https://www.longdom.org/open-access/determination-of-caffeine-in-soft-and-energy-

drinks-available-in-market-byusing-uvvisible-spectrophotometer-2327-4972-

1000206.pdf
 Retrieved on 21/03/2019 at 1226hours.

4. http://cssf.usc.edu/History/2011/Projects/S0611.pdf

Retrieved on 21/03/2019 at 1239hours.