73 SERIES

SPECTROPHOTOMETER Application note: A09-010A

The quantitative determination of caffeine in beverages
and soft drinks using UV wavelength spectroscopy
Introduction
Caffeine is a naturally occurring alkaloid which Caffeine can be extracted from aqueous
is found in the leaves, seeds or fruits of over 63 solutions with chlorinated solvents such as
plants species worldwide. The most common dichlormethane and chloroform, a technique
sources of caffeine are coffee, cocoa beans, commonly employed commercially to de-
cola nuts and tea leaves and the worldwide caffeinate coffee and tea. After the caffeine is
consumption of products derived from these extracted it can be analysed directly by
natural materials means that caffeine is one of measuring the absorbance of the solvent
the most popular and commonly consumed solution at 260nm.
drugs in the world. Caffeine’s popularity stems
mainly from the fact that it is a Five samples were chosen to include the
pharmacologically active substance and a mild commonly consumed beverages and soft drinks
central nervous system stimulant. including instant coffee (Nescafe), brewed tea
(PG Pyramid Tea Bags), Coca Cola, Pepsi Cola
and Red Bull.

The analysis is performed on a Jenway 7305

spectrophotometer controlled using the free-of-
charge PC software, supplied with each model
in the 73 series. The software allows the user to
emulate all measurement tasks normally
performed on the instrument with the additional
benefit of allowing data to be seamlessly
Caffeine (1, 3, 5-trimethylxanthine) transferred to external Microsoft office
applications.
It is generally agreed that there is little risk of
harm when a person consumes less than 300
mg of caffeine a day1,2. However at times of
Methods
Reagents
anxiety or stress, or during pregnancy, the FSA
Caffeine
recommends consumption of less than 200 mg
Dichloromethane
a day3. While there are no regulatory
Purified water
requirements to control or label food products
with their caffeine content, numerous studies
Standard Preparation:-
have been carried out to determine the typical
A 1000ppm stock standard of caffeine was
caffeine content of commonly consumed
prepared by dissolving 198.2mg of caffeine in
beverages. A wide variety of methods have
200.0ml purified water. Working standards were
been employed with High Pressure Liquid
prepared by pipetting 25, 12.5, 10, 7.5, 5 and
Chromatography (HPLC) being the method of
2.5ml aliquots of the stock standard solution into
choice in many analytical studies as it
separate 50.0ml volumetric flasks and diluting to
commonly is subject to fewer interferences than
volume with purified water.
alternative methods.
Sample Preparation:-
HPLC is an expensive and resource-hungry
200ml aliquots of boiling purified water was
technique that is not typically found in the
added to each of two 250ml beakers containing
scientific teaching labs of schools and colleges.
2g of instant coffee and a single PG pyramid tea
Therefore this application note will investigate
bag (3.2g of dried tea leaves) respectively. The
an alternative analytical method that uses UV
coffee and tea preparations were stirred for 30
spectroscopy to analyse and quantify the
seconds using a magnetic stirrer (500rpm) and
caffeine content of some common beverages
allowed to cool to room temperature. The soft
and soda drinks.
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Tel: +44 (0)1785 810433
drink samples were used as supplied by the (1) y = 0.0181x
manufacturer.
A linear regression of concentration vs
Caffeine Extraction Procedure:- absorbance allowed the factor of 55.358,
A 50ml aliquot was taken from each working included in equation 2, to be determined.
standard or sample solution. This aliquot was Equation 2 was then used to calculate the
placed into a separating funnel and 25ml of concentration of caffeine in the extracted
dichloromethane was added. The caffeine was sample solution, from the solution’s measured
extracted by inverting the funnel at least three absorbance value.
times, venting the funnel after each inversion.
The dichloromethane layer was removed to a (2) Conc (ppm) = 55.358 x Abs
clean flask and the extraction procedure was
repeated twice more and the solvent layers The caffeine content of the extracted sample
combined. solutions are shown in Table 2.

Sample Measurement:- Concentration of

Aliquots of the extracted standards were placed Sample Sample Abs Extracted
into quartz cuvettes (part code 035 028) and Sample (ppm)
analysed using a 7305 spectrophotometer. The
Nescafe 1.352 74.8
Photometrics mode was accessed by selecting
the Photometrics icon from the main menu PG Tea 0.889 49.2
screen in the PC software and measurements Coca Cola 0.457 25.3
were performed according to the procedure Pepsi Cola 0.504 27.9
described in the instrument and PC software Red Bull 1.275 70.6
operating manuals. Microsoft Excel was used to
tabulate the measurement data and perform a Table 2. The caffeine concentration of the
linear regression analysis. This allowed a extracted sample solutions.
concentration factor to be calculated which was
then input into the settings in the Concentration The final caffeine content of the beverage under
mode of the 7305 PC software. The test is then calculated from the extracted
concentration mode was then used to quantify sample solution’s concentration using equation
the caffeine concentration of the sample 3. Dividing this value by the volume of the drink
solutions with measurements performed against gives the caffeine content per ml.
a dichloromethane blank.
⎛ (Total Sample Vol (ml))2 ⎞
Results (3) Caffeine content (mg) = Conc (ppm) x ⎜ ⎟
⎜ (Measured Sample Vol (ml)) x 1000 ⎟
⎝ ⎠
The absorbance values of the six working
standard solutions were measured; the
Caffeine
measured values are shown in Table 1.
Total Measured Content Caffeine
Sample Sample Sample Per Content
Caffeine Std Vol (ml) Vol (ml) Serving (mg/ml)
Absorbance
Conc. (ppm) (mg)
100 1.806 Nescafe 200 50 59.8 0.30
50 0.899 PG Tea 200 50 39.4 0.20
40 0.724 Coca
330 50 55.1 0.17
Cola
30 0.545
Pepsi
20 0.365 330 50 60.8 0.18
Cola
10 0.183 Red
250 50 88.2 0.35
Bull
Table 1. Standard absorbance values.
Table 3. The calculated caffeine content of
A linear regression of absorbance vs standard beverages and soft drinks.
concentration, forced through the origin, gave
equation 1. Of the five samples tested the sample of Red
Bull had the highest total and per ml content of
caffeine. When ranking the drinks in terms of
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Tel: +44 (0)1785 810433
caffeine content per serving the order was Red 4. http://www.nescafe.com/worldwide/en/well-
Bull ⇒ Pepsi Cola ⇒ Instant Coffee ⇒ Coca being/Pages/Myth+to+Facts.aspx
Cola ⇒ PG Tea. Instant coffee and PG Tea
rise from third and fifth places to second and 5. http://www.food.gov.uk/science/surveillance
third respectively when the drinks are ranked in /fsis2004branch/fsis5304
order of the caffeine content per ml.
6. http://www.cspinet.org/new/cafchart.htm
Conclusions
When the results obtained in this application 7. http://www.timesonline.co.uk/tol/life_and_st
note are compared to data published in yle/health/article4537831.ece
previous reports, or by the drinks manufacturer,
it can be seen that the results obtained using
the 7305 spectrophotometer are broadly in line
with expectations with the exception of the two
cola drink samples.

Published
Sample Caffeine Content
Per Serving
Nescafe
64mg4
(2g)
Instant Tea
40mg5
(1 bag)
Coca Cola
35mg6
(330ml)
Pepsi Cola
38mg6
(330ml)
Red Bull
80mg7
(250ml)

Table 4. Published Caffeine Content of

Beverages and Soft Drinks

The higher than expected values obtained for

the cola drink samples may be the result of
additional compounds, that absorb light at the
wavelength used in this application note, being
extracted into the dichoromethane sample
solution. The resulting solution would give a
higher than expected absorbance reading and
calculated caffeine content. Alternative
extraction procedures could be investigated to
see of the interfering compounds could be
excluded from the extraction solution.

References
1. Smith AP., "Caffeine at Work." Hum
Psychopharmacol. 2005 Aug;20(6):441.

2. Rogers PJ, Dernoncourt C. "Regular

caffeine consumption: a balance of adverse
and beneficial effects for mood and
psychomotor performance." Pharmacol
Biochem Behav. 1998 Apr;59(4):1039-45.

3. http://www.food.gov.uk/news/newsarchive/2
008/nov/caffeinenov08
[email protected]
www.jenway.com
Tel: +44 (0)1785 810433