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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
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,
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
drugs in the world. Caffeine’s popularity stems
pharmacologically active substance and a mild
commonly consumed beverages and soft drinks
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
transferred to external Microsoft office
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 Reagents
anxiety or stress, or during pregnancy, the FSA
recommends consumption of less than 200 mg
a day3. While there are no regulatory
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
prepared by dissolving 198.2mg of caffeine in
beverages. A wide variety of methods have
200.0ml purified water. Working standards were
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
separate 50.0ml volumetric flasks and diluting to
commonly is subject to fewer interferences than
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
drink samples were used as supplied by the
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
times, venting the funnel after each inversion.
The dichloromethane layer was removed to a
clean flask and the extraction procedure was
repeated twice more and the solvent layers
The caffeine content of the extracted sample
Sample Measurement:- Concentration of
Aliquots of the extracted standards were placed
Sample Abs Extracted
into quartz cuvettes (part code 035 028) and
Sample (ppm)
analysed using a 7305 spectrophotometer. The
Photometrics mode was accessed by selecting the Photometrics icon from the main menu
screen in the PC software and measurements
were performed according to the procedure
described in the instrument and PC software
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
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
(3) Caffeine content (mg) = Conc (ppm) x ⎜
The absorbance values of the six working
Caffeine Total Measured Content Caffeine Sample Sample Sample Caffeine Std Absorbance Vol (ml) Serving (mg/ml) Conc. (ppm) 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
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
caffeine content per serving the order was Red
4. http://www.nescafe.com/worldwide/en/well-
Bull ⇒ Pepsi Cola ⇒ Instant Coffee ⇒ Coca
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
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
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
Published Caffeine Content Per Serving 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
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
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