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 plants species worldwide. The most common sources of caffeine are coffee, cocoa beans, cola nuts and tea leaves and the worldwide consumption of products derived from these natural materials means that caffeine is one of the most popular and commonly consumed drugs in the world. Caffeine’s popularity stems mainly from the fact that it is a pharmacologically active substance and a mild central nervous system stimulant. Caffeine can be extracted from aqueous solutions with chlorinated solvents such as dichlormethane and chloroform, a technique commonly employed commercially to decaffeinate coffee and tea. After the caffeine is extracted it can be analysed directly by measuring the absorbance of the solvent solution at 260nm. Five samples were chosen to include the 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-ofcharge 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 applications.
Caffeine (1, 3, 5-trimethylxanthine) 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 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 have been carried out to determine the typical caffeine content of commonly consumed beverages. A wide variety of methods have been employed with High Pressure Liquid Chromatography (HPLC) being the method of choice in many analytical studies as it commonly is subject to fewer interferences than alternative methods. HPLC is an expensive and resource-hungry technique that is not typically found in the scientific teaching labs of schools and colleges. Therefore this application note will investigate an alternative analytical method that uses UV spectroscopy to analyse and quantify the caffeine content of some common beverages and soda drinks.
Reagents Caffeine Dichloromethane Purified water Standard Preparation:A 1000ppm stock standard of caffeine was prepared by dissolving 198.2mg of caffeine in 200.0ml purified water. Working standards were prepared by pipetting 25, 12.5, 10, 7.5, 5 and 2.5ml aliquots of the stock standard solution into separate 50.0ml volumetric flasks and diluting to volume with purified water. Sample Preparation:200ml aliquots of boiling purified water was added to each of two 250ml beakers containing 2g of instant coffee and a single PG pyramid tea bag (3.2g of dried tea leaves) respectively. The coffee and tea preparations were stirred for 30 seconds using a magnetic stirrer (500rpm) and allowed to cool to room temperature. The soft
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drink samples were used as supplied by the manufacturer. Caffeine Extraction Procedure:A 50ml aliquot was taken from each working standard or sample solution. This aliquot was placed into a separating funnel and 25ml of dichloromethane was added. The caffeine was 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 combined. Sample Measurement:Aliquots of the extracted standards...