Caffeine is a naturally occurring molecule found in various plants worldwide, and found in beverages such as tea, coffee and soft drinks.
The amount of caffeine present in drinks can be measured by UV spectroscopy.
The full method below, as originally outlined by the Jenway team at Bibby Scientific, can be used to measure caffeine in drinks for quality control, comparison across different brands or as an educational introduction to working with UV-Vis spectroscopy.
- Analytical balance
- Weighing boats
- 200ml volumetric flask x1
- Micropipette – ideally 1ml to 10ml size
- Compatible Pipette tips
- 50ml volumetric flasks x6
- Hotplate stirrer
- Stirrer bar
- 250ml beakers (one per hot drink sample to be tested)
- 100ml measuring cylinder
- Separating funnel
- Retort stand with separating funnel ring
- 250ml Conical flasks (one per sample to be tested)
- Dropper pipettes
- Quartz cuvette with 10mm path length
- UV range Spectrophotometer (with concentration mode preferred)
- Prepare a standard 1,000 ppm stock solution of caffeine; use an analytical balance to weigh 198.2mg of caffeine and make this up to 200ml using purified water in a volumetric flask.
- Prepare calibration standards; using a pipette add 25ml, 12.5ml, 10ml, 7.5ml, 5ml and 2.5ml to each of 6x 50ml volumetric flasks. Make the standards up to the 50ml volume using purified water. These amounts will create 100ppm, 50ppm, 40ppm, 30ppm, 20ppm and 10ppm calibration standards respectively (ppm = mg/L)
- Prepare the samples;
- For instant coffee; add 2g of granules to a 250ml beaker and add 200ml boiling purified water. Stir at 500 rpm on a magnetic stirrer for 30 seconds, then leave to cool to room temperature without further stirring.
- For tea; add 3.2g of dried tea leaves to a 250ml beaker and add 200ml boiling purified water. Stir at 500 rpm on a magnetic stirrer for 30 seconds, then leave to cool to room temperature without further stirring.
- For pre made soft drinks (e.g. cola, energy drinks) no preparation is required, skip ahead to step 4
- Extract caffeine from the samples; take 50ml of the calibration standard or sample and it to a separating funnel. Use the measuring cylinder to add 25ml of dichloromethane.Invert the separating funnel 3 times, then vent to avoid pressure build-up. Put the funnel in a stand and allow the layers to separate, before removing the dichloromethane layer to a labelled conical flask.Return the calibration standard or sample to the separating funnel and repeat twice more, until 3x 25ml dichloromethane layers have been combined in the conical flask.
- Measure the calibration curve; use a dropper pipette to add the calibration standards to the quartz cuvette for measurement.First measure purified water only as a blank, then measure each of the calibration standards in increasing order of concentration.Tabulate the results of caffeine concentration in ppm vs. absorbance at 260nm.
- Calculate the calibration curve; use a spreadsheet to create a line graph of the calibration curve results. Find the linear regression equation of the calibration curve, y = mx +c (where y = absorbance and x = concentration)
- Measure the samples; use a dropper pipette to add your first sample to the (cleaned and dried) cuvette. Take a measurement and record the absorbance at 260nm. Repeat for each sample, taking care to clean and dry the cuvette carefully between samples.
- Calculate the results; using the y = mx + c equation from your calibration graph, you can calculate the caffeine concentration of your samples. Substitute Y for the absorbance value recorded for that sample, keep M and C constant, and rearrange to solve for X.For some models of spectrophotometer, such as the Jenway 7305, a concentration mode is available which allows the instrument to do this calculation for you based on your y = mx + c equation, so the readout on the spectrophotometer will be in ppm directly.
For more information on this test and the equipment needed to perform it, just follow the links above or contact us with any questions;