Caffeine in Soft Drinks by High Performance Liquid


Liquid column chromatography is essentially a separation process in which sample components are dissolved in a liquid mobile phase and are selectively retained by a stationary phase. Differences in the equilibrium distribution between the two phases will cause the components in the sample to be separated. High Performance Liquid Chromatography (HPLC) employs packed columns containing small particles of stationary phase and uses high pressure to force the mobile phase through the column.


The most common solid-phase material is silica and the process is described as normal phase or straight phase chromatography if the stationary phase is more polar than the mobile phase. The solid-phase can also be chemically altered, where polar or non-polar groups are covalently bonded to the microporous silica particles. In cases where the stationary phase is less polar than the mobile phase, this is known as reverse phase chromatography. The use of an auto-sampler and injection loop (injects volumes from 0.1 to 200 μl) gives accurate and highly reproducible sample introduction into the HPLC system.

Caffeine, a stimulant, is present in many soft drinks and is often found as a constituent of street drugs. The following experiment is designed to analyse and quantify caffeine in soft drinks. You will be introduced to how samples are prepared for analysis, analysis of samples by HPLC and interpretation of the data obtained.

Experimental Method (remember to make note of any changes in the parameters used).

Standards and Sample Preparation:

A stock solution of 100 ppm caffeine in mobile phase (1:1 mixture of methanol and 1 % aqueous acetic acid)is provided. Use this stock solution to prepare standards containing 2, 4, 6, 8 and 10 ppm of caffeine by dilution with the mobile phase solution (total volume 10 mL). Take 5.0 mL of one of the soft drinks and dilute to 100 mL with the mobile phase solution in a standard volumetric flask. Repeat this sample preparation procedure with the other soft drink samples. Degas the standards and the soft drink samples by standing them in an ultrasonic bath for 5 minutes.

HPLC analysis

The standard solutions and samples will be run on the HPLC instrument in triplicate. A blank sample (just mobile phase) will be ran on the HPLC instrument between each standard and sample analysed. The standards, drink samples andblank will beran using the instrumental parameters given below:

Instrumental parameters


Include an exemplar chromatogram of one of the samples containing caffeine and comment on the peak shape of the caffeine peak. (2 marks)


Caffeine which is obtained from decaffeination is majorly applied in soft-drink industries and can be detected by HPLC isolation using UV detector. Caffeine appears as a sharp and defined peak that elutes at around 3.19 min when the detector is set at 270 nm. The peak area and height is dependent on the amount of caffeine present.

Tabulate the results from the HPLC analysis of caffeine in the standards and drink samples below.(8 marks)


Using Excel, plot a calibration graph using the average peak area results for the caffeine standards and derive the linear regression equation. Embed your calibration graph below. (6 marks)

 standards and derive

Using the linear regression equation derived from the calibration graph and the results presented in the table above, calculate the concentration of caffeine in each of the diluted drink samples where caffeine was present. (2 marks)

For this problem, we apply the linear regression equation from the curve y = 5158.2x - 4828.2 ; where

Y = peak height and X = concentration.

Our slope of the curve is y = 5158.2 and the intercept of the y-axis is -4828.2

Calculate the concentration of caffeine in each of the undiluted drink samples where caffeine was present. (2 marks)

HPLC Practical Discussion

Using the calibration curve above and the deduced linear regression equation, it is possible to find the concentrations of caffeine in each individual soft-drink samples. Caffeine peak from the HPLC spectrum appears as a sharp peak that elutes at time 3.19 minutes. The height as well as the area of this peak is used to calculate the individual quantities of caffeine in different soft drinks.

HPLC Practical Limitations and Recommendations

Lower separation power – This leads to a spectrum with very close peaks that could at times overlap. This makes it very difficult to make out the peak heights and area of the targeted caffeine sample. To alleviate this problem, longer columns should be used in order to space out the individual components.

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Complex procedures when using the HPLC increases the chances of error. This originates from solvent preparation, sample preparation and even the injection process. An error in any of the process multiplies the effect as the experiment proceeds. The solution to this is to automate crucial procedures such as solvent and sample measurements and mixing processes when preparing HPLC solvents.


Snyder, L.R., Kirkland, J.J. and Glajch, J.L., 2012. Practical HPLC method development. John Wiley & Sons.

Meyer, V.R., 2013. Practical high-performance liquid chromatography. John Wiley & Sons.

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