Determination of caffeine in beverages by high-pressure liquid chromatography (HPLC)


The aim of the practical is to introduce students to the operation of the Department’s high pressure liquid chromatography apparatus.

Hazards and safety precautions

All chemicals should be treated with caution. Absence of specific comments does not mean that a substance is harmless. You must wear eye protection and a lab coat and avoid ingestion of solids or liquids whilst in the lab.


Caffeine Toxic if swallowed. Wear gloves when handling

Risk assessment:

Hazard category – low, exposure potential – low, use in open lab

Acetonitrile Toxic by inhalation and ingestion. Wear gloves when handling

Risk assessment:

Hazard category – low, exposure potential – medium, use in fume cupboard

Methanol Toxic by inhalation and ingestion.

Risk assessment:

Hazard category – low, exposure potential – medium, use in fume cupboard


Experimental procedure


Instrument.      Beckman Coulter HPLC.  32 Karat software.

Column.           C18 reverse phase column (column 250 mm x 4 mm, guard column 4 mm x 4 mm).

Solvent A                     HPLC water

Solvent B                     HPLC acetonitrile

Program                      25 % B, 1 mL/ min

Injection volume         100 mL

Detection                    UV at 254 nm


After the standard solutions of caffeine have been prepared, the Demonstrator will describe the operation of the chromatograph, the detector and the 32 Karat software. It is important that students take careful notes during the discussion, you will be working unassisted during the experiment.

Preparation of standards:

You are provided with a 1000 ppm solution of Caffeine in methanol.  Prepare 10 mL of the following standard solutions of caffeine in methanol in the volumetric flasks provided: 10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm.  The method for analysis of samples is described below; you will also be shown how to do this by the Demonstrator. Analyse all five standards twice (which one first?) and the unknowns three times each.

  1. On the computer, select Control, Single Run
  2. Enter the Sample ID and Data File (if required, see the Demonstrator for specific instructions for different instruments)
  3. Press Start
  4. The instrument will now go through its initialisation procedures
  5. When a message appears “Waiting for Trigger”, an injection can be made
  6. Wash the syringe 5 times with the sample to be analysed
  7. Fill the syringe with sample (g. 50 L for a 50 L syringe) ensuring no air bubbles are present
  8. With the injector set to Load, insert the syringe as far as it will go and inject the sample
  9. Turn the injector to the Inject position – this will start the acquisition
  10. When at least 2 minutes have elapsed, the syringe can be withdrawn, but the injector MUST be put back into the Load position BEFORE withdrawing the syringe
  11. Allow at least two minutes to elapse after the caffeine peak has eluted, then select the STOP icon to stop the run.

Analysis of the unknowns – determination of caffeine concentration in beverages

Dilute the three beverages five times in deionised water (not methanol). Filter the samples using the syringe filters provided (the Demonstrator will show you how).  Use a fresh syringe and filter for each unknown.  Analyse the samples for caffeine content using the procedures outlined above.



Complete and upload the report/ presentation/ journal article/ poster as instructed.