Caffeine HPLC Method

Caffeine can be detected using Reversed Phase High Pressure Liquid Chromatography with UV Detection.

In order to accomplish caffeine analysis the HPLC system requires the following:

• C18 column
• UV Detector Monitoring 275nm
• Mobile Phase
  • Acetonitrile
  • 0.01% Acidified Water

Samples should be filtered through 45 μm syringe filters into HPLC sample vials and loaded into the HPLC auto-sampler carousel.

Trial Configuration Once prepared, the samples can be injected in 10 μl increments and trials can range in length from 15-30 minutes depending upon the complexity of one's sample.

High Pressure Liquid Chromatography Setup (HPLC)

HPLC Pump Gradient Flow Settings	HPLC Pump Solvent Settings	HPLC UV Detector General Settings
HPLC UV Detector Channel 1 Settings	HPLC UV Detector Channel 2 Settings

Results

Caffeine should be detected as sharp peaks which elute at approximately 11 minutes when monitoring 275nm on the UV Detector.

The image below is the spectra of a caffeine standard run with the above method.

Caffeine Standard Spectra