Caffeine HPLC Method
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Caffeine can be detected using Reversed Phase High Pressure Liquid Chromatography with UV Detection. (Chris Knutson, 2015)
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, capped, labeled, and loaded into the HPLC auto-sampler carousel.
High Pressure Liquid Chromatography Setup (HPLC)
Pump Gradient
| Time | Flow | %A | %B | Curve | |
| 1 | 1.00 | 0.0 | 100.0 | ||
| 2 | 3.00 | 1.00 | 0.0 | 100.0 | 6 |
| 3 | 15.00 | 1.00 | 60.0 | 40.0 | 6 |
| 4 | 20.00 | 1.00 | 60.0 | 40.0 | 6 |
| 5 | 25.00 | 1.00 | 0.0 | 100.0 | 6 |
Solvents
- Solvent A: Acetonitrile
- Solvent B: 0.01% Acidified Water
UV Detection
- Dual Wavelength Detection
- Channel 1: 275nm
- Channel 2: 325nm
Trial Configuration
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.
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.
