HPLC Method for Analysis of Caffeine, Theophylline and Theobromine on Primesep SB Column on Alltesta™

HPLC Method for Caffeine, Theophylline, Theobromine on Primesep SB by SIELC Technologies


High Performance Liquid Chromatography (HPLC) Method for Analysis of Caffeine, Theophylline, Theobromine

Caffeine is a natural stimulant and methylxanthine alkaloid. with the molecular formula C6H10N4O2. Caffeine can be found in a variety of plants, including tea, coffea, cocoa, kola nuts, and guarana. Ingestion of it can increase alertness and cognitive function. It can also cause worsening anxiety, heart palpitations, and headaches.

Theophylline is a methylxanthine compound with the molecular formula C7H8N4O2. It is a caffeine metabolite and is often used in forensic analyses. Besides analytical use, it is often used in treating respiratory conditions.

Theobromine is a methylxanthine alkaloid with the molecular formula C7H8N4O2. It is a derivative of caffeine. It can be found in many foods, including chocolate and tea. Despite that, it is toxic to dogs.

You can find detailed UV spectra of Caffeine and information about its various lambda maxima by visiting the following link.

You can find detailed UV spectra of Theophylline and information about its various lambda maxima by visiting the following link.

You can find detailed UV spectra of Theobromine and information about its various lambda maxima by visiting the following link

Caffeine, Theophylline, Theobromine can be retained and analyzed using the Primesep SB stationary phase column. The analysis utilizes an isocratic method with a simple mobile phase consisting of water and ethanol (EtOH) with a phosphoric acid buffer. Detection is performed using UV.

ColumnPrimesep SB, 4.6 x 150 mm, 5 µm, 100 A, dual ended
Mobile PhaseEtOH- 10%
BufferPhosphoric Acid
Flow Rate1.0 ml/min
DetectionUV 275 nm

Class of CompoundsAlkaloid
Analyzing CompoundsCaffeine, Theophylline, Theobromine

Application Column

Primesep SB

Column Diameter: 4.6 mm
Column Length: 150 mm
Particle Size: 5 µm
Pore Size: 100 A
Column options: dual ended

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Application Analytes:
Caffeine
Theobromine
Theophylline
Application Detection:
UV Detection
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.