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. Caffeine, Theobromine, Theophylline 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.
The HPLC Method for Analysis of Caffeine (1), Theophylline (2) and Theobromine (3) on Alltesta™ Gradient is designed for Caffeine (1), Theophylline (2) and Theobromine (3), a alkaloid compound. Caffeine is a natural stimulant and methylxanthine alkaloid.
Run the separation on Primesep SB (4.6 x 150 mm, 5 um, 100 A, dual ended), SKU SB-46.150.0510. Typical mobile phase: EtOH, 10% 10% H3PO4, 0.3%; H2O 90%.
Operate on Alltesta™ Gradient with 275 nm. Flow rate: 1. Injection volume: 5. Run time: 10.
Our application team can help you adapt this method or develop a new one for your specific needs.
Last updated: Dec 3, 2025
