Analysis of Caffeine in BSA

Analysis of caffeine in BSA by the column switching method is shown. Caffeine is adsorbed to MSpak PK-2A at pretreatment (eluent : 10mM CH₃COONH₄(pH7.0)). After switching valve, caffeine is separated from PK-2A and is carried to Asahipak ODP-50 2D (eluent : 10mM CH₃COONH₄(pH7.0)/CH₃CN =90/10). After elution of caffeine, the analytical column is washed by CH3COONH4(pH 7.0)/CH3CN =50/50.
Please refer to System diagram.

Sample : 1ppm Caffeine in 10% BSA, 100micro-L

Pretreatment
Column         : Shodex MSpak PK-2A (2.0mmID*10mm)
Eluent         : 10mM CH3COONH4(pH7.0)
Flow rate      : 0.5mL/min
Column temp.   : Room temp.
Switching time : 5min

Analysis 
Column       : Shodex Asahipak ODP-50 2D (2.0mmID*150mm)
Eluent       : 10mM CH3COONH4(pH7.0)/CH3CN
               (step gradient) 0 to 10min : 90/10, 10 to 20min : 50/50, after 20min : 90/10 
Flow rate    : 0.5mL/min
Detector     : UV(254nm)
Column temp. : Room temp.