Determination of Theanine by HPLC-ELSD 1) Reagents a. Nanopure water b. Acetonitrile (HPLC) c.TFA(C.R)
2) Apparatus HPLC (Shimadzu) Analytical balance (1/10000) Ultrasonic cleaner 0.45μm PTFE filter 50ml volumetric flask Screw cap vials ELSD detector(Alltech)
3) HPLC condition Column: Polaris C18-A 5 m 4.6250mm Mobile Phase: A:0.1% TFA of Water B: 0.1% TFA of Acetonitrile Flow Rate: 0.5ml/min Injection: 10μl Drift tube temperature :95℃ Gas flow:2.5L/min Impactor: Off Gain:2 Gradient: Time %A %B 0.01 100 0 5 100 0 20 60 40 25 60 40 28 100 0 30 100 0
4) Procedure Samples Preparation Accurately weigh 45mg extraction into 50ml volumetric flask. Add Water to volume and sonicate to mix, filter through a 0.45μm PTFE filter into a screw cap vial and proceed with the analysis. Standard preparation /calibration Prepare a standard solution (approximately 0.2mg/ml in Water using a standard of Theanine,Sonicate to mix, filter through a 0.45μm PTFE filter into a screw cap vial and proceed with the analysis.
5) Calculation Determine the peak area’s logarithm of Theanine the on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the standard solution.
|