Theanine by UV
Determination of Theanine by UV
1) Reagents
a.Phosphoric acid b. Acetronitrile(HPLC) c. Nanopure water d.Sodium acetate,trihydrate
e.EDTA f.NaN3 g.TEA h.Acetone i.ACQ-Tag reagent
2) Apparatus
HPLC (SHIMADZU) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
Vortex mixer Oven capable of maintaining 110+1℃
3) HPLC condition
Column: Hypersil C18 5 m 4.6200mm
Detector: UV248nm
Mobile Phase:A: (1) Dilute Phosphoric acid 200ml ：Add 100ml concentrated Phosphoric acid to 100ml waret
(2)EDTA solution: weigh 100mg EDTA ,add 100ml Milli-Q water sonicate to dissolve
(3)Weigh 19.04g sodium acetate ,add 1liter water ,stir bring pH to 5.2 with dilute phosphoric acid ,add 1ml EDTA solution ,0.1g NaN3,2.37ml TEA, titrate to pH4.95 with dilute phosphoric acid ,filter through aqueous solvent filter
B: Filter 1Lof HPLC grade acetonitrile through an organic filter ,combine 300ml of filtered acetonitrile and 200ml of Milli-Q water (measure separately ),stir ,add 50μl
Of acetone,degas under vacuum in a sonic bath for 20 seconds
Gradient:
Time 0 5 9 20 22 23 25 26 33 38
%A 100 93 90 66 66 0 0 100 100 stop
%B 0 7 10 34 34 100 100 0 0
Flow Rate: 1.0ml/min
Injection: 10μl
4) Procedure
Samples Preparation
Accurately weigh 30mg 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 .Pipette 10μl sample solution into screw cap vials, add 70μl of borate buffer, vortex 10 seconds, add 20μl of AQC, vortex immediately and thoroughly, heat the vial in a reaction block or oven for ten minutes at 55℃。
Reference preparation /calibration
Prepare an reference solution (approximately 4.36mg/10ml in water) using a commercial reference of theanine，Pipette 10μl sample solution into screw cap vials, add 70μl of borate buffer, vortex 10 seconds, add 20μl of AQC, vortex immediately and thoroughly, heat the vial in a reaction block or oven for ten minutes at 55℃。
5) Calculation
Compare the peak areas of the theanine on the sample chromatogram and the corresponding peaks in the chromatogram for the reference solution.
1) Reagents
a.Phosphoric acid b. Acetronitrile(HPLC) c. Nanopure water d.Sodium acetate,trihydrate
e.EDTA f.NaN3 g.TEA h.Acetone i.ACQ-Tag reagent
2) Apparatus
HPLC (SHIMADZU) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
Vortex mixer Oven capable of maintaining 110+1℃
3) HPLC condition
Column: Hypersil C18 5 m 4.6200mm
Detector: UV248nm
Mobile Phase:A: (1) Dilute Phosphoric acid 200ml ：Add 100ml concentrated Phosphoric acid to 100ml waret
(2)EDTA solution: weigh 100mg EDTA ,add 100ml Milli-Q water sonicate to dissolve
(3)Weigh 19.04g sodium acetate ,add 1liter water ,stir bring pH to 5.2 with dilute phosphoric acid ,add 1ml EDTA solution ,0.1g NaN3,2.37ml TEA, titrate to pH4.95 with dilute phosphoric acid ,filter through aqueous solvent filter
B: Filter 1Lof HPLC grade acetonitrile through an organic filter ,combine 300ml of filtered acetonitrile and 200ml of Milli-Q water (measure separately ),stir ,add 50μl
Of acetone,degas under vacuum in a sonic bath for 20 seconds
Gradient:
Time 0 5 9 20 22 23 25 26 33 38
%A 100 93 90 66 66 0 0 100 100 stop
%B 0 7 10 34 34 100 100 0 0
Flow Rate: 1.0ml/min
Injection: 10μl
4) Procedure
Samples Preparation
Accurately weigh 30mg 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 .Pipette 10μl sample solution into screw cap vials, add 70μl of borate buffer, vortex 10 seconds, add 20μl of AQC, vortex immediately and thoroughly, heat the vial in a reaction block or oven for ten minutes at 55℃。
Reference preparation /calibration
Prepare an reference solution (approximately 4.36mg/10ml in water) using a commercial reference of theanine，Pipette 10μl sample solution into screw cap vials, add 70μl of borate buffer, vortex 10 seconds, add 20μl of AQC, vortex immediately and thoroughly, heat the vial in a reaction block or oven for ten minutes at 55℃。
5) Calculation
Compare the peak areas of the theanine on the sample chromatogram and the corresponding peaks in the chromatogram for the reference solution.