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 mm  4.6′200mm

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:

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.