Analysis of L-Theanine in Tea by HPLC with Post-Column Derivatization

Theanine is a neurologically active amino acid found in tea plants. L-Theanine is a dominant amino acid in green tea and is responsible for its unique pleasant taste as well as known relaxation effect. Theanine also has been proven to reduce physical and mental stress, and improve cognition and mood.

Figure 1: Chromatogram of green tea sample.

Cation-exchange chromatography using post-column Ninhydrin reagent Trione and UV detection has shown unmatched reproducibility and selectivity in the analysis of free amino acids in complex matrices. The Pinnacle PCX post-column derivatization system allows shortening run times by utilizing column temperature gradients.

Figure 2: Chromatogram of white tea sample.

We introduce a simple and robust method for analysis of L-Theanine in addition to the other free amino acids in tea leaves.

Table I: Column oven program

Method

Sample Preparation

Homogenize 2 g of dry tea leaves with 25 mL of Li220 for 5 min. Centrifuge and filter through 0.45 µm nylon filter.

Table I: HPLC gradient

Analytical Conditions

Column: High-efficiency Lithium cation-exchange
column, 4.6 × 75 mm, Catalog number 0354675
Flow rate: 0.55 mL/min
Mobile phase: See method

Post-Column Conditions

Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Reagent: Trione Ninhydrin reagent
Flow Rate: 0.3 mL/min
Detection: UV-vis 570 nm for primary amino acids,
440 nm for secondary amino acids
Run time: 72 min
Equilibration time: 12 min

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