2.4. Methods2.4.1. Pre-staining pap

2.4. Methods
2.4.1. Pre-staining paper chromatographic conditions
The improved method was conducted as follows. Appropriate 2 μL of samples were spotted onto the chromatography paper. The PC was developed at 30 °C with n-butanol–acetic acid–water (5:3:2) containing ninhydrin (ascending technique). After development, the paper was directly dried for color yield in the convection oven. Then the GABA spots were cut out from the paper and were extracted with 5.0 mL of 75% alcohol (v/v):0.6% cupric sulfate (w/v) (38:2, v/v) at 40 °C in a shaker (50 rpm). The absorptions were read in a UV–vis spectrophotometer at 512 nm. The effects of ninhydrin concentration, color temperature, color time and Cu2+ concentration on the ninhydrin reaction were optimized.