I want to quantify 2 specific amino acid form my plant leaf samples. Preferably through spectrophotometric pathway or LC-MS. any leads for choosing which buffer? pathways? or any lead?
Amino acids in plant leaf samples can be quantified spectrophotometrically by extracting with buffers like phosphate buffer and using derivatization agents such as ninhydrin or OPA for colorimetric detection. LC-MS offers sensitive and selective quantification, often using MS-compatible buffers and internal standards, with sample preparation involving homogenization, extraction, and optional derivatization for accurate results.
1. Extract amino acids from your plant leaves using a solvent like methanol/water/chloroform.
2. Separation on the basis of Charge by using Ion exchange Chromatography or size by using Gel-filtration/molecular sieve Chromatography, if you not successfully carry the above two chromatography then instead of this you directly perform the thin layer chromatography.
3.Apply a Ninhydrin reagent (Ninhydrin assya) to quantitative analysis of separated amino acids (colored products) by spectrophotometer.
4. Measure the absorbance at a specific wavelength (often 570 nm) using a spectrophotometer to determine the amino acid concentration.
Sample can be prepared by Ultrasonication method with raw leaf samples.
You can use Sodium Acetate buffer, pH 6.7 and Acetonitrile:Water (60:40) as mobile phase A and B.
Column you can use Reverse phase-C18, 100 mm optimal length.
Complete sample preparation and Gradient method conditions, already published. Search on Waters website as well as you can search in research papers from CSIR-IHBT.
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