PRECOLUMN LABELING FOR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF AMINO-ACIDS WITH 7-FLUORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE AND ITS APPLICATION TO PROTEIN HYDROLYSATES
PRECOLUMN LABELING FOR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF AMINO-ACIDS WITH 7-FLUORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE AND ITS APPLICATION TO PROTEIN HYDROLYSATES
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DOI：
10.1016/s0021-9673(00)82032-4
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发表时间：
1982-01-01
期刊：
影响因子：
--
通讯作者：
IMAI, K
中科院分区：
文献类型：
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作者：
WATANABE, Y;IMAI, K
A high-performance liquid chromatographic (HPLC) method for the determination for minute amounts of eighteen amino acids of the presumed components of protein hydrolysates is presented. The method consists of derivatization of the amino acids with a new fluorigenic reagent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F), in ethanol-0.1Mphosphate buffer (pH 8.0) (50:50) at 60°C for 1 min, separation of the resultant fluorophores, including ε-aminocaproic acid as an internal standard, on a reversed-phase column (μBondapak C18) by (i) isocratic elution by solvent A [methanol-tetrahydrofuran (THF) in 0.1Mphosphate buffer (pH 6.0) (3.75:1.6:94.65)] for 24 min, (ii) a linear gradient of 100% solvent A to 100% solvent B [methanol-THF in 0.1Mphosphate buffer (pH 6.0) (25:15:60)] over 30 min, (iii) isocratic elution using solvent B for 6 min and (iv) an isocratic elution by solvent C [methanol-water (40:60)] for 12 min, and detection at 530 nm with excitation at 470 nm. The detection limit for each amino acid isca.10 fmol. Profile analysis was achieved forca.1.5 μg of protein hydrolysates (rabbit pyruvate kinase-M1, rabbit aldolase A and papain) in the final pre-column labelling reaction mixture for HPLC.
