Glutamate using HPLC

HPLC does not give a good response to long-chain derivatives of folate, therefore, conversion of the natural form of folate, polyglutamates, to mono- or di-glutamate is required. The enzyme used is y-glutamylcarboxypeptidase, extracted from chicken pancreas, hog kidney, or to a lesser extent from... 

In this assay the asparagine, aspartate, glutamine, and glutamate are separated by reversed-phase HPLC (Cj8) using a mobile phase of 70% sodium acetate buffer (pH 5.9), and 30% methanol as shown in Figure 9.44. 

Glutamine synthetase and glutamic acid decarboxylase catalyze the conversion of glutamic acid to glutamine and y-aminobutyric acid (GABA), respectively. Since both activities are involved in the metabolism of this amino acid, it would be useful to be able to study both simultaneously. And indeed, an HPLC method was developed to measure both activities in crude extracts. 

Eckstein, J.A. et al. Analysis of glutamine, glutamate, pyroglutamate, and GABA in cerebrospinal fluid using ion-pairing HPLC with positive electrospray LC/MS/MS. J. 

Fig. 11.2.10. HPLC of human leucocyte interferon hydrolysate using post-column fluorescence detection with fluorescamine. Chromatographic conditions column, DC-4A (Dumim) (500 x 4.6 mm I.D.) mobile phase, 0.175 N sodium citrate, 2.5% isopropanol, pH 3.5 (Buffer A), 0.175 N sodium citrate, pH 3.45 (Buffer B), 0.2 N sodium citrate, 0.05% thiodiglycol, pH 4.1 (Buffer C), 0.52 N sodium citrate, 1 N sodium chloride, 0.05% thiodiglycol, pH 7.9 (Buffer D), elution was achieved with the following gradient Buffer A (15 min). Buffer B (17 min). Buffer C (24 min). Buffer D (41 min), flow rate, 18 ml/h temperature, 57 ° C. Peaks 1, aspartic acid 2, threonine 3, serine 4, glutamic acid 5, cysteine 6, proline 7, glycine 8, alanine 10, valine 11, methionine 12, isoleucine 13, leucine 14, norleucine 15, tyrosine 16, phenylalanine 17, histidine 18, lysine 19, ammonia 21, arginine. Reproduced from Stein and Brink (1981), with...

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