Glutamic acid, chromatographic separation

OW Lau, CS Mok. Indirect conductometric detection of amino acids after liquid chromatographic separation. Part II. Determination of monosodium glutamate in foods. Anal Chim Acta 302 45 -52, 1995. 

A mixture of alanine, glutamic acid, and arginine was chromatographed on a weakly basic ion-exchange column (positively charged) at pH 6.1. Predict the order of elution of the amino acids from the ion-exchange column. Are the amino acids separated from each other Explain. 

The first synthesis of a 3-deaza analogue, 3-deazaFA (70), was accomplished, albeit in poor yield, via the Waller condensation of 2,3,6-triamino-4-hydroxypy-ridine hydrochloride, 2,3-dibromopropionaldehyde and p-aminobenzoyl-L-glutamic acid, as shown in Scheme 3.14. This approach suffered from its equivocal nature as well as from a lengthy work-up involving repeated centrifugations and column chromatographic separations which were necessary to obtain (70) sufficiently pure to be characterized and evaluated [71]. 

D/L enantiomeric ratios for alanine, valine, glutamic acid, leucine, proline, and phenylalanine in the remaining half of the desalted amino acid fraction were obtained by gas chromatography (15). Hie N-trifluoroacetyl-L-prolyl-DL-amino acid esters were synthesized, then separated on a Hewlett-Packard Model 5711A Gas Chromatograph with flame ionization detector and a 12 foot colunn of Chromasorb W-AW-DMCS solid support coated with 8% SP 2250. 

Figure 1. Typical HPLC chromatograms of the OPA-NAC (II) derivitized amino acids detected from the spark discharge reactions. Chromatograms labeled with roman numerals I.) Amino acid standard, II.) CO2/N2 not sparked, III.) CO2/N2 + CaCOs, sparked, hydrolyzed- ascorbate. IV.) CO2/N2, sparked, hydrolyzed - ascorbate V.) CO2/N2, sparked + CaCOs, hydrolyzed + ascorbate. Amino acids I.) DL aspartic acid 2.) DL glutamic acid 3.) DL serine 4.) glycine 5.) P-alanine 6.) DL alanine 7.) a-amino isobutyric acid 8.) DL norleucine (internal standard). The D and L enantiomers of glutamic acid and serine are not separated under these chromatographic conditions.

Fig. 11.2.11. Isocratic separation of PTH-amino adds. Chromatographic conditions column, Ultrasphere ODS (250 X 4.6 mm I.D.) mobile phase, 0.01 M sodium acetate (pH 4.9)-acetonitrile (62.2 37.8) flow rate, 1 ml/min temperature, ambient. Peak identity corresponding to the single letter code for amino acids D, aspartic acid E, glutamic acid N, asparagine Q, glutamine T, threonine G, glycine A, alanine Y, tyrosine M, methionine V, valine P, proline W, tryptophan F, phenylalanine K, lysine I, isoleucine L, leucine S, serine. Reproduced from Noyes (1983), with...

Fig. 11.2.12. Normal phase separation of amino acids. Chromatographic conditions column, Zorbax NH2 (250 x 4.6 mm I.D.) mobile phase, 10 mM potassium phosphate, pH 4.3 (A), acetonitrile-water 50 7 (v/v) (B) flow rate, 2 ml/min temperature, 35 °C. Peaks 1, phenylalanine 2, leucine 3, isoleucine 4, methionine 5, tyrosine 6, valine 7, proline 8, alanine 9, hypro 10, threonine 11, glycine 12, serine 13, histidine 14, cysteine 15, arginine 16, lysine 17, hydroxylysine 18, glutamic acid 19, aspartic acid. Reproduced from Smolensk et al. (1983), with permission.

Fig. 2. The elution pattern of a standard mixture of OPA-derivatized primary amines, separated on a 5 (Jim Nucleosil C-18 column (200 X 4.6 mm id). The flow-rate was 1 mL/min employing the indicated gradient of metlianol and Na phosphate buffer (50 mA4, pH 5.25). Each peak represents 39 pmol except for those indicated below. 1, glutathione 2, cysteic acid 3, O-phosphoserine (19.5 pmol) 4, cysteine sulfinic acid 5, aspartic acid 6, asparagine (19.5 pmol) 7, glutamic acid 8, histidine 9, serine 10, glutamine 11, 3-methyl-histidine 12, a-aminoadipic acid (9.8 pmol) 13, citrulline (9.8 pmol) 14, carnosine 15, threonine,glycine 16, O-phosphoethanolamine 17, taurine (19.5 pmol) 18, p-alanine (19.5 pmol) 19, tyrosine 20, alanine 21, a-aminoisobutyric acid 22, aminoisobutyric acid 23, y-amino-ii-butyric acid 24, p-amino-u-butyric acid 25, a-amino-butyric acid 26, histamine 27, cystathione (19.5 pmol) 28, methionine 29, valine 30, phenylalanine 31, isoleucine 32, leucine 33, 5-hydroxytryptamine (5-H i ) 34, lysine. The chromatographic system consisted of a Varian LC 5000 chromatograph and a Schoeffel FS 970 fluorimeter.

Solvents employed in paper chromatographic separation of PTH-amino acids [182, 190—192] cannot be used on silica gel G. We have, however, achieved good separations using the solvents given in Table 190. Chloroform-methanol-formic acid (70 + 30 + 2) is suitable for separating PTH-aspartic acid and PTH-glutamic acid (Fig. 218). 

Subjecting pteroyl-di-y-glutamylglutamic acid and also pteroyl-y-glutamyl-glutamic acid to the steps used in s mthesis of foHnic acid resulted in three cmd two active compounds, respectively. Folinic acid was formed from each pteroyl derivative, and the corresponding di- and triglutamates which were separated chromatographically account for the other factors . 

A mixture of seven amino acids (glycine, glutamate, leucine, lysine, alanine, isoleucine, and aspartate) is separated by TLC. Explain why only six spots show up when the chromatographic plate is sprayed with ninhydrin and heated. 

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