Aspartic acid, chromatographic separation

Blumenfeld and Gallop (1962b) have used lithium borohydride reduction, with subsequent chromatographic separation of the amino alcohols produced, to identify the carboxyl donor of the ester links previously found by Gallop et al. (1959) using hydroxylamine and hydrazine. The peaks obtained on the chromatogram for the two products in question, namely homoserine and /3-amino-7-hydroxybutyric acid, are very small, but nonetheless seem to establish that a- and /3-carboxyl groups of aspartic acid participate in the hydroxylamine-sensitive links. 

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). 

Aminolysis of the intact rings with taurine leads to the formation of poly(2-sulfoethyl aspartamide) silica and the reaction with ethanolamine to the formation of poly(2-hydroxyethyl aspartamide) silica. Poly(succinimide)-based silica phases are manufactured by PolyLC (Columbia, MD, USA) under the trade names of PolyCAT A for poly(aspartic acid) silica, PolySulfoethyl A for poly(2-sulfoethyl aspartamide) silica, and PolyHydroxyethyl A for poly(2-hydroxyethyl aspartamide) silica. All three poly(succinimide)-based columns have a pore size of 200 A and a surface area of 188 m /g. Various poly(succinimide)-based columns have been used for the separation of carbohydrates, phosphorylated and nonphosphorylated amino acids, petides and glycopeptides, oligonucleotides, and various other polar analytes under HILIC conditions, but lately lost some of their momentum due to a lower chromatographic efficiency in comparison to more modern HILIC phases and column bleed [44]. 

Bhushan and Ali [25] were the first to propose the use of silica gel G layers coated with acidic L-amino acids for the resolution of racemic alkaloids such as hyoscyamine (or atropine) and colchicine. Preparation of the plates, chromatographic development, and solute detection were performed as reported previously. The slurry of silica gel G was prepared in 100 ml of water containing 0.3 g of L-aspartic acid and chromatograms were developed at 0°C for 3.5 h. Migration distance was 10 cm eluting with n-butanol/chloroform/acetic acid/water (3 6 4 1, v/v/v/v). The extremely low temperature necessary for the enantiomeric separation should be noted as no resolution was observed at 5°C. Based on the results obtained (see Table 5.9), layers impregnated with this chiral selector were suitable for the resolution of the two racemates and particularly of atropine a = 1.86). 

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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