Dinitrophenyl -amino acids separation

SOLVENTS FOR THE SEPARATION OF DINITROPHENYL-AMINO ACIDS WHICH ARE SOLUBLE IN DIETHYL ETHER... 

Very recently, pressurized gradient capillary electrochromatography was used to separate seventeen 2,4-dinitrophenyl amino acid derivatives using a C18 reversed-phase column (3 pm, 130 mm x 75 pm i.d.) using 50 mM sodium acetate, pH 6.4, as... 

It has been applied to the separation and enrichment of organic compounds such as indole auxins, bromoacetyl thyroxine and its analogs, dinitrophenyl amino acids. 

Park et al. reported a method to modify zirconia by adsorption with BSA and, subsequently, crosslinked by glutaraldehyde. The BSA-zirconia showed good enantios-electivity for some enantiomers and could be used for RPLC separation in mobile phases of alkaline pH. They also developed a carboxymethyl-p-cyclodextiin-coated zirconia stationary phase for the separation of racemic 2,4-dinitrophenyl amino acids. 

Park, S.Y. Park, J.K. Park, J.H. McNeff, C.V. Carr, P.W. Separation of racemic 2,4-dinitrophenyl amino acids on carboxymethyl-beta-cyclodextrin coated zirconia in RPLC. Microchem. J. 2001, 70, 179-185. 

In the early prototype instrument [1], the columns were fabricated from relatively large-bore PTFE tubing of 4.6 mm inner diameter (i.d.) with PTFE disk inserts having 0.8-mm-diameter holes. These disks were spaced in 3-mm intervals to form 47 locules in each unit. A number of column units were connected in series to provide 5000 locules with a total capacity of 100 mL. The capability of the system was demonstrated with the separation of DNP (dinitrophenyl)-amino acids using a two-phase solvent system composed of chloroform-acetic acid-0.IM HCl at a 2 2 1 volume ratio. In this system, nine DNP-amino acids were resolved within 70 h at about 3000 theoretical plates. 

J. L. Sandlin and Y. Ito, Gram quantity separation of DNP (dinitrophenyl) amino acids with multi-layer coil countercurrent chromatography (CCC), /. Liquid Chromatogr. 7(2) 323-340 (1984). 

Li, S and WC Purdy (1991). Liquid chromatographic separation of the enantiomers of dinitrophenyl amino acids using a j8-cyclodextrin-bonded stationary phase. Journal of Chromatography, 543,105-112. 

S. Li and W.C. Purdy, Liquid Chromatographic Separation of the Enantiomers of Dinitrophenyl Amino Acids Using a P-Cyclodextrin-bonded Stationary Phase, /. Chromatogr., 543(1991)105. 

Fig. 2.2. Effects of coil revolution speed and flow rate upon separation efficiency of the dinitrophenyl amino acids, DNP-Glu and DNP-Ala. HSES mode is represented by rpm = 0 other separations represent HDES mode (169)...

Amino acids and their derivatives] resolution of amino acids and dieir derivatives on impregnated layers, 406-408 resolution of enantiomeric mixtures of amino acids and their derivatives, 411-419 separation of amino acid derivatives, 398-406 DABITC derivatives of amino acids, 403-406 dansyl amino acids, 402 dinitrophenyl-amino acids, 406 PTH amino acids, 399-402 separation of amino acids, 389-398 chromatographic techniques, 393-395 preparation of test materials, 389-392 TLC system for amino acids, 395-398 Aminoglycosides, 457-461 Amino-modified precoated silica layers, 114,117-119... 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

Applications. Various N-derivatives of amino acids (qv) are resolvable on BSA columns. These A/-amino acid derivatives include benzenesulfonyl-, phthalimido-, 5-dimethylamino-l-naphthalenesulfonyl- (DANSYL-), 2,4-dinitrophenyl- (DNP-), and 2,3,6-trinitrophenyl- (TNP-) derivatives (30). Amines such as Prilocain, (+)-2-(propylamino)-0-propiono-toluidide, a local anesthetic (Astra Pharm. Co.), are also resolved on BSA. The aromatic amino acids DL-tryptophan, 5-hydroxy-DL-tryptophan, DL-kynurenine [343-65-7], C10H12N2O3, and 3-hydroxy-m.-kynurenine [484-78-6], and dmgs such as warfarin, phenprocoumon, and benzodiazepine derivatives can be separated on BSA as well. 

These amino-acid derivatives can be separated from the ordinary amino acids resulting from hydrolysis of the peptide because the low basicity of the 2,4-dinitrophenyl-substituted nitrogen (Section 23-7C) greatly reduces the solubility of the compound in acid solution and alters its chromatographic behavior. The main disadvantage to the method is that the entire peptide must be destroyed in order to identify the one A-terminal acid. 

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

Wang and Porter [92] resolved the enantiomers of oxazepam, lorazepam, and temazepam using /1-cyclodextrin as the CMPA by CEC. The authors varied separation parameters such as voltage and mobile phase. Wei et al. [93] resolved the enantiomers of phenylephrine and synephrine by varying the concentration of /1-cyclodextrin (CMPA), pH, electrolyte concentration, and temperature. Lelievre et al. [99] separated the enantiomers of chlorthalidone using hydroxypropyl /1-cyclodextrin as the CMPA. Lammerhofer and Lindner [90] resolved the enantiomers of N-derivatized amino acids (e.g., 3,5-dinitrobenzoyl, 3,5-dinitro-benzyloxycarbonyl, 2,4-dinitrophenyl, and 9-fluorenylmethoxycarbonyl amino... 

Benzyloxycarbonyl, N-(3,5-dinitrobenzyloxy carbonyl), 9-fluorenylmethoxycarbonyl, benzoyl, acetyl and N-(2,4-dinitrophenyl) derivatized amino acids and profens WAX (weak anion-exchange) type CSP tert. -butylcarbamoylquinine as chiral selector on Hypersil silica gel), 3 pm Acetonitrile-methanol (80 20)+400 mM acetic acid+4 mM triethylamine 335 mm x 100 pm i.d. 250 mm effective length, chiral separation... 

FDNB reacts with all free -NH2 groups, including e-amino groups. Following acid hydrolysis of the dinitrophenylated protein, a separation of the dinitrophenylated N-terminal amino acid from the rest of the hydrolysis products can be achieved by organic solvent extraction of the hydrolysate. A more modern procedure utilizes dansyl chloride instead of FDNB. This method requires less protein, because dansylated amino acids can be identified via fluorescence measurements. 

In 1965, Raban and Mislow [90] postulated that nuclei placed in an asymmetric magnetic field should show NMR nonequivalence. In 1966, Pirkle [91] first reported the validity of the prediction when it was shown that (5)-l-phenylmethylamine caused F-NMR nonequivalency of 2,2,2-trifluoro-1-phenylethanol in a carbon tetrachloride solution. In later studies, 2,2,2-trifluoro-l-(9-anthryl)ethanol, an NMR shift reagent, was used as a mobile-phase additive to separate 2,4-dinitrophenyl methyl sulfoxide on a silica gel column [92]. Later, one enantiomer of this fluoroalcohol was covalently attached to silica gel and used for resolution of a large number of solutes including sulfoxides, lactones, derivatives of alcohols, amines, amino acids, hydroxy acids, and mercaptans [93]. 

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