Amino acids derivatizing reagents

Amino acid derivatization reagents Performic acid... 

F. Lai and T. Sheehan, BioTecbniques 14, 642-649 (1993). A comparison of reagents for amino acid derivatization. 

P Grossman andj Colburn, Editors, Capillary Electrophoresis Theory and Practice (1992), Academic Press (San Diego). An up-to-date and complete review of CE E Heimer,/. Chem. Educ. 49, 547 (1972) Paper chromatography of amino acids F Lai and T Sheehan, BioTechniques 14, 642-649 (1993) A comparison of reagents for amino acid derivatization... 

A chip-based integrated precolumn microreactor with 1 nl reaction volume has been explored by Jacobson et al. [67]. The reactor is operated in a continuous manner by electrokinetically mixing of sample (amino acids) and reagent (o-phthaldialdehyde) streams. The reaction time is adjusted via the respective flow velocities. By switching of potentials, small plugs of the reaction product were injected into a 15.4 mm separation channel in a gated injection scheme (< 1.8% RSD in peak area). The separation efficiency achieved was relatively poor, however, electrokinetic control of reaction time (and yield) permitted to monitor the kinetics of the derivatization under pseudo first-order conditions. A similar integrated precolumn reactor operated in a stopped flow mode has been described by Harrison et al. [68]. 

One of the most popular methods of single-stage amino acid derivatization at present is their conversion to N,0 S) err-butyldimethylsilyl derivatives [the reagent rert-butyldimethylsilyl trifluoroacetamide (MTBSTFA) or its A-Me analog]. Another way, which was proposed at the beginning of the 1970s is based on amino acid interaction with dimethylformamide di-alkylacetals (CH3)2NCH(OR )2 (R = Me, Et, Pr, iso-Pr, Bu, Am) with formation of A-dimethylaminometh-ylene derivatives of amino acids esters ... 

The precolumn technique that is most frequently employed today was developed during the early 1980s [32,33]. For this method, The classical Edman reagent phenylisothiocyanate (PITC) is used for amino acid derivatization after hydrolysis. Separation of the PTC amino acids i then accomplished by HPLG, with detection at 254 nm. Although standard Cig columns available Irom a variety of vendors are suitable for separation of the PTC-derivatized amino acids, there are specific columns that have bqen optimized for this purpose (e.g.. Waters). Approximately 0.5 /ug of peptMe should be hydrolyzed for analyses using precolumn derivatization. ... 

A number of groups have shown how enantiomeric resolution of amino acids derivatized with non-chiral reagents is possible in SFC with chiral stationary phases. N-Acetylamino acid t-butyl ester racemates were rapidly resolved [17] on (N-formyl-L-valylamino)propyl silica with CO2 modified with methanol, acetonitrile and diethyl ether. A similar stationary phase allowed (18 rapid (< 5 min) separation of racemic N-4-nitrobenzoyl-amino acid isopropyl esto-s with methanol-modified CO2 the enantioselectivity in SFC was comparable with that in HPLC with isopropanol/n-hexane as mobile phase. Capillary column SFC on polysiloxane stationary phases containing chiral side chains has been employed... 

FMOC is a reagent that reacts quickly with both primary and secondary amino acids. Excess reagent must be removed rapidly because it undergoes hydrolysis. The excitation and emission wavelengths are 270 and 315 nm, respectively. An increasingly popular method for amino acid analysis is to measure the primary amino acids with OPA and the secondary amino acids with FMOC. Samples are derivatized first with OPA and then with FMOC. Primary amines are detected using the fluorescence properties of OPA while the secondary amines are detected using those of FMOC. 

The mechanism was further supported by the separation behavior of certain amino acids derivatized with chiral variants of FDAA in which L-Ala-NH2 was replaced with L-Val-NH2, L-Phe-NH2, L-Ile-NH2, and l-Lcu-NH2 since the retention time became longer, and the resolution became better with the increase of their length of alkyl side chains in the amino acid amides [14], The amino acid derivatized with the reagent of D-alaninamides showed the completely opposite elution order. These results indicated that the a-carboxyl group of an amino acid was not always essential for the resolution and the separation behavior could be explained without consideration of intramolecular H-bonding (between the carboxyl of analyte and the carboxamide of the reagent). 

Fig. 7-6). Two unichiral amides which have been known capable of this reaction are 1-phenylethylamine [15] and l-(l-naphthyl)ethylamine [16]. Marfey s reagent [N-a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide] was introduced as a reagent to deriva-tize amino acids with cyclopentane, tetrahydroisoquinoline or tetraline structures [17]. Simple chiral alcohols such as 2-octanol can also be used to derivatize acids such as 2-chloro-3-phenylmethoxypropionic acid [18]. 

Excess of the reagent hydrolyses to a non-fluorescent residue and the reagent itself does not fluoresce. The optimum wavelength of the excitation light is 390 nm and that of the emitted light 475 nm. This regent is, however, less sensitive than Fluoropa and the derivative is unstable consequently, it must be injected onto the column immediately after formation if used in pre-column derivatization. It has been used successfully in the separation and analysis of polyamines (32), catecholamines (33) and amino acids (34). 

The versatility of the reagent system in the assay of small peptides is nicely illustrated in Figures 3 through 5. Neurotensin, a polypeptide composed of 13 amino acids, and three fragments from partial hydrolysis were derivatized with NDA/CN and separated by... 

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... 

Dansyl chloride is the most widely used of the derivatizing reagents. It forms derivatives with primary and secondary amines readily, less rapidly with phenols and imidazoles, and very slowly with alcohols. The reaction medium is usually an aqueous-organic sixture (e.g., 1 1 acetone-water) adjusted to a pH of 9.5-10. Dansyl chloride has two major application areas. It is used to determine small amounts of amines, amino acids and phenols, as... 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

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