Amino acids precolumn derivatization

Fig. 7 Typical reversed-pbase separation of amino acids. Precolumn derivatization of a standard amino acid mixture was achieved employing FMOC. Resolution was achieved by gradient elution with acetonitrile, methanol, and acetate buffer (pH 4.2) on a C,8 column. Standard three-letter abbreviations for amino acids are used also, CySO H = cysteic acid. (From Ref. 164. Copyright 1983 Elsevier Science.)...

The most frequent application of precolumn derivatization is in amino acid analysis. Here, the amino acids are derivatized for instance with 9-fliiorenyl methoxycarbonyl chloride (FMOC) or o-phthalaldehyde (OPA) to form strongly fluorescing compounds, which can be detected easily with a fluorescence detector [93], [94], Another typical application for precolumn derivatization is in the analysis of fatty acids. Fatty acids have no chromophore, so UV detection is impossible. Even highly unsaturated fatty acids can be detected only in the very far UV range (< 200 nm) with poor sensitivity. Other detectors, such as the refractive index detector, have a disadvantage because they cannot be used in gradient elution, which is essential for fatty acid analysis. Therefore, the fatty... 

Heinrikson, R.L. and Meredith, S.C. 1984 Amino acid analysis by reverse-phase high-performance liquid chromotography Precolumn derivatization with phenylisothiocyanate. Analytical Biochemistry 136 65-74. 

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

Cooper, J. D. H., Ogden, G., McIntosh, J., and Tumell, D. C., The stability of the o-phthaldehyde/2-mercaptoethanol derivatives of amino acids an investigation using high-pressure liquid chromatography with a precolumn derivatization technique, Anal. Biochem., 142, 98, 1984. 

Albin, D. M., Wubben, J. E., and Gabert, V. M., Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate,. Agr. Food Chem., 48, 1684, 2000. 

Arnold, U., Ludwig, E., Kuehn, R., Moeschwitzer, U., Analysis of free amino acids in green coffee beans, Part 1, Determination of amino acids after precolumn derivatization using 9-fluorenylmethylchloroformate, Z. Lebensm.-Forsch. 199(1), 22, 1994. (CA121 132515y)... 

Many times an analyte must be derivatized to improve detection. When this derivatization takes place is incredibly important, especially in regards to chiral separations. Papers cited in this chapter employ both precolumn and postcolumn derivatization. Since postcolumn derivatization takes place after the enantiomeric separation it does not change the way the analyte separates on the chiral stationary phase. This prevents the need for development of a new chiral separation method for the derivatized analyte. A chiral analyte that has been derivatized before the enantiomeric separation may not interact with the chiral stationary phase in the same manner as the underivatized analyte. This change in interactions can cause a decrease or increase in the enantioselectivity. A decrease in enantioselectivity can result when precolumn derivatization modifies the same functional groups that contribute to enantioselectivity. For example, chiral crown ethers can no longer separate amino acids that have a derivatized amine group because the protonated primary amine is... 

Wen-Chen Z, Ling-Jun L, Xian-En Z et al (2008) Application of 2-(l l//-benzo[ i]carbazol-11 -yl) ethyl carbonochloridate as a precolumn derivatization reagent of amino acid by high performance liquid chromatography with fluorescence detection. Chin J Anal Chem 36 1071-1076... 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

Lindroth, P., and K. Mopper. 1979. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Analytical Chemistry 51 1667-1674. 

Direct and indirect chromatographic methods were developed and compared in systematic examinations for the enantioseparation of P-amino acids direct separation of underivatized analytes involved the use of commercially available Crownpak CR(-I-), teicoplanin, and ristocetin A CSPs [148], while indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-G-acetyl-f)-D-glucopyranosyl isothiocyanate (GITC) or A - a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (EDAA, Marfey s reagent), with subsequent separation on a nonenantioselective column. 

Nimura N, Fujiwara T, Watanabe A, Sekine M, Furuchi T, et al. 2003. A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay. Anal Bio-chem 315 262-269. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

Diverse analytical methods have been proposed for the analysis of amino acids, including GC, HPLC, and capillary electrophoresis. The preferred method at present is RP-HPLC with precolumn derivatization, which has the advantages of requiring short analysis time, simple instrumentation, and low cost [192]. 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

The earliest approach to amino acid analysis involved postcolumn reaction. This scheme offers several advantages compared to precolumn reaction. First, it simplifies the sample preparation necessary. Often, precolumn derivatizations require sample cleanup steps to eliminate sample... 

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