Quantitative analysis of amino acids

The method of choice is ion exchange chromatography by automatic analysis (S14). Urine and cerebrospinal fluid can be applied directly to the column, but plasma must be first deproteinized. Since the accurate estimation of gultamine is of paramount importance in all inborn errors of the urea cycle, care must be taken to avoid the breakdown of glu- 

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The column chromatography technique using Dowex 50 ion-exchange resin, introduced in 1951 (M2) and improved in 1954 (M3) by Moore and Stein, first made possible the precise quantitative analysis of amino acids liberated in the course of acid hydrolysis of urine. Similar results were also obtained by Muting in 1954 (M4), who used paper chromatography methods. In this procedure amino acids were quantitatively determined after staining on the paper and elution of the resulting spots. 

H Frank, GR Nicholson, E Bayer. Enantiomer labelling, a method for the quantitative analysis of amino acids. J Chromatog 167, 187, 1978. 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

Soga, T., Kakazu, Y., Robert, M., Tomita, M., andNishioka, T. (2004). Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry. Electrophoresis 25, 1964—1972. 

CR Krishnamurti, AM Heindze, G Galzy. Application of reversed-phase high-performance liquid chromatography using pre-column derivatization with o-phthalaldehyde for the quantitative analysis of amino acids in adult and fetal sheep plasma, animal feeds and tissues. J Chromatogr 315 321-331, 1984. 

Amino acids react with ninhydrin to give a violet dye. This reaction is useful for detection and quantitative analysis of amino acids. 

Jungas RL, Halperin ML, Brosnan JT. Quantitative analysis of amino acid oxidation and related gluconeogenesis in humans. Physiol Rev 72 419-448, 1992. 

Another widely used method for qualitative and quantitative analysis of amino acid mixtures is high-performance liquid chromatography (HPLC) (see Experiments 2 and 6). The mixture of amino acids is first subjected to reaction with phenylisothiocyanate (PITC) to convert them to the phenylthiocarbamyl-amino acid derivatives, which are then subjected to chromatographic separation. The derivativatization of the amino acids serves two purposes it attaches a UV-absorbing tag, which makes their quantitative determination easy, and it converts them to a more hydrophobic form, which is necessary for good separation on the reverse-phase system commonly used with this technique. This method of amino acid analysis will be used in Experiment 6. 

The present chromatographic methods for the quantitative analysis of amino acids in protein hydrolyzates are sufficiently precise that the methods employed for preparation of hydrolyzates often limit the accuracy of the analytical methods (Hill et al., 1959). The experience gained from several studies indicates that total acid hydrolysis can best be achieved by treatment of a protein or peptide for 24 hr with 6 N HCl at 110°C, under conditions that rigidly exclude oxygen, nonprotein substances, and metals. Conditions which meet these requirements have been described by Moore and Stein (1963). Present evidence suggests that the composition of acid hydrolyzates prepared in this manner correctly reflects the amounts of most... 

This topic is included in order to contribute brief details of the principles involved in the quantitative analysis of amino acids and of some oligopeptides in aqueous solutions. The simple equipment needed so that an electrical response owing to the presence of these compounds will be generated and measured is based on particular enzyme-catalysed reactions. These create net changes of electrical potential generated in various ways from the reaction products that are created and the magnitude of the electrical response is proportional to the concentration of the amino acid or peptide. 

Research in New York. In 1954 he was promoted to a professorship therein. Stein and Moore developed methods for the quantitative analysis of amino acids, their automation, and the application in peptide and protein chemistry. 

Recently, m our laboratories, an improved method for quantitative analysis of amino acids has been developed (Yeung, Baker, and Courts, manuscript in preparation). The carboxylic acid moiety IS derivahzed quantitatively by adding one drop of concentrated HCl to an isobutanol solution of the amino acids, rather than bubbling HCl gas in the conventional manner, prior to heating. Benzoyl chloride is known to be more reactive chemically than acid anhydrides, and pentafluorobenzoyl derivatives are highly sensitive to electron-capture detection (Moffat et al., 1972, Matin and Rowland, 1972, McCallum and Armstrong, 1973 Midha et al., 1979 Cristofoli et al, 1982, Nazarali et al., 1983). Pentafluorobenzoyl chloride (PFBC), therefore, was chosen to derivatize the other reactive groups of the amino acid isobutyl esters. Pentafluorobenzoylation (Fig. 7) was conducted at room temperature in aqueous conditions. 

However, although it is possible to detect very low concentrations of dansyl amino acids fluorimetrically, use of the dansyl reaction for the quantitative analysis of amino acids has proven difficult in the past. Many factors influence the yield of the dansylation reaction In addition, side reactions are known to occur. During dansylation, a second molecule of dansyl chloride may combine with the acidic group of the ammo acid and the product then readily hydrolyzes in the alkaline conditions of the reaction medium (Boulton, 1968) This situation is potentially even more complex when mixtures of ammo acids are to be derivatized. In such cases, the yield of the dansylation reaction for each ammo acid constituent of the mixture must be estimated. This has required, in the past, time-consuming parallel experiments involving dansylation of standard ammo acid mixtures... 

Boulton A. A. and Bush I E. (1964) Qualitative and quantitative analysis of amino acids and their dansyl derivatives Biochem J. 92, UP. 

This reaction is commonly used in both qualitative and quantitative analysis of amino acids. Nineteen of the 20 protein-derived a-amino acids have primary amino groups and give the same purple-colored ninhydrin-derived anion. Proline, a secondary amine, gives a different, orange-colored compound. 

The first step in determination of primary structure is hydrolysis and quantitative analysis of amino acid composition by ion-exchange chromatography. 

Difficulties of this sort have been reported, e.g., in the extraction of phenols which had been converted to azo dyes on the adsorbent layer [623]. The reaction of sulphonamides with a diazonium reagent has been taken to completion by adding excess reagent again after having extracted the coloured product [69]. The determination of monosaccharides with benzidine-glacial acetic acid has been carried out similarly [38]. The method has been used also for the quantitative analysis of amino acids, after colour reaction with ninhydrin [42, 95] corticosteroids, after reaction yielding formazans [64] and of cannabinols after conversion into azo compounds [370]. 

Studies by Heathcote (1979) indicate that quantitative densitometric TLC analyses of amino acids will provide results equivalent to that obtained from automated amino acid analysis procedures. In a study by Mack et al. (1979) on amino acids in mosquitoes, qualitative TLC procedures were first used to determine amino acid profiles in the tissue and hemolymph, and, subsequently, the automated amino acid analyzer was applied for quantification. The study of Mack et al. (1979) should be useful to those interested in qualitative and quantitative analysis of amino acids from biological samples. 

Ninhydrin (Section 23.3) The hydrate of indan-l,2,3-trione, a molecule that reacts with amino acids to give the purple dye used in quantitative analysis of amino acids. 

Quantitative analysis of amino acids can be performed by an amino acid analyzer with ion-exchange column, a HPLC system with a reverse phase column, or gas chromatography. The amino acid analyzer has been the most popular method in clinical laboratories. Most of the normal and pathological values in the literature were obtained by this technique. Analysis by HPLC is now being used by an increasing number of laboratories. 

Much of the remarkable progress in protein chemistry over the past years has been due to the development of accurate and convenient methods for the qualitative and quantitative analysis of amino acids. The most common procedure for amino acid analysis of a protein involves acid hydrolysis in the absence of oxygen, resolution of the amino acids by ion-exchange chromatography, and colorimetric estimation with ninhydrin (369). 

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