Amino photometric determination

Moore S and Stein WH (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J Biol Chem 211, 907-913. 

Among the analytical methods presently used for the characterization of natural and synthetic peptides and proteins, the primary value of amino acid analysis is the determination of absolute peptide and protein content in solids and solutions and the quantitation of their amino acid composition and stoichiometry. It involves two steps, i.e. complete hydrolysis of peptides and proteins, followed by photometric determination of the released amino adds. The steps are laborious and time-consuming, and there is a continuous need for improvement of the techniques to increase precision and sensitivity. 

The pKd of 2-amino-4-methyl-6//-l,3-thiazine-6-one has been spectro-photometrically determined and is high (13.30) similar to that of disodium sulfide (80CCC732). 

In the photometric determination of copper, a coupling product formed between the diazonium salt from 2-amino-pyridine and resorcinol, or 4-(2-pyridinylazo)-l,3-benzenediol 21, has been used. Here the formed copper complex under acetate buffer exhibits an absorption peak at 520 nm, which is measured photometrically <2003KPU28>. Similarly for photometric determination of iron(ll), a coupling product formed between the diazonium salt of 2-amino-4,6-dihydroxypyrimidine and 8-hydroxyquinoline, or 6-hydroxy-2-(8-hydroxy-7-quinolinyl)azo-4(l//)-pyrimidinone 22, has been used. This reagent forms a blue complex with iron(n) ions with an absorption maximum at 625 nm that does not interfere with the presence of other metals <2003KD95>. 

The common disadvantage of crown ethers as extractants of amino compounds is the interference from hard metal ions, such as alkali and alkaline earth cations. This may cause problems in extraction-based analytical and technological applications, as these metals, particularly sodium and potassium, are often present in (bio)media of interest. For illustration. Fig. 1 shows the influence of alkali metals on the extraction-photometric determination of benzylamine with DC18C6 and picrate [56,70]. 

Horseradish peroxidase was also modified with activated PEG2 [25]. PEG-peroxi-dase, in which 60% of the amino groups were coupled with PEG, had 70% of the original enzymic activity in aqueous solution and was found to be active in benzene. Using PEG-peroxidase and PEG-cholesterol oxidase, cholesterol could be photometrically determined in transparent benzene solution by measuring the absorbance increase at 490 nm [37] as shown by the following equations ... 

Gravimetric, photometric, chromatographic, enzymatic, and microbiological methods for the determination of amino acids are reviewed and discussed. Marked advances have been made during the present decade in methods applicable to the determination of amino acids, and with the development of new analytical methods it should soon be possible to determine all the amino acids of biological importance with a degree of accuracy sufficient for practical as well as many theoretical purposes. 

Table IV. Photometric Methods First Used to Determine Amino Acids...

The percentages of amino acids in silk fibroin which Poison et al. (224) found by direct visual and indirect photometric analysis of ninhydrin paper-partition chromatograms are shown in Table VII. The percentages obtained for alanine, glycine, and serine appear to be reasonably accurate, inasmuch as they agree closely with those found by other methods. It would be of interest to determine alanine by the microbiological method reported recently by Sauberlich and Baumann (238), in view of the widely different values found for this amino acid by the described ninhydrin-chromatographic procedure and the selec-... 

Kataoka, H., Nagao, K., Nabeshima, N., Kiyama, M., and Makita, M., Selective determination of secondary amino acids as their A-dimethylthiophosphoryl methyl ester derivatives by gas chromatography with flame photometric detection, J. Chromatogr., 626, 239-243, 1992. 

There are several recorded determinations of the absorption curves of the aromatic amino-acids. Most of these were obtained with photographic methods of spectrophotometry which have been superceded by more accurate photoelectric methods. It will be shown that in the spectrophotometric analysis of tyrosine and tryptophan in proteins, the photometric error is magnified in the final estimate of tyrosine and tryptophan contents. This fact is inevitably bound up with the form of the equations of mixture analysis. It is therefore important that the absorption constants be measured as accurately as possible. 

The present authors with co-workers studied extraction with readily available DC18C6 (common mixture of isomers) and picrate [56,69,70]. Extraction efficacy for primary amines was found to follow the expected series methyl < /-butyl < benzyl < octyl < nonylamine. Secondary and tertiary amines are recovered with crown ethers much less efficiently, as are amines with hydrophilic groups (e.g., noradrenaline). Among the chlorinated hydrocarbons studied as diluents, tetrachloroethane appeared the best [71] (Table 2). Tetrachloroethane is known to be the best in some other systems probably, its bulky molecules could not effectively solvate the host, which makes complexation with the guest easier [72]. We also developed an extraction-photometric method for the determination of benzylamine (1 — 10 jug/mL, relative standard deviation 5%) in the presence of less hydro-phobic primary amines, secondary/tertiary amines, and amino acids. 

The colorimetric methods recommended for the determination of total fusel oil contents in beverages are based on a color reaction. In this reaction, 2-methyl-l-propanol, 2-methyl-l-butanol, and 3-methyl-l-butanol lose water during heating in a strongly acidic solution and the unsaturated hydrocarbons formed yield colored complexes with vanillin, salicylaldehyde, and 4-dimethyl-amino-benzaldehyde. The absorbance is measured spectro-photometrically at 445 and 560 nm. 

Primary amines and amino acids selectively react with carbonyl compounds such as benzaldehyde, furfural, and pentafluorobenzaldehyde in aqueous medium at room temperature, and give Shiff s bases in good yields. This reaction is used for the determination of aliphatic diamines and aromatic amines by GC with a flame photometric detector or an electron capture detector. However, a cleanup step for sample preparation is required to remove the excess reagent. 

Gas chromatography Acesulfame-K, aspartame, cyclamate, saccharin, and stevioside are determined by gas chromatography, but the main drawback of this technique is that a derivatization is required. Acesulfame-K is methylated with ethereal diazomethane, aspartame is converted into its N- 2-methylpropoxycarbonyl) methyl ester derivative, menthol and isobutyl chloroformate are used to convert aspartame to 3-[(isobutoxycarbonyl)amino]-4-[[a-(methoxycarbonyl)phenethyl]amino]-4-oxobutyric acid, cyclamate is determined as cyclohexene resulting from the reaction with nitrite, saccharin is converted to N-methylsaccharin, and stevioside is hydrolyzed. Detection is carried out utilizing flame-ionization, flame-photometric electron-capture detectors or nitrogen-phosphorus detection. 

Squibb [154, 155] has separated the free amino acids in bird liver extracts, using silica gel G layers on plastic sheets. The coloured reaction products with ninhydrin were evaluated photometrically at 525 nm, directly on the layer. Lysine, histidine, arginine, asparagine, alanine, valine and leucine were determined in this way. Recoveries lay between 90 and 106%. 

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