Detection of amino acids

The post-column derivatization with ninhydrin introduced by Spackman, Stein, and Moore [13] represents even today the most customary detection method for quantitative 

An aldehyde shorter by one C-atom and hydrindantin, the reduced form of ninhydrin, are formed in this reaction. Hydrindantin reacts with ammonia and a second molecule of ninhydrin to form a red dye called Ruhemann s purple. This dye has an absorption maximum at 570 run (see Fig. 4-25). 

Secondary amino acids, imino acids , such as proline and hydroxyproline, do not possess an a-amino group, and react with ninhydrin to form a yellow product which is usually detected at 440 nm. Therefore, amino acid analyzers are equipped with a photometer capable of measuring at two different wavelengths (570 nm and 440 nm). The sensitivity of this detection method is about 200 pmol. 

The subsequent fluorescence detection is carried out at an excitation wavelength of 340 nm, the emission is measured at 455 nm. The addition of thiols such as 2-mercaptoetha-nol increases the fluorescence yield. However, OPA only reacts with primary amino acids. Secondary amino acids can only be detected after their oxidation (for example with hypochlorite or chloramine T) [34]. In practical applications, this results in significant difficulties. 

An interesting derivatization method for cysteine was recently described by Jenke and Brown [35], They mixed the column effluent with a buffered solution of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), yielding a strongly yellow-colored chromophore, which can be detected photometrically at 412 nm  

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The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

Differences in the materials employed for the layers can also become evident when chemical reactions are performed on them. Thus, Macherey-Nagel report that the detection of amino acids and peptides by reaction with ninhydrin is less sensitive on layers containing luminescent or phosphorescent indicators compared to adsorbents which do not contain any indicator [7]. 

Progress has been made in developing electrochemical methods for detection of amino acids without derivatization.74 75 Evaporative light scattering (ELSD) is also a promising detection method.76 Flow-injection analysis... 

Clarke, A. R, Jandlik, P., Rocklin, R. D., Liu, Y., and Avdalovic, N., An integrated amperometry waveform for the direct, sensitive detection of amino acids and amino sugars following anion-exchange chromatography, Anal. Chem., 71, 2774,1999. 

Watanabe Y, Imai K (1981) High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole. Anal Biochem 116 471—472... 

Hu S, Li PCH (2000) Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,l,3-benzoxadiazole. J Chromatogr A 876 183-191... 

In the context of the discovery of amines and oxygen-containing organic compounds, the question arises as to whether the presence of atomic carbon in olivine or MgO crystals could have led to the formation of amino acids. Knobel et al. (1984) reported the detection of amino acids in liquid extracts of the reaction mixtures at the 1983 ISSOL conference yields were, however, extremely low, the total yield being 1.5-3.0 x 10-7 g per gram of MgO. These results were the subject of considerable attention in the media. 

Proteins are detected and localized by ToF-SIMS on account of the detection of amino acid fragments, as described previously. A similar approach is used for polysaccharide residues. Fragments at m/z 45, 59, 71 and 99 are used for their identification and localization (Figure 15.13). The attribution of these small fragments to polysaccharides is ensured... 

Femtomol levels of detection limits were also achieved in the determination of stimulant amines with the benzofurazan derivative 4-(Af,Af-dimethylaminosul-phonyl)-7-fluoro-2,l,3-benzoxadizole (DBD-F) [73], DBD-F was successfully applied to the PO-CL detection of amino acids and epinephrine [74] and a P-blocker, metoprolol [75], 4-(Af,Af-Dimethylaminosulphonyl)-7-hydrazino-2,l,3-benzoxadizole (DBD-H) has also been used for PO-CL determination of a neuronal cell protective compound, propentofylline. The method was applied for the first time to determine propentofylline concentration in the dialysate obtained from the rat hippocampus [85],... 

Table 10.8 Reagents for the colorimetric detection of amino acids... 

Carbon paste and graphite epoxy electrodes modified with RUO2 can be used for detection of amino acids and peptides in F1A systems. Optimal conditions are in strongly alkaline solutions at +0.45 V vs Ag/AgCl electrode, with a fast and linear response. Carbon paste electrodes can be modified also with C03O4371. Colorimetric methods for the determination of amino groups attached to a solid support may give erroneous values... 

A simple and efficient alternative to the traditional UV detection of amino acids and related compounds is nowadays represented by the evaporative light scattering (ELS) detector, which allows the direct chromatographic separation, with no need for preliminary derivatization. In the field of glycopeptides-based CSPs, it was applied for the first time in the chromatographic resolution of carnitine and 0-acylcarnitine enantiomers on a TE CSP [61]. The considered compounds are nonvolatile solids and gave optimal ELS response under a variety of experimental conditions (buffered and unbuffered mobile phases, flow-rates from 0.5 to 1.5 mL/min, different kind and... 

Several kit solutions for indirect detection of amino acids and carbohydrates and fluorescence detection of amino acids after derivatization. 

The electrophoresis is run at 1000 V cvfor 30-45 min. After finishing, the cellulose sheet is dried in a stream of warm air. For visual detection of amino acids the cellulose sheet is sprayed with Soln. B and incubated in a drying oven at 110-120 °C until blue spots appear. Autoradiography or a Phospholmager detects P-labled amino acids. 

In many kinds of research it is important to have simple and sensitive means for analysis of amino acids, particularly in small quantities. Detection of amino acids can be achieved readily by the ninhydrin color test, whereby an alcoholic solution of the triketone, ninhydrin, is heated with an amino acid and produces an intense blue-violet color. The sensitivity and reliability of this test is such that 0.1 micromole of amino acid gives a color intensity reproducible to a few per cent, provided that a reducing agent such as stannous chloride is present to prevent oxidation of the colored salt by dissolved oxygen. 

Since the early detection of amino acids with ninhydrin, many derivatization procedures have investigated these solutes. 4-Dimethylaminoazobenzene-4 -sulfonyl chloride (DABS-C1) is performing well, but the best seems to be FMOC (9-fluorenyl chloroformate) (63). More generally, each class of reactions replaces the active hydrogens of the OH, NH, and SH groups. 

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. 

Refractive-index detection is seldom used for many of the same reasons just mentioned for the spectroscopic detection of amino acids in their native forms. In fact, these problems are even more severe. Refractive-index detection has almost no selectivity whatsoever. Nearly every sample component passing the detector will register a signal. Also, this makes refractive index entirely incompatible with gradient elution. Even for isocratic separation, and detection of only a select few amino acids, refractive index can be very troublesome because of the detector s tendency to drift due to temperature changes in the laboratory (perhaps newer models have fixed this problem ). Finally, detection limits tend to be very poor for refractive-index detection. 

Relative to the UV-spectroscopic and/or fluorometric determination of derivatized amino acids, electrochemical detection of amino acids seems to be more complex and problematic. Furthermore, electrochemical detection has not gained sufficient popularity to warrant an extensive examination here. For those readers specifically interested in electrochemical detection of amino acids, an excellent review article has been written by Dou et al. (143). 

OW Lau, CS Mok. Indirect conductometric detection of amino acids after liquid chromatographic separation. Part II. Determination of monosodium glutamate in foods. Anal Chim Acta 302 45 -52, 1995. 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

S. J. Setford, S.F. White and J.A. Bolbot, Measurement of protein using an electrochemical bi-enzyme sensor, Biosens. Bioelectron., 17 (2002) 79-86. P. Sarkar and A.P.F. Turner, Application of dual-step potential on single screen-printed modified carbon paste electrodes for detection of amino acids and proteins, Fresenius J. Anal. Chem., 364 (1999) 154-159. 

Amino acids (isoleucine, phenylalanine, arginine and alanine) have been analysed on a microchip with a post-channel reaction with amino acid oxidase reaction [144], Pre-channel derivatisation of amino acids with naphthalene-2,3-dicarboxyaldehyde (NDA) has been described for facilitating its amperometric detection [145]. Separation and direct detection of amino acids without derivatisation have also been achieved in microchips [89,109,122,132,146-148]. 

J. Wang, G. Chen and M. Pumera, Microchip separation and electrochemical detection of amino acids and peptides following precolumn de-rivatization with naphthalene-2,3-dicarboxyaldehyde, Electroanalysis, 15 (2003) 862-865. 

Leland and Powell also studied ECL obtained from reaction of [(bpy)3Ru]3+ with trialkylamines [47], Since the mechanism involves an electron transfer from the amine to Ru3+, there exists an inverse relationship between the first ionization potential of the amine and ECL intensity. The relative intensity of [(bpy)3Ru]2+ ECL was found to be ordered tertiary > secondary > primary. Quaternary ammonium ions and aromatic amines do not produce ECL with Ru(II) diimine complexes. Brune and Bobbitt subsequently reported the detection of amino acids by [(bpy)3Ru]2+ ECL [28,29], Employing capillary electrophoresis for separation, the presence of various amino acids can be detected directly by reaction with [(bpy)3Ru]3+ generated in situ with up to femtomo-lar sensitivity and with a selectivity for proline and leucine over other amino acids. The formation of an amine radical cation intermediate is characteristic of proposed mechanisms of both aliphatic amines and amino acids. 

Michel, W. C., Trapido-Rosenthal, H. G., Chao, E. T., and Wachowiak, M., Stereoselective detection of amino acids by lobster olfactory receptor neurons, J. Comp. Physiol. A, 171, 705, 1993. 

Munro, N.J., Huang, Z., Finegold, D.N., Landers, J.R, Indirect fluorescence detection of amino acids on electrophoretic microchips. Anal. Chem. 2002, 72, 2765-2773. 

B. Basak, U. K. Bhattacharyya, and S. Laskar, Spray reagent for the detection of amino acids on thin layer chromatography plates, Amino Acids, 4 193(1993). 

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