Amino acid analysis absorbance detection

The detection of M. aeruginosa toxins is readily achieved by HPLC and ultraviolet absorbance. Provisionally, components with identical retention times are considered identical. Confirmation is made by preparative isolation, amino acid analysis, and toxicity testing. The frequent, but not invariable, presence of toxin-LR as a principal toxin provides a useful HPLC marker. The wavelength used for detection of the toxins, 238 nm, is the principal absorbance maximum of toxin-LR. The 238 nm absorbance is probably... 

The visible absorption spectrum of a solution containing a known concentration of nitrated protein is measured in a solution buffered at pH 9.0, and the absorbance at the maximum (near 428 nm) used to calculate the nitrotyrosine content ( 428nm for the nitrophenoxide ion is 4200). The tyrosine and nitrotyrosine content of the modified protein should also be determined by amino acid analysis. If the sum of these values does not add up to the tyrosine content of the unmodified protein, intra- or intermolecular cross-linking may have occurred. The amino acid analysis may also reveal whether other side-reactions have taken place. Particular attention should be paid to the half-cystine, cysteine, methionine, histidine and tryptophan contents of the modified proteins. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate offers a rapid and highly sensitive way of detecting products of intermolecular cross-linking. Such products are readily removed by gel filtration. 

Fig. 11.2.9. Amino acid analysis using a conventional analyser. Chromatographic conditions colunm, Dionex DC-6A (300x4.6 mm I.D.) mobile phase, sodium citrate, three-buffer programme detection, post-column reagent ninhydrin (absorbance 1 full scale). Peaks 1, cysteic acid 2, aspartic acid 3, methionine sulphone 4, threonine 5, serine 6, glutamic acid 7, proline 8, glycine 9, alanine 10, half cystine 11, valine 12, methionine 13, isoleucine 14, leucine 15, Y-leucine 16, tyrosine 17, phenylalanine 18, ammonia 19, lysine 20, histidine 21, arginine. Reproduced from Beckman information catalogue, with permission.

The second approach involves post-column derlvatization of the amino acids, which improves the sensitivity for detection by creating either a highly fluorescent derivative or one with a strong chromo-phore. The traditional approach for amino acid analysis uses post-column derlvatization in which the reagent nlnhydrln mixes with the column effluent and reacts with the amino acids to form a derivative that can be detected by absorbance at 570 nm and 440 nm. 

Derivatization of a racemic compound with an achiral group may play an important role in the analysis of a chiral compound (Fig. 7-15). In the case of substances with low or no UV-activity, the compounds can be rendered detectable by introducing an UV-absorbing or fluorescent group. If the racemate itself shows selectivity on a chiral stationary phase (CSP), this method can be applied to reduce the limit of detection. Examples have been reported in the literature, especially for the derivatization of amino acids which are difficult to detect using UV detection. Different derivatization strategies can be applied (Fig. 7-16). 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Detection of peptides in HPLC can be achieved by measuring natural absorbance of peptide bonds at 200-220 nm. Unfortunately at these wavelengths a lot of food components and also the solvents used for analysis absorb, demanding an intensive sample pretreatment and clean-up [129]. Peptides with aromatic residues can be detected at 254 nm (phenylalanine, tyrosine, and tryptophan) or 280 nm (tyrosine and tryptophan). Taking advantage of the natural fluorescence shown by some amino acids (tyrosine and tryptophan), detection by fluorescence can also be used for peptides containing these amino acids [106]. 

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. 

The TLM detection method has been applied to detect labeled amino acids. For instance, after separation in a conventional capillary, DABSYL-labeled amino acids were detected in a Pyrex TLM detector chip. The LOD was 4.6 x l() 8 M, as compared to the LOD of 5.2 X 10-6 M obtained by the conventional absorbance method [343]. TLM has also been applied for the analysis of metal ions such as Co [319,734], Ni [735], Pb [736], Fe [734], and of organic molecules such as o-toluidine after its oxidation [737],... 

Chemiluminescence is a very sensitive and selective technique. Reagent types, analytes, and detection limits have been summarized in a review by Imai.56 Chemiluminescence has been applied to the analysis of compounds that exhibit low UV absorbance, including metal ions, amino acids, fatty acids, and bile acids. Other detectors include detectors for radioactivity, nuclear magnetic resonance (NMR), and surface-enhanced Raman spectroscopy. Radioactivity detection is one of the most selective detectors, as only components that have been radiolabeled will be detected. The interface of NMR with HPLC and has been discussed in detail by Grenier-Loustalot et al.57 Surface-enhanced Raman spectroscopy is another technique that... 

Peptides are commonly detected by absorbance at 200-220 nm. However, most of the compounds present in wine may interfere in the ultraviolet detection of peptides when low wavelengths are used. Thus, for the analysis of these compounds it is useful to apply sensitive and selective detection methods. To this end, it is possible to form derivates of the peptides that can be detected at higher and more specific wavelengths. Detection by fluorescence can also be used to detect peptides containing fluorescence amino acids (tyrosine and tryptophan). For peptides without this property, the formation of derivates with derivatizing agents have been proved to be very useful (Moreno-Arribas et al. 1998a). 

A. Routine analysis of PTH-amino acids using Micellar Electrokinetic Capillary Chromatography with Thermo-optical Absorbance Detection (MECC-TOAD)... 

Thermooptical detection has been combined with CE and used for protein and peptide Edman degradation sequencing detection, native protein detection, as well as the analysis of derivatized amino acid mixtures. " Frequency-doubled argon-ion lasers have been employed to supply an UV (257 nm) pump beam for the analysis of dansylated amino acids as well as the analysis of etopside and etopside phosphate in human blood plasma. In addition, two-color thermooptical absorbance detectors have been constructed, where each laser serves a dual role of pump and probe this system can be used to detect analytes that absorb in differing regions of the electromagnetic spectrum. 

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