Amino acids chromatographic methods

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

The 2-D TLC was successfully applied to the separation of amino acids as early as the beginning of thin-layer chromatography. Separation efficiency is, by far, best with chloroform-methanol-17% ammonium hydroxide (40 40 20, v/v), n-butanol-glacial acetic acid-water (80 20 20, v/v) in combination with phenol-water (75 25, g/g). A novel 2-D TLC method has been elaborated and found suitable for the chromatographic identification of 52 amino acids. This method is based on three 2-D TLC developments on cellulose (CMN 300 50 p) using the same solvent system 1 for the first dimension and three different systems (11-IV) of suitable properties for the second dimension. System 1 n-butanol-acetone -diethylamine-water (10 10 2 5, v/v) system 11 2-propanol-formic acid-water (40 2 10, v/v) system 111 iec-butanol-methyl ethyl ketone-dicyclohexylamine-water (10 10 2 5, v/v) and system IV phenol-water (75 25, g/g) (h- 7.5 mg Na-cyanide) with 3% ammonia. With this technique, all amino acids can be differentiated and characterized by their fixed positions and also by some color reactions. Moreover, the relative merits of cellulose and silica gel are discussed in relation to separation efficiency, reproducibility, and detection sensitivity. Two-dimensional TLC separation of a performic acid oxidized mixture of 20 protein amino acids plus p-alanine and y-amino-n-butyric acid was performed in the first direction with chloroform-methanol-ammonia (17%) (40 40 20, v/v) and in the second direction with phenol-water (75 25, g/g). Detection was performed via ninhydrin reagent spray. 

The techniques presented below are restricted to determinations of carbohydrates and amino acids by methods that have been found to be reproducible and suitable for use in field work. While the less specific techniques generally do not require sophisticated equipment and hence are adapted easily to shipboard work, chromatographic techniques are, from an instrumentation point of view, more complex. However, modem instruments are usually rugged enough to withstand conditions at sea, at least for some time. 

For resolution of amino acids, the method of ligand-exchange chromatography has received by far the most emphasis. The utility of this chromatographic technique in areas other than the resolution of race-mates has been reviewed (Davankov and Semechkin, 1977). Only results pertinent to the use of this chromatographic method in the resolution of racemates will be discussed here. 

Phosphorus and Silicon in Waters, Effluents and Sludges [e.g. Phosphorus in Waters, Effluents and Sludges by Spectrophotometry-phosphomolybdenum blue method. Phosphorus in Waters and Acidic Digests by Spectrophotometry-phosphovanadomolybdate method. Ion Chromatographic Methods for the Determination of Phosphorus Compound, Pretreatment Methods for Phosphorus Determinations, Determination of silicon by Spectrophotometric Determination of Molybdate Reactive Silicon-1 -amino-2-naphthol-4, sulphonic acid (ANSA) or Metol reduction methods or ascorbic acid reduction method. Pretreatment Methods to Convert Other Eorms of Silicon to Soluble Molybdate Reactive Silicon, Determination of Phosphorus and Silicon Emission Spectrophotometry], 1992... 

One method that combines the good chromatographic properties with improved limit of detection is the separation of isoindole derivatives of amino acids that may be detected fluorimetrically. This method may be applied to protein hydrolysates, and used in automated format in routine analyses [22]. 

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. 

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-... 

Many are distinguished from Hb-A by electrophoretic or chromatographic methods The number and types of abnormal hemoglobins that have been discovered thus far are Indeed overwhelming At the latest count (December, 1974) at least 135 3-chaln variants, 72 a-chaln variants, 8 6-chaln variants, and 11 y-chaln variants have been found These Include variants with single amino acid substitutions (the majority), variants with two substitutions (the 6-chaln variants, Hb-C-Harlem and Hb-Arllng-ton Park), variants with deletion of one or more residues... 

Fou, M. F., A split stream ion exchange chromatographic method for isolating amino acids and peptides, Anal. Biochem., 55, 51, 1973. 

Mass spectrometry (MS) is also being used to add another dimension of analysis to achiral-chiral analysis. Recently, an achiral-chiral column-switching LC/LC-MS/MS method was reported for the pindolol enantiomers in human serum (Motoyama et al., 2002) and phenprocoumon metabolites (Kammerer et al., 1998). For analytes that have very poor chromophores or cannot naturally fluoresce, MS detection can be more sensitive for the underivatized form of the analyte. Also, MS detection can be particularly useful when very similar analytes that differ in mass (such as some amino acids and metabolites) cannot be satisfactorily separated chromatographically,... 

A GC analysis of amino acids requires a derivatisation step to increase the volatility of the amino acids. Generally, norleucine and/or norvaline are the internal standards added to the hydrolysate to check the derivatisation yield. According to the experimental method applied, the limits of detection (LOD) vary in the range 10 100 pg for each amino acid. Regarding the chromatographic columns, as most of the derivatives are esters barely polar compounds the most commonly used are fused-silica capillary columns with a low... 

Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side chains of the amino acids tyrosine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their content of tyrosine and tryptophan vary to any significant extent. Hence, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process. 

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