Gas-liquid chromatographic separation of N-trifluoroacetyl iso-butyl esters of 20 protein amino acids on a single column

Summary A procedure is established for the separation and quantitation of 20 protein amino acids by gas-chromatography in which the N-trifluoroacetyl-iso-butyl esters of these acids were chromatographically separated on a single mixed phase column OV-17 OV-210 (2%1% on 100/120 Supelcoport). The problems faced by previous investigators for separation and analysis of histidine, arginine, tryptophane and cystine have been solved. The procedure enables rapid, accurate and sensitive amino acid analysis and might fill an important need of scientists who are faced with the problem of amino acid analysis of biologically important substances.

---

Gas-flüssig-chromatographische Trennung der N-Trifluoracetyl-isobutylester von 20 Protein-Aminosäuren auf einer einzigen Säule

Zusammenfassung Die Trennung erfolgt auf einer einzigen Mischphasensäule (OV-17OV-210; 2%1% an 100/120 Supelcoport). Die Probleme, die sich früher für die Trennung und Analyse von Histidin, Arginin, Tryptophan und Cystin ergaben, konnten dadurch überwunden werden. Das Verfahren ermöglicht eine schnelle, genaue und empfindliche Trennung und ist nützlich für die Untersuchung von Substanzen von biologischem Interesse.

     

High-performance liquid chromatography of amino acids, peptides and proteins CXXV. Molecular dynamics simulation of n-butyl chains chemically bonded to silica-based reversed-phase high-performance liquid chromatography sorbents

The molecular dynamics method has been applied to investigate the conformations of n-butyl ligands immobilised onto an amorphous silica surface analogous to those utilised with silica-based RP-HPLC sorbents. Three systems were constructed which corresponded to ligand densities of 1.64, 2.67 and 3.69 μmol/m². A number of parameters related to the structure of the sorbent materials were derived in order to characterise the molecular properties of each system. These parameters included the hydrocarbon layer thickness, the frequency of gauche conformations, the distance distribution for carbon atoms and the diffusion coefficients of individual atoms in the n-butyl ligands. From these properties, the positions of chains with respect to the surface as well as their mobility were estimated. It was found that at higher densities, the n-butyl chains are predominantly perpendicular to the surface while at low density they are highly tilted or lying almost parallel to the surface. The degree of ligand flexibility decreased with increasing surface density. Mobility of individual carbon atoms as well as chain disorder increased with distance from the surface for all ligand densities. The simulated properties of n-butyl chains immobilised to a silica surface correlated well with results obtained by Fourier transform IR and ¹³C-cross polarisation magic angle spinning NMR experimental methods and statistical predictions of the behaviour of immobilised chains.     

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters.

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.     

Gas-liquid chromatography of amino acids: Columns and methodology as a basis for routine amino acid analysis using glass capillary gas chromatography

A carefully Standardized technique is described for the preparation of glass capillary columns which can be used successfully for routine quantitative amino acid analysis. Comparison is made between two different modes of sample injection. Preliminary quantitative results from “split” injection and “on-column” injection techniques are evaluated statistically and it is concluded that the “on-column” system is a prerequisite for quantitative amino acid analysis by glass capillary gas chromatography. An analysis of fish muscle protein hydrolyzate illustrates an application of this technique and results are compared with those from a packed column analysis.     

Capillary gas chromatography of n-butyl and isobutyl-, n-amyl and isoamyl polyethylene glycol ethers and their derivatives

The influence of a branching and increase in the length of alkyl and polyoxyethylene chain in homologous series of n-butyl and isobutyl-, n-amyl and isoamylpolyethylene glycol ethers on the retention indices at linearly programmed temperatures of a capillary column was studied. Alkylpolyethylene glycol ethers were converted by derivatization reactions into acetates, trifluoroacetates, and trimethylsilyl ethers. The influence of the structure of the alkylpolyethylene glycol molecule and the influence of the functional groups introduced into a molecule of studied compounds were examined by means of increments of retention index. Calculated retention indices were used to identify residues of individual oligomers in the products of biodegradation.     

Adsorption and reversed-phase high-performance liquid chromatography of p-nitrobenzyl esters of monohydroxy fatty acids

To enhance the UV detectability of hydroxy fatty acids, p-nitrobenzyl (PNB) esters of twenty-two different monohydroxy fatty acids of various chain-lengths (C16-C22) and differing positional isomers were formed using O-(p-nitrobenzyl)-N,N-(diisopropyl)-isourea (PNBDI) as alkylating agent. Reversed-phase and adsorption high-performance liquid chromatography (HPLC) of the twenty-two monohydroxy fatty acid PNB esters were studied. The PNB group did not dominate the chromatographic properties of monohydroxy fatty acids and it did not interfere with the HPLC separation of positional isomers. PNBDI was, however, found to be less than ideal for formation of PNB derivatives of monohydroxy fatty acids because UV absorbing contaminants of PNBDI interfered with the HPLC analyses.     

Rapid determination of amino acids by high-performance liquid chromatography: release of amino acids by perfused rat liver.

Perfused rat liver can be considered as one of the most suitable ex vivo models for studies of liver metabolism. To assess the possible effect of L-carnitine and some of its acyl esters on proteolysis in the rat liver, the amino acid derivatization and high-performance liquid chromatographic separation of Tapuhi et al. [Anal. Biochem., 115 (1981) 123] was modified.     

Assessment of fatty acids in cardiac tissue as 9-anthryldiazomethane esters by high-performance liquid chromatography.

A high-performance liquid chromatographic technique for the rapid assessment fatty acids in cardiac tissue is described. A level of 50.4 +/- 14.9 nmol fatty acids per g wet weight of rat myocardial tissue could be monitored. The content of the individual fatty acids C14:0, C16:0, C16:1, C18:0, C18:1, C18:2 and C20:4 amounted to 1.9, 13.5, 0.6, 14.4, 6.1, 6.5 and 7.2 nmol/g wet weight, respectively. A comparison of this method with a well established gas chromatographic technique yielded good agreement. In contrast with time-consuming gas chromatographic techniques, there is no need to isolate (unesterified) fatty acids from the other lipid classes with column chromatography or thin-layer chromatography, because the derivatizing reagent 9-anthryldiazomethane reacts highly specifically with fatty acids.     

Properties of 2,4-dinitrophenyl derivatives of amino acids as analytical forms for high-performance liquid chromatography

Dissociation constants of 2,4-dinitrophenyl derivatives of α-amino acids in micellar solutions of sodium dodecyl sulfate used as a micellar mobile phase in reversed-phase liquid chromatography were determined. The method of micellar liquid chromatography was used to determine the composition of polypeptide fractions of animal origin.