Separation Of Phenylthiohydantoin-amino Acids Research Articles

Separation of phenylthiohydantoin-amino acids by fast protein liquid chromatography

An optimization strategy for the separation of 22 phenylthiohydantoin (PTH)-amino acids is described. The separation parameters systematically investigated were the ionic strength, pH, type of organic solvent, column temperature, gradient shape and addition of different ion-pairing agents. Two different separation systems for two different column packing materials, PepRPC™ and ProRPC™, were developed. Reproducibility and detection limits are reported.

Separation of phenylthiohydantoin-amino acids by high-performance liquid chromatography and some applications in dansyl Edman sequence analysis

Separation of phenylthiohydantoin-amino acids by high-performance liquid chromatography and some applications in dansyl Edman sequence analysis

Electrokinetic chromatography with micellar solutions : Separation of phenylthiohydantoin-amino acids

A mixture of 22 phenylthiohydantoin-amino acids was separated by electrokinetic chromatography with a sodium dodecyl sulphate (SDS) solution and a 650 × 0.05 mm I.D. fused-silica tube. This chromatography is based on micellar solubilization and electrokinetic migration in an open-tubular capillary. The retention times of the solutes could be controlled by changing the SDS concentration, the applied voltage or the length of the separation tube. Dodecyltrimethylammonium bromide (DTAB) solutions were also employed. The separation characteristics with SDS solutions were completely different from those with DTAB solutions. The resolutions were better with the former than with the latter solution, but for some solutes the latter gave better results.

The Use of Large Volume Injections for the Isocratic Separation of Phenylthiohydantoin Amino Acids by Microbore Liquid Chromatography

Abstract The use of microbore columns in high performance liquid chromatography (HPLC) has remained limited primarily due to difficulties in adapting conventional HPLC analysis to the smaller volumes and lower flow rates of microbore chromatography while maintaining high quality chromatographic results. Although conventional HPLC techniques are the method of choice for PTH-amino acid analysis, these techniques are rapidly approaching their useful limits in the field of microsequencing. A suitable method for the isocratic room temperature separation of PTH-amino acids employing microbore liquid chromatography is discussed herein. As described, the system employed for this type of analysis makes it readily adaptable to the field of protein sequencing. A comparison of a large volume mobile phase injection and a large volume “non-eluting” solvent injection with microbore columns is also presented.

Examination of theoretical principles of gradient elution as applied to reversed-phase high-performance liquid chromatography separation of phenylthiohydantoin amino acids

More than twenty different phenylthiohydantoin (PTH)-derivatized amino acids were used in the examination of the relevance and the applicability of theoretical principles that have been postulated earlier to describe gradient elution in high-performance liquid chromatography (HPLC). Reversed-phase HPLC conditions representative of PTH-amino acid assays, i.e. chemically modified silica microspheres in conjunction with an aqueous-organic mobile phase, were utilized. As predicted by theory, inevitable compromises between resolution and retention time were observed empirically. However, an a priori assumption of equivalent linear solvent strength conditions was shown to be inappropriate for selected PTH-amino acid solutes. These findings were supported by the experimentally determined variability among the different solutes of solvent strength values, S, i.e. the slope of the plots of the logarithm of isocratic capacity factor versus the fraction of organic modifier.

Separation of phenylthiohydantoin-amino acids by overpressured-layer chromatography

Separation of phenylthiohydantoin-amino acids by overpressured-layer chromatography

Optimization of complex separations in high-performance liquid chromatography : Application to phenylthiohydantoin amino acids

Graphical methods are described for determining suitable experimental conditions for optimum chromatographic resolution of complex mixtures. Plotting the logarithm of retention time for individual components or retention time ratios for close eluite pairs as a function of an experimental variable such as solvent strength or temperature allows direct visual evaluation of the best resolution attainable with a given column of known efficiency. Optimization of temperature and solvent strength for the separation of phenylthiohydantoin amino acids is demonstrated for two reversed-phase columns.

On the analysis of phenylthiohydantoin amino acids by high performance liquid chromatography

The key experimental variables and the corresponding separations of synthetic mixtures of phenylthiohydantoin amino acids achieved by high performance liquid chromatography have been abstracted from the literature and are listed in tabular form. From this table it is seen that the large majority of separations reported since 1977 have been performed utilizing stainless steel tubing of large internal diameter (i.d.≥3.9 mm). The reason for the widespread adoption of this particular practice is that a number of earlier studies have indicated that use of small diameter columns leads to significant loss of chromatographic resolution. An experimental study, employing two commercial chomatographic systems and tubings of differing internal diameter (i.d. 4.5 to 1.5 mm), leads to the conclusion that columns of reverse phase support material packed in stainless steel micro-bore tubing (i.d.≤2 mm) can effect separation of phenylthiohydantoin amino acids with similar efficiency as larger diameter columns. The practical advantages of employing micro-bore columns in general are discussed and adoption of micro-bore columns for the routine separation of this particularly important group of amino acid derivatives is recommended.

Separation of phenylthiohydantoin-amino acids by high-performance liquid chromatography

Separation of phenylthiohydantoin-amino acids by high-performance liquid chromatography

Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography for sequence determination of radiolabeled proteins

Published procedures for the separation of ethyl acetate-extractable phenylthiohydantoin-amino acids by high-pressure liquid ehromatography with organic solvent gradients have been modified to facilitate sequence determination of proteins labeled with several radioactive amino acids at a time.

Separation of phenylthiohydantoin-amino acids by highpressure liquid chromatography

A method for the separation of all phenylthiohydantoin (PTH)-amino acids except PTH-arginine and PTH-histidine by high-pressure liquid chromatography on a silica column is described. Elution is performed with a concave solvent gradient from hexane—methanol—propanol (3980:9:11) to methanol—propanol (9:11). A complete run is achieved in 40 rain with a pressure drop of 1000 p.s.i. over the 250 mm × 2.1 mm column. Eluted peaks of 2–5 nmole are easily detected by their ultraviolet absorption at 254 nm. This method is superior to existing gas—liquid and thin-layer chromatographic techniques since all PTH-amino acids except PTH-arginine and PTH-histidine may be both separated and quantitated in a single run of 40 min. The use of the technique in conjunction with an automated peptide sequence analyser is illustrated.