Retention prediction of phenylthiohydantoin amino acid derivatives in reversed-phase liquid chromatography

Summary The concept of retention prediction for the separation of phenylthiohydatoin-amino acid derivatives in isocratic reversed-phase liquid chromatography is described. A novel retention-solubility parameter, R, is defined, for the retention prediction strategy and the performance of this R value is evaluated by comparing measured and predicted retention data. Excellent agreement between these values were observed. It is concluded that the R value has a very high potential in describing the retention of phenylthiohydantopinamino acid derivatives withdifferent types of separation systems consisting of C-18, C-8 and phenethyl bonded stationary phases and various mobile phases.     

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.     

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.     

Identification of the electrophoretic peaks of the phenylthiohydantoin derivatives of amino acids

Two techniques for identifying the peaks of phenylthiohydantoin (PTH) amino acids separated under the conditions of micellar electrokinetic chromatography were compared. The first technique is linear regression analysis, in which the retention time of an amino acid is a function of the retention times of two retention-time standards. The second technique takes into account hydrophobicity constants logD′, which were calculated using the ACD/LC Simulator 8.0 program package from ACDLabs (Canada). These constants provide an opportunity to calculate the relative migration times of PTH amino acids taking into account the velocity of the electroosmotic flow. The first technique allows us to identify the electrophoretic peaks of all 16 amino acids separated; the second procedure allows us to predict the elution order of the electrophoretic peaks; the use of a correlation equation gives better results.     

Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.     

Separation of phenylthiohydantoin amino acids by capillary electrochromatography

Capillary electrochromatography (CEC) was employed as a rapid and high-efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N-terminal protein sequencing. For this purpose, 75 microm ID fused-silica capillaries were packed with standard 3 microm Hypersil octadecyl silica (ODS) particles using a two-step column fabrication process, which represents a fast, reliable and efficient means of producing long-term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same-day and day-to-day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase-high performance liquid chromatography (RP-HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.     

Resolution of nucleic acids by high-performance liquid chromatography

High-performance liquid chromatography is a very powerful technique for the separation and isolation of nucleic acids. Nucleic acids can be generally characterized as long, negatively charged polymers with hydrophobic nucleobases. Chromatographic processes which use electrostatic interactions (anion-exchange), hydrophobic interactions (reversed-phase or salting-out) or both types of interactions (mixed-mode) are most effective for resolution of these materials.     

Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography

A high-performance liquid chromatographic method (HPLC) with combined diode array and fluorescence detection of amino acids and amines in various cheese samples is described. The proposal is based on acidic deproteinization, derivatization and gradient optimization studies, resulting in the identification and quantification of 21 amino acids and 9 amines from a single solution, by one injection. The optimized, simple protocol consists of deproteinization (1M perchloric acid), centrifugation, filtration and the subsequent derivatization with the o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate (OPA-ET-FMOC) reagent. The method can be characterized with a linearity of wide concentration range (6.25-1000pM/injection), a good chromatographic reproducibility (average: 2.69% RSD) and an excellent recovery (average: 100.2%; average 3.84% RSD). The developed method was successfully applied in the determination of the amino acid and amine contents of port salut cheese, blue cheese and smoked cheese samples.