Pre Column Research Articles

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry.

An on-line method has been developed for the derivatization and coupled liquid chromatography (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments.

High Performance Liquid Chromatographic Determination of Free Amino Acids in Shrimp

Abstract A reverse phase high performance liquid chromatographic method for the analysis of total free amino acids in shrimp, utilizing pre column fluorescence derivatization is described. The primary amino acids were treated with o-phthalaldehyde (OPA). The reaction products were separated on a Microsorb Short-ones 3-μm reversed-phase column with gradient elution development. 15-OPA amino acids were separated in 21 min. Secondary amino acids were reacted with 4-chloro-7-nitrobenzofurazan (NBD). The separation was carried out on a Lichrosorb RP-C18, 5μm column. Wild and cultured Mexican shrimp species (Penaeus vannamei) were analyzed. Free amino acid contents of glycine, alanine and proline were higher than those of other amino acids. Total Free amino acid content was significantly higher in cultured than wild shrimp. The OPA-retention time could be measured within ±0.1 relative standard deviation and the relative peak areas, based on the internal standard calculation methodology, were within ±3% or less....

Stereoisomeric Flavor Compounds XXXVII: Enantiomer Separation of l-Alken-3-yl Esters and their Chirality Evaluation from Essential Oils Using Multidimensional Gas Chromatography (MDGC)

ABSTRACT Heptakis (3-0-acetyl-2, 6-di-0-pentyl)-β-cyclodextrin (Lipodex D), and nickel (II)-bis [3-heptafluorobutanoyl-(lR)-camphorate] respectively are proved to be powerful tools to differentiate chiral l-alken-3-yl esters. From commercially available lavender oils l-octen-3-yl acetate and linalyl acetate are directly stereoanalyzed by multidimensional gas chromatography (MDGC) employing heart-cutting techniques from DB-1701 as the pre column onto the chiral main column. Optical purity control of linalyl acetate from some lavender oils is discussed as an indicator of their natural origin.

High performance liquid chromatography of plasma aminoacids using orthophthalaldehyde derivatisation

A modified high performance liquid chromatography method for measuring plasma aminoacids using pre column orthophthaladehyde derivatisation is described. Protein-free samples were obtained by ultrafiltration. The method has been used to determine reference values in fasting and fed normal individuals.