Cereal Chem 65:192-198 | VIEW
The Effect of Injection Volume on the Quantitative Analysis of Wheat Storage Proteins by Reversed-Phase High-Performance Liquid Chromatography.
B. A. Marchylo and J. E. Kruger. Copyright 1988 by the American Association of Cereal Chemists, Inc.
The relationship between injection volume and the binding of gliadins and glutenins to reversed-phase high- performance liquid chromatography (RP-HPLC) columns was studied. Significant quantitative changes were observed, concomitant with increasing injection volumb (5-80 microliters), in the amount of protein and in relative proportions of protein peaks eluted in chromatograms. Rapid decreases in the recovery of proteins, in particular the least hydrophobic gliadins, were observed as injection volume increased. Analysis of solvent peaks by RP-HPLC and sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis indicated that as injection volume increased, progressively more gliadin and glutenin proteins did not bind to the column upon injection and were eluted in the solvent peak (i.e., void volume). The effects of column temperature, extraction solvent, and type of column on this phenomenon were examined. A remedy to the problem was devised entailing the use of mutliple 5-microliter injections of protein extract prior to initiation of the gradient program. Virtually 100% of proteins were bound to the RP-HPLC column using this technique.