Cereal Chem 72:504-507  |  VIEW ARTICLE

Rapid and Sensitive Wheat Protein Fractionation and Varietal Identification by Narrow-Bore Reversed-Phase High-Performance Liquid Chromatography.

F. R. Huebner and J. A. Bietz. Copyright 1995 by the American Association of Cereal Chemists, Inc. 

For wheat varietal identification by reversed-phase high- performance liquid chromatography (RP-HPLC), gliadins are typically extracted from approximately 50 mg of flour with 70% ethanol. Aliquots 10-15 microliters are analyzed on 15- or 25-cm x 4.6-mm (i.d.) columns at 1.0 ml/min for approximately 1 hr. A gradient of increasing acetonitrile concentration, containing trifluoroacetic acid, is commonly used to elute proteins. However, such solvents are expensive, and present a disposal problem. Smaller RP-HPLC columns offer shorter analysis times, reduced flow rates, decreased disposal problems, and lower cost per analysis. The ability of such columns to fractionate gliadins and to differentiate cultivars was investigated. Using a 3.2-mm x 15-cm C4 column, flow rate, sample size, analysis time, and solvent consumption were reduced by half, while preserving excellent resolution of extracted proteins. Gliadins can also be extracted from as little as 1-2 mg of flour or ground endosperm. With a 2.1-mm x 15-cm C4 column (Vydac), further reductions of flow rates and analysis time (about one fourth that of a standard column) are possible, while maintaining excellent resolution. Under these conditions, solvent use is about one eighth that of standard RP-HPLC analyses. To further reduce analysis time and sample consumption, gradient times can be reduced to as little as one tenth that of standard columns, and separations can be done in only 9-10 min. Some resolution is sacrificed for such rapid analyses; while this is often acceptable, it may sometimes prevent differentiation of genetically similar cultivars that are otherwise identifiable. Similar RP-HPLC procedures can be used to analyze reduced subunits of glutenins. The ability to perform such rapid, high-resolution analyses of small samples, with low solvent consumption, suggests that narrow-bore RP-HPLC may become especially useful for selection and identification during wheat breeding.