RAPID AND SENSITIVE WHEAT-PROTEIN FRACTIONATION AND VARIETAL IDENTIFICATION BY NARROW-BORE REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

For wheat varietal identification by reversed-phase high-performance l iquid chromatography (RP-HPLC), gliadins are typically extracted from approximate to 50 mg of flour with 70% ethanol. Aliquots 10-15 mu l ar e analyzed on 15- or 25-cm x 4.6-mm (i.d.) columns at 1.0 ml/min for a pproximate to 1 hr. A gradient of increasing acetonitrile concentratio n, 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 r ates, decreased disposal problems, and lower cost per analysis. The ab ility of such columns to fractionate gliadins and to differentiate cul tivars was investigated. Using a 3.2-mm x 15-cm C-4 column, flow rate, sample size, analysis time, and solvent consumption were reduced by h alf, while preserving excellent resolution of extracted proteins. Glia dins can also be extracted from as little as 1-2 mg of flour or ground endosperm. With a 2.1-mm x 15-cm C-4 column (Vydac), further reductio ns of flow rates and analysis time (about one fourth that of a standar d 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 stand ard columns, and separations can be done in only 9-10 min. Some resolu tion is sacrificed for such rapid analyses; while this is often accept able, 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 p erform such rapid, high-resolution analyses of small samples, with low solvent consumption, suggests that narrow-bore RP-HPLC may become esp ecially useful for selection and identification during wheat breeding.