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Title: RAPID AND SENSITIVE WHEAT PROTEIN FRACTIONATION AND VARIETAL IDENTIFICATIONBY NARROW-BORE REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Author
item Huebner, Floyd
item Bietz, Jerold

Submitted to: Cereal Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/23/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: In recent years it has become possible to identify wheat varieties based on their proteins. One method, high-performance liquid chromatography (HPLC), has become especially useful for this purpose. It can, however, take more than 1 hour to analyze a sample. We now find that new smaller HPLC columns are up to six times faster than ones used previously, and that they still give excellent results. Much less expensive solvent is used, and less waste for disposal is produced. Also, a small part of a kernel can be analyzed, so breeders can accurately identify lines used to develop new varieties. Kernels that have been tested can be planted and grow new plants. These rapid tests may also make it possible to identify specific varieties during marketing. This could aid wheat's utilization, while increasing returns to producers.

Technical Abstract: For wheat varietal identification using reversed-phase high-performance liquid chromatography (RP-HPLC), gliadins are typically extracted from 50- 60 mg flour, and 10-15 mul aliquots are analyzed on 15-25 cm x 4.6 mm i.d. columns at 1.0 ml/min during ca. 1 hour. A gradient of increasing acetonitrile concentration, in the presence of trifluoroacetic acid, is commonly used to elute proteins. Such solvents are expensive, however, an 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 varieties was investigated. Using a 3.2 mm i.d. 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 flour or rground endosperm. With a 2.1 mm i.d. x 15 cm, further reductions of flow rates and analysis time (to ca. 1/4 that of a standard column) are possible, while maintaining excellent resolution. Under these conditions, solvent use is about 1/8 that of standard RP-HPLC analyses. For specific limited variety identification even quicker analyses can be performed, with greater savings of time and solvents. 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.