Quantitation and Reproducibility Problems in Reversed-Phase and Size-Exclusion HPLC Analyses of Wheat Proteins!

Cereal Chern. 76(2):299-302 Reversed-phase (RP-) and size-exclusion (SE-) high-performance liquid chromatography have become important methods for rapid identification of wheat and other cereal cultivars and for revealing quality differences. Accuracy and reproducibility are essential for good results. Due to recent changes in these methods, however, such as using smaller columns, lower flow rates, and smaller samples, small procedural errors become more critical for final results. We therefore further studied the causes and magnitude of problems involving quantitation and reproducibility in RPFractionation of cereal proteins by high-performance liquid chromatography is still a relatively young method. The first practical separations of wheat and maize proteins by reversed-phase (RP-) and size-exclusion (SE-) HPLC were reported in 1983-1984 (Bietz 1983, 1984). Improvements in columns and techniques came rapidly, as noted in the first book devoted to HPLC of cereal and legume proteins (Kruger and Bietz 1994). Because of its improved reproducibility and speed, and the ability to accurately quantify results, especially as compared to gel electrophoresis, HPLC quickly became one of the most important methods used by many researchers to analyze cereal proteins. In spite of these advances, some problems remain with these methods. For example, during our analysis of a large sample set for which quantitative data were of utmost importance, breakdowns necessitated reanalyzing many samples. The second sets of results often differed significantly from the first. Analytical SEand ionexchange HPLC confirmed this reproducibility problem. We also became aware of a study (Dolan 1996) that showed that proteins could adsorb to HPLC injector seals or other system components, affecting quantitative precision. We have thus further investigated the causes of this irreproducibility. We here show that various factors (many of which have not previously been well recognized) can cause problems in quantitative results from HPLC of cereal proteins.