CAPILLARY ELECTROPHORESIS OF WHEAT GLIADINS

Identification of wheat varieties by their gliadin spectrum with acidic polyacrylamide gel electrophoresis has been in use for decades. Since the early 70s electrophoresis has been one of the most frequently used analytical methods for separation and characterisation of gluten proteins. Electrophoresis separates the analytes according to the difference in their charge distribution and/or Stoke's radii. Capillary electrophoresis (CE), the miniaturised instrumental version of electrophoresis, uses similar separation principle as the traditional technique, with several advantages. These are: high electric field, thus fast separation; low sample amount (nl); low buffer consumption (5 ml/day) thus low running costs; oncolumn detection, thus quantitative analysis; use of aqueous buffers thus no environmental wastes. Due to the several advantages capillary electrophoresis is gaining popularity in a number of fields, as opposed to the standard electrophoretic techniques. In our study a capillary zone electrophoretic method has been developed to separate the gliadin fraction of wheat proteins. The effect of the buffer composition on the resolution of the separation is shown. Various wheat types have been analysed for their gliadin spectra using both the traditional and the capillary electrophoretic method. Comparing the gliadin spectra obtained by means of the two methods, capillary electrophoresis seems to be a suitable alternative to the traditional method for identification / quality control of wheat species according to their gliadin spectra

APPLICABILITY OF CAPILLARY ELECTROPHORESIS IN PROTEIN SEPARATIONS WITH REGARD TO WHEAT AND WHEAT ANALOGOUS PROTEINS

High-performance capillary electrophoresis (HPCE), among other analytical techniques (e.g. acid polyacrylamide gel electrophoresis, sodium dodecyl sulphate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography) [1]-[4] is more and more widely used also in the field of separation of cereal proteins [5]-[18]. HPCE is versatile, easy to automate, does not need toxic reagents or long analysis time, but requires only small sample size, small amount of buffer and provides high-resolution separations. So this analytical technique is particularly applicable for studying the fine structure of the composition of wheat proteins. Capillary electrophoresis has been used at our department for the determination of the fine structure of different wheat protein fractions [19]. Differences between the various wheat cultivars and changes during grain maturation process have also been studied using a home-built capillary electrophoretic system [20]. The same system has been used to investigate the electrophoretic properties of a gliadin analogous protein (BM180 - a basement membrane protein with a potential autoantigen role) [21]. The more strictly controllable nature of an automated system (especially the temperature control of the capillary) allows the use of higher voltages, so separation time decreases and resolution increases. Purchasing an automated capillary electrophoretic system we gained an opportunity to compare the two electrophoretic systems also in the field of wheat protein analyses. The aim of present work was to give an impression of the work done by capillary electrophoresis at our department

Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2μm particles

A new size exclusion chromatography column packed with 1.7 µm particles articles and possessing 200 ˚A pore size has been critically evaluated for the determination of proteins and monoclonal antibody aggregates. In a first instance, the kinetic performance of this column was compared with that of a conventional column packed with 5 µm particles and with a recently launched 3 µm material, also possessing 200 ˚A pore size. In average, 2–5 times lower plate height were achieved on the 1.7 µm packing, compared with the conventional 5 µm particles. It was also demonstrated that elevated mobile phase temperature (up to 50 or 60 ◦C) allows improving the kinetic efficiency by 20-40% in size exclusion chromatography, compared to 30 ◦C. On the other hand, the new 3 µm material performed only slightly lower kinetic efficiency than the 1.7 µm one. When considering the upper pressure and temperature limits of these three columns, the 1.7 µm column systematically outperforms the 5 and 3 µm materials in the “practical” plate number range (N < 30,000) and analysis times could be cut by 2–4 times. The column packed with 5 µm particles was only beneficial for plate counts beyond 100,000 plates, while the 3 µm packing could be considered as a good compromise between speed, efficiency and pressure. Besides the excellent kinetic performance of 1.7 µm size exclusion material under high temperature conditions, some artifacts were observed when quantifying protein aggregates. Indeed, both high pressure observed with 1.7 µm particles (shear forces, frictional heating) and elevated temperature produce some non negligible amount of on-column additional protein aggregates