Quantitative Determination of Gluten Protein Types in Wheat Flour by Reversed‐Phase High‐Performance Liquid Chromatography

Abstract

A combined extraction‐HPLC procedure was developed on a microscale to determine the amounts of the different gluten protein types (ω5‐, ω1,2‐, α‐ and γ‐gliadins; high molecular weight [HMW] and low molecular weight [LMW] glutenin subunits) in wheat flour. After preextraction of albumins and globulins from flour (100 mg) with a salt solution (2 × 1.0 mL), extraction of gliadins was achieved with 60% aqueous ethanol (3 × 0.5 mL). Subsequently, the glutenin subunits were extracted under nitrogen and at 60°C with 50% aqueous 1‐propanol containing Tris‐HCl (0.05 mol/L, pH 7.5), urea (2 mol/L) and dithioerythritol (1%). The separation and quantitative determination of gliadins and glutenin subunits was then performed by reversed‐phase HPLC on C8 silica gel at 50°C using a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. The flow rate was 1.0 mL/min, and the detection wavelength was 210 nm. Temperature and flow rate were modified for the quantitation of single underivatized HMW subunits. To determine the absolute amounts of protein types, different protein standards (gliadin, LMW and HMW subunits, bovine serum albumin) with known protein contents were compared to HPLC absorbance areas. The calibration curves were almost identical and linear over a broad range (20–220 μg). This extraction‐HPLC procedure allows an accurate, reproducible, sensitive, and relatively fast quantitative determination of all gluten protein types in wheat flour, and can be applied to quality evaluation of cereals as raw materials or in processed products.

The influence of Fusarium infection and growing location on the quantitative protein composition of (Part II) naked barley (Hordeum vulgare nudum)

“…1), 150 lL of sample solution were injected and separated by using the following gradient: 0 min, 20% B; 20 min, 60% B; 21 min, 90% B, 26 min, 90% B; 37 min 20% B; whereas for prolamins and glutelins ( Fig. 1), 50 and 100 lL were injected, respectively, and the separation was performed by applying the following gradient: 0 min, 24% B; 50 min, 56% B; 51 min, 90% B, 56 min, 90% B; 67 min 24% B [24]. The external PWG (Prolamin Working Group) gliadin standard was used for the quantification of the protein fractions [25].…”

The influence of Fusarium infection and growing location on the quantitative protein composition of (part I) emmer (Triticum dicoccum)

“…The Xow rate was 1 ml/min with the column temperature maintained at 50°C. For the detection of albumins/globulins, 150 l of sample solution was injected and separated using the following gradient: 0 min, 20% B; 20 min, 60% B; whereas for the gliadins and glutenins, 50 l and 100 l were injected, respectively, and the separation was performed by applying the following gradient: 0 min, 24% B; 50 min, 56% B [19]. For the quantiWcation of the protein fractions the external PWG (Prolamin Working Group) gliadin standard was used [20] (Fig.…”

Effects of sulphur fertilizer on glutenin macropolymer content and particle size distribution in wheat grain

“…A Waters 1525 Binary HPLC pump + 2998 diode-array detector with Nucleosil 300-5 C8 column (4.6 mm × 250 mm) was used according to Wieser et al (1998).…”