Multiwave 3000

Grains of barley (Hordeum vulgare L. cv. Golden Promise) were harvested from greenhouse-grown plants cultivated in a mixture of soil and sand supplemented with 0.15 kg m⁻³ Osmocote plus (Scotts Company, UK). The grains were rapidly rinsed three times with milli-Q water to remove surface contaminants and thereafter freeze dried. A subsample of 50 g, i.e. >1000 grains, was pulverized in a titanium mill and used as whole grain control material. For determination of total elemental concentrations in different grain tissues, 35 randomly selected grains were divided into five batches and their hull and embryo removed manually by use of a teflon-coated scalpel. Thereafter, each batch of seven grains was polished by high-speed shaking at 30 Hz in a ball mill (MM301, Retsch, Germany) mounted with a rack containing microcentrifuge tubes loaded with 200 mg of acid-washed quartz sand (Fluka 84878, 40–150 mesh SiO₂). Before use, the sand had been additionally purified with 5% HNO₃ and dried. Polishing was performed in six repeated cycles each of 150 s duration. Between each cycle, the remaining weight of the seven grains was recorded and they were moved to new tubes. In parallel, the mixture of abraded material and sand was weighed. This procedure allowed collection of six fractions of abraded material consisting of different proportions of pericarp, testa, aleurone, and endosperm in mixture with sand. After the final cycle of polishing, the rest of the grain, representing the core endosperm, was rapidly washed three times with milli-Q water in order to remove surface dust. Following drying, all samples, together with control samples of pure sand and pulverized whole grain, were digested for 80 min at 140 °C in a closed microscaled microwave digestion system (Multiwave 3000, Anton Paar GmbH, Graz, Austria) equipped with a 64 position rotor (64MG5, Anton Paar GmbH, Graz, Austria). The digestion medium consisted of a mixture of 125 μl of 30% H₂O₂ and 250 μl of 65% HNO₃. All digests were diluted to 3.5% HNO₃ and analysed by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7500ce, Agilent Technologies, UK). The results were validated using certified reference material NIST 8436 (durum wheat) and true blanks. Results for pure sand were subtracted from those for the abraded samples. Five independent replicates were analysed for each of the nine grain fractions (hull, embryo, pure endosperm, and six samples representing a gradient across the testa–aleurone–endosperm interface). The satisfactory micronutrient recoveries shown in Table 1 indicate that neither significant micronutrient contamination nor losses occurred during the process, in agreement with previous validations (Hansen et al., 2009 (link)).