The next step of phospholipid extracted 2 weeks ago is wet digestion with sulfuric acid. Firstly, wet digestion tubes were prepared and then soaked in solution of nitric acid overnight. After that, they were washed with deionized and milli-Q water respectively. When the tubes were dry, they were transferred to oven overnight. Secondly, sample solution was prepared by adding phospholipid from extraction process in to the tube. Solvent mixtures, chloroform and methanol, were also added to phospholipid tubes. Blank tubes were prepared to compare with previous samples, and then transferred sample and blank tubes into heat block at 100 degree Celsius. After 30 minute added sulfuric acid (H2SO4) and covered with grass ball at the same temperature about 1 hour and then adjust the temperature at 200 degree Celsius about 3 hour. In tubes we could see black sediment by observation. We then dropped hydrogen peroxide (H2O2) and transferred to in heat block about 15 minute. After 15 minutes in heat block the result would show light yellow to clear solution and then added H2O2 again and continuously heat about 2 hour. Tubes then were taken out from heat block and placed at room temperature. After digestion, we added milli-Q water with shaking and collected samples into vial.
|Heat block for wet digestion with sulfuric acid.|
Standard solution was prepared by using various concentration values such as 1.0, 0.5, 0.25, 0.125 and 0 ppm of phosphorus solution. H2SO4 is solvent dilution in serial dilution. Samples from wet digestion were used and then added milli-Q water for 10 time dilution. After 10 minutes, we added 1.75%(w/v) ammonium molybdate-4H20 and wait for 30 minutes. Lastly, we added 0.035%(w/v) malachite green solution containing 0.35% polyvinyl alcohol.
Standards and samples were measured absorbance at 610 nm by spectrophotometer. In the result, root of barley has more phospholipid concentration than rice. This is consistent with hypothesis of experimentation, Barley is aluminium(Al) sensitive plant have higher phospholipid proportion than rice in the plasma membrane.
|Comparison between rice (yellow) and barley (green) root for phospholipid concentration.|