Pepsin (1 wt. % of the estimated collagen) was added to the supernatant, stirred for another 2 days. Soluble collagen was purified by a repeated process of salting-out with 1 M NaCl, condensation by centrifugation at 14000 ×g for 20 minutes and subsequent solubilization in 0.5 M acetic acid. The collagen solution thus obtained was dialyzed in dialysis tubes (VISKING dialysis tubing, MWCO 12 000 - 14 000 RC, diameter 21 mm) against sterile 0.1 M acetic acid and sterile distilled water, respectively and
The mixture was shaken using shaking incubator at 200 rpm for 120 min at 5000C. The mixture was then centrifuged at 3000 rpm for 15 min at room temperature and the supernatant was taken. This supernatant was stored at -2000C until further analysis. Preparation of watermelon extract Ten grams (10 g) of dehulled powdered sample were mixed with 150 ml of respective solvents, aqueous methanol (50%) and methanol and placed on water bath at 40 °C for 18 h with stirring/shaking. The extracts were filtered and concentrated by rotary evaporator.
Dialysis membrane having a pore size 2.4 nm and molecular weight cut off between 12,000 and 14,000 was used. The membrane was soaked in double distilled water for 12 hr. before mounting in a Franz diffusion cell. About 1ml of semisolid preparation of NLC was applied to the donor compartment, and the receptor compartment was filled with phosphate buffer, pH 7.4 (14 ml). During the experiment, the solution in the receptor side was maintained at 370C and stirred at 800 rpm with Teflon coated magnetic stirrer bar.
Determination of fatty acids composition: Lipid Extraction of fatty acids 10g sample was weight out in conical, then 10 ml of conc hydrochloric acid was added and immersed in boiling water until the sample was dissolved. At this stage, the mixture should be brown or violet in colour and the fat will be collected on the surface. Conical was cooled and the fat was extracted by shaking with 30 ml of diethyl ether and the extract was blowed, after allowing the layers to separate in to a weighed flask. Extraction process was repeated three times more and the solvent was distilled then the fat was dried at 100ºC, cooled and weighed . Fatty acids methylation 50 mg of lipid was weighed in a glass tube.
33) A sixty-minute timer was started to see how the 9 Urea (mM) solvent diffuses through the 100 (MWCO) Dialysis Membrane. 34) The outcome is cataloged. 35) The 100 (MWCO) Dialysis Membrane was removed from between the beakers and put back into the original membrane container. 36) The left and right beaker are emptied and clean to begin the next trial. 37) The 200 (MWCO) Dialysis Membrane was placed between the left and the right beaker.
The making of the Si standard solution done by dissolving 0.1 grams of The metal in solution 20 ml HCl 37% and added aquades to the volume of 100 ml of The solution thus obtained 1000 ppm. The solution is diluted so that its concentration is obtained several times with variations 0.2; 0.4; 0.8; 1.2; 1.6 and 2.0
A sample was withdrawn at appropriate times, weighed and dissolved in methanol to get solution of 1000 µg/ml. 6 µl of the resulting solution was applied to HPTLC. . RESULTS AND DISCUSSION Optimization of chromatographic conditions The primary objective in developing this stability indicating HPTLC method is to achieve the resolution of Boceprevir and its degradation products. The chromatographic separation was achieved by linear ascending development in 10 cm × 10 cm twin trough glass chamber using Ethyl Acetate: Toluene (6:4, v/v) as mobile phase and detection was carried out at 215 nm.
The pH was adjusted to 9.5 with 2M NaOH. 20 g of NaOCl (in which 4% active chlorine) was added drop-wise to the slurry over a period of 30 min, while maintaining pH range 9.0-9.5, with constant stirring 30±2°C. The reaction proceeded for 10 min after addition of NaOCl. After the reaction, the pH was adjusted to 7 with 1M H₂SO₄ and the oxidized starch was filtered, washed four times with distilled water by using centrifuge and dried in oven at 30±2.0°C for 48h. Dried starch was powdered with morter and pestle and passed through 200 mm sieve and the powdered oxidized starch was kept in a polythene bag.
Add 100 mL of distilled water to each beaker in the ratio of 1:0.4 (fish: water) and mix the mixture well with the aid of magnetic stirrer. After that, measure the pH value of the mixture using a pH meter and adjust it to pH 6 by adding 1 N hydrochloric acid, HCl. Cover all beakers, then place them in a water bath shaker operating at 30°C with 140 rpm and keep for 30 minutes. The temperature is measured by using a thermometer. The enzymatic hydrolysis is started by adding 0.5% (by weight of raw material) Sea-B Zyme L200 to each beaker.
Then, the solution was loaded into the well. The gel then was run at 100 volt for 30 minutes. After that, the agar was cleaned and put into the EtBr solution for 10 minutes. The agar then washed by using tap water. Next, the agar was observed under UV transilluminator to see the formation of band.