First the solution of 1 mL of extract was added to deionizer water (10 mL), then Folin–Ciocalteu phenol reagents (1.0 mL) added to the mixture. The mixture was left for 5 minutes, and solution of 20% sodium carbonate (2.0 mL) was added to the mixture. This mixture diluted until 50 mL with deionizer water. And after that kept in total darkness room for 1 hour. The mixture absorbance was measured at 750 nm using a spectrophotometer.
This solution was prepared in a 250-mL volumetric flask using distilled water. The solution was made using the following method: The volumetric flask was approximately filled 75% full with distilled water through the funnel. To the volumetric flask, sodium hydroxide pellets were added. The cap/stopper was put on the mouth of the volumetric flask and the solution was gently shaken. Once the sodium hydroxide pellets had dissolved in the solution the flask was again filled with distilled water till the 250-mL mark was reached.
Apparatus- Chromatography column: C18 (10 microns particle size), with Guard column Flow rate: 1.2ml/min Pressure: 30-40kgf Wavelength: 326nm Mobile phase: methanol : water (95:5 v/v) Internal standard: retinyl acetate Injection volume: 20µl Procedure for Retinol extraction from serum samples- 1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins. 2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec. 3) Centrifuged at 2500 rpm for 12 mins.
The resin was washed well with DMF and DCM until the resin appeared colourless. The strongly red-coloured filtrate was concentrated down as much as possible and then purified by RP-HPLC using a ?? column. Retention time ~ 20 min. The column was eluted with a gradient of 40-70% acetonitrile-water containing 0.1% TFA, over 50 min at 10 ml/min.
Subsequently extracted by Microwave at power level 70 for 16 minutes and then filtered. The filtrate obtained, was added 10% HCl (until pH 2-3). Then do the bleaching with NaOCl diluted with water 1: 1 to white. Then converted to sodium alginate by adding 20 g of Na2CO3 and stirred in a mixer. The resulting solution is then etched with ethanol to form sodium alginate fibers.
Then, the flask is put on shaker table and mixed at 150 rounds per minute before allowing them to settle for 10 minutes. After settling, the water sample is poured from side spout which is connected to the bottom of the flask. Some researchers reported better reproducibility with modified flask where stopcock is installed at the bottom of the flask, instead of side spout to pour the water sample (Blondina, et al., 1997) (Sorial, et al., 2004a) (Sorial, et al., 2004b). The dispersed oil in the removed water sample is extracted into methylene chloride for further analysis. Then, the oil concentration is evaluated using 340, 370 and 400 nm light absorbance (Environmental Protection Agency,
Step-III: Synthesis of Cr(II) Complexes: The Schiff's base complexes were synthesized by mixing the Schiff's base (1.5 g) in ethanolic solution of Chromium chloride [CrCl2]. This reaction is refluxed in a waterbath for two hours and their volume were reduced to 70% of it’s original volume and residue was obtained. The coloured product obtained was filtered under suction, washed with ethanol. The product were recrystallized from ethanol. Their yields ranges from 50-55%, the product obtained were light green colour and melting point was 2100C.
Stored in light glass bottle. 2.5.2: Formation of PANI(CoFe2O4) 0.1g of Nano material and 50ml of Aniline hydrochloride were mixed in a beaker. Then 50ml of Ammonium peroxydisulphate was added drop wise in reaction mixture with constant stirring below 20 oC. After 24 hours the coated sample was filtered, washed and dried at 60 oC in oven and then grinded into a fine powder in agate
This diluted solution will be used in the assay as duplicate samples. Then, 1.0mL of standard glycine solution containing (7.5mg/mL) was diluted to 100mL with water using a volumetric flask. This solution contains 1.0µmole/mL of glycine. 8 tubes were set up according to the following protocol and 2.0mL of ninhydrin reagent was then added to each of the 8 tubes and were placed in a boiling water bath for 20 minutes. After 20 minutes, the tubes from the bath was carefully removed, cooled in a beaker of cold water, then 8.0mL of 50% ethanol was added and mixed well.