Standardization of NaOH solution The prepared solution in part A was used to determine the acidity of the two different brands of soft drinks. But before it, the NaOH solution was standardized first. A 0.15 g of potassium acid phthalate was dissolved in 0.05 L of water in an Erlenmeyer flask. Afterwards, 3 drops of phenolphthalein was added. A 50 mL buret was obtained and was washed with NaOH solution.
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.
One millilitreer of the lichen extract (1 mg/mL) in a volumetric flask was diluted with distilled water (46 mL), and the content was mixed in a volumetric flask after adding. oOne millilitreer of Folin-Ciocalteu reagent was added and the content of the flask was mixed. thoroughly . After 3 min, 3 mL of 2% sodium carbonate (2 %) was added and then was allowed to standleft for 2 h with intermittent shaking. The reaction absorbance of prepared mixture absorbance was measured at 760 nm in a spectrophotometer (Jenway UK).
(ii) Acidify to methyl orange end point with concentrated H2SO4. (iii) Add 5 ml concentrated HNO3. (iv) Add 2 ml 30% H2O2 . (v) Evaporate on a hot plate to 15 to 20 ml. (vi) Transfer the concentrate and any precipitate to a 250 ml conical flask using 5 ml concentrated HNO3.
Characterization of Crude Extract 3.1 Physical Test 3.1.1 Organoleptic Test The color, odor and physical state of extract will be determined. 3.1.2 Solubility Test The solubility test for extract will be performed using distilled water, 80% alcohol, chloroform, hexane and ether. 3.2 Chemical Tests The crude extract will be dissolved in 20mL of 80% ethyl alcohol. It will be filtered and divided into 5 test tubes and will be labeled as A, B, C, D and E. The test tube A will be kept blank. 3.2.1 Ferric Chloride Test The sample extraction will be dissolved in 2 ml ethanol.
Absorbance of the solution was measured at 238 nm. Drug content was calculated by the formula absorbance/ slope* dilution factor. Drug loading Microsphers equivalent to 100 mg of DLTZH/ND taken and washed with 3 x 10 ml of methanol, which removes the free unloaded drug. Filtrate was diluted suitably to Beer’s concentration range and free drug concentration was determined spectrophotometrically at 238 nm and 236 nm for DLTZH and ND respectively. Further microspheres were crushed, dissolved in methanol and made up to 100 ml, which was further diluted suitably to Beer’s rangeand drug concentration was determined.
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.
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.
1.25mL of acetic acid and ferric chloride mixture was added to 2.5mL of the pure extract. Occurrence of blue-green color indicates the presence of glycosides. Test for Saponins. 10mL of distilled water was added to 5 mL of the pure extract the mixture was shaken in a graduated cylinder for 15minutes. Occurrence of layer of foam or bubbles indicates presence of saponins.
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. The absorbance of each tube was determined at 570nm and the absorbance were plotted against amount of standard glycine. For determination of tyrosine content using Folin & Ciocalteu’s reagent, seven tubes were set up. Then, 1.6mL of 0.5M Na2CO3 was added to each of the tubes and were mixed thoroughly. 0.4mL of the Folin & Ciocalteu’s reagent then were pipette in, mixed thoroughly, and allowed to stand for 10 minutes.