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.
In the round-bottom flask (100 mL), we placed p-aminobenzoic acid (1.2 g) and ethanol (12 mL). We swirled the mixture until the solid dissolved completely. We used Pasteur pipet to add concentrated sulfuric acid (1.0 mL) to the flask. We added boiling stone and assembled the reflux. Then, we did reflux for 75 minutes.
In 10 g dried sediment sample added 7 ml 0.2 M NH4Cl solution. A mixture of 100 ml hexane: acetone (1:1) was used as a solvent to extract pesticides with overnight shaking for 12 h on reciprocal or wrist action shaker at 180 rpm. The extract was carefully decanted through activated florisil column (2-3 cm), giving twice wash with25 ml hexane: acetone (1:1) to the sediments. The elute was then washed with 200 ml water and then again aqueous layer was extracted with 50 ml hexane. Finally the hexane layer was washed with 100 ml water and then evaporated to dryness with a vacuum rotary evaporator.
The precipitate was dialyzed and used for enzyme purification using column chromatography. The dialyzed sample was loaded on DEAE-cellulose column, which was equilibrated previously with the buffer A (50 mM sodium phosphate buffer, pH 7.4). The column was washed with the five bed volumes of buffer A, and the enzyme was eluted with buffer A
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).
The above solution converts of colorless to yellow suspension solution which produced TiO2 nanopowder by drying process at 85°C in anstove for 15 hours. Finally, TiO2 nanopowder obtained were treated in furnace at different temperatures (400°C-800°C) for 2 hours. The initial heating rate was maintained at 5 °C/min. 2.3. Characterization 2.3.1 UV- Visible diffuse reflectance spectroscopy (DRS)
Absorption was read by spectrophotometer at wave length (510CHOL) and (505TG). 6. After this we applied the following equation Result = ×CON. standard Extraction of tissue The extract one gram of fat tissue using n hexane, resolve to dissolve tissue homogenizer homogeneity by adding 1 ml of the same solution to become a 3: 1 and continued homogeneity of the sample to become a solution homogeneity.
The supernatant was then used as enzyme extract. • Hydrogen peroxide (H2O2, 2mM) in phosphate buffer (3.0ml) was taken in an experimental cuvette, followed by the rapid addition of 40μl of enzyme extract and mixed thoroughly. • The time required for a decrease in absorbance by 0.05 units was recorded at 240nm in a spectrophotometer (Genesys 10-S, USA). • The enzyme solution containing H2O2-free phosphate buffer
Part 2: Preparing the Acetic acid Wash the 100mL-graduated cylinder and dry it with paper towels. Then get the Acetic acid and pour exactly 100mL of it into the graduated cylinder (This should almost fill it up). When that is completed repeat steps 1 and 2 four more times, which would mean five graduated cylinders of 100mL of Acetic acid.
Total essential oils analysis method is conducted based on Ali  with modification. 0,1 gr of kaffir lime oil nanocapsule is weighed and diluted to 100 ml using aquadest, taken as much as 1 ml (100x dilution) to put in the reaction tube then 1ml of saturated NaCO3 solution is added to the test tube and incubated for 10 min at room temperature. Then 0.5 ml of the folinciocalteu (Chemix CV, Yogyakarta) reagent and 7.5 ml aquadest were added, the mixture homogenized using vortex and then incubated for 30 min at room temperature under dark environmental conditions. Absorbance of the sample was then measured using a UV-vis spectrophotometer at a wavelength of 770 nm. The total phenol content of the sample was interpreted to be equivalent to gallic acid based on the standard curve of obtained gallic
The silver ion TLC was prepared through the following procedure: Silver nitrate was dissolved in 10 ml of distilled water. This aqueous solution of silver nitrate was absolutely mixed with 9 g of silica gel (10 ~ 40 μm particles). Then, a 10 × 5 cm TLC plate was coated with the above slurry and activated for 1 h at 90 °C before use. They were immediately transferred into a desiccator in dark for storage after cooling. 32 100 μL of afore-prepared sample solution and the mixed reference standard were diluted 100 times with ethyl acetate.
Then carefully measure 25ml of methanol and 25ml of ethyl acetate using a measuring beaker 12. Pour each solvent into its respective labelled mortar 13. Stir each sample for 10 minutes using a stirring rod 14. Leave solutions in the sun for 12 hours, this is to allow for the active ingredients bond with the solvent and form a solution and to allow some of the Methanol and Ethyl acetate to evaporate. 15.