2. FORMATION OF HYDRAZONE FROM ESTER Materials required: * The ester which was synthesized in the previous reaction. The total weight of ester obtained was 230mg. * Methanol – 20 ml * P-toluene sulfonylhydrazide (1.2 equivalent ) Procedure: * The ester was transferred in a round bottom flask and it was mixed with about 20ml of methanol and stirring was done on a magnetic stirrer till the ester dissolves in it completely. * In the above RB, a calculated amount of 1.2 equivalent amount of PTSH was added during continous stirring.
200 µl of lysis buffer (2 % Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl), 1mM EDTA, pH 8.0 and 0.2 g of glass beads were added to each Eppendorf tube. Then 200 µl of the solution phenol/chloroform/isoamyl alcohol (25:24:1) was added to the tubes under the fume hood and tubes were placed on rotator and left to mix for 3 min. 200 µl of TE buffer was added and spun for 5 min at maximum speed, the water phase was transferred to new tubes. 1 ml of cold 96 % ethanol was added, mixed and then spun for 5 min at maximum speed at 4°C. the supernatant was discarded and the pellet re-suspended in 400 µl of TE buffer (40 mM Tris-Base, 20 mM acetic acid, 1 mM EDTA, pH 8.0).
We added sodium carbonate until the pH of the mixture was 8. After neutralize, we collected benzocaine by vacuum filtration. We used a Buchner funnel to collect benzocaine. We used three 10 ml of water to wash the product. After the product was dry, we weighed, calculate the percent yield and determined the melting point of the product.
A sample of fish oil (50 mg) was transferred into screw-cap vial. 2 ml benzene and 10 ml sulfuric acid (1%) in absolute methanol were added. The vial was covered under a stream of nitrogen before heating in an oven 90 °C for 90 minutes. Ten ml of distilled water were added to the cooled vial and the methyl esters in each vial were extracted with 5 ml of petroleum ether for three times. The three petroleum ether extracts were combined and concentrate to its minimum volume by using a stream of nitrogen.
The volumetric flask was then filled up to its 100 mL mark with deionized water. The buret was washed out with dionized water and then with the strong base NaOH before being filled up with NaOH. About 20 mL of the unknown weak acid was pipetted into a beaker. The starting volume of the NaOH in the buret was recorded before about 4 mL of the strong base was titrated into the weak acid solution. The final volume was recorded.
The aqueous extract was prepared by dissolving 1g of dry extract with 20 ml of sterilized distilled water, so the final concentration of extract would be 0.05 g/ml, from this solution other concentration were prepared (0.1-0.2) g/ml. the solutions were shaken for 30 min. The extract was centrifuged (30,000 rpm; 15 min) and the supernatant was Separated. To hydroalcoholic extract, 80 g of the powder was extracted with aqueous methanol (75%). The other two concentrations were prepared from soaking sixfold aqueous methanol (75%) with different amounts of powder.
Add 160µL of double distilled water maintained at the same temperature to the transparent solutions formed, these upon cooling change to yellow translucent liquid/gel or white creamy proliposomal gel. Proliposomal gel formulations with positive and negative charge were prepared in above mentioned manner by adding 10 mol% of total lipid of stearylamine
5-aminotetrazole monohydrate: In a 250 ml round-bottom flask equipped with a condenser for refluxing (90 °C) and a magnetic stirring bar, 5.00 g (5.95 mmol) dicyandiamide (three times crystallized), 7.47 g (11.9 mmol) sodium azide and 11.00 g (17.8 mmol) boric acid and 100 ml of water is added and allowed to reflux for 24 hours, after the completion of the reaction, until the solution pH to about 2 to 3 as hydrochloric acid 37% is added (about 12 ml) Then the reaction mixture was cooled in a refrigerator for 18 hours and the white crystals formed. The mixture was filtered and washed three times with 10 ml of water and and dried in 60 °C for 5 hours and finally 45.8 g of product by it will be obtained. 5-Aminotetrazol monohydrate: Yield:,
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.
In a 217 total volume of 1.5 mL, the reaction mixture contained 1 mL of the eluate, 400 mL of 218 12.5 mM 3-(dimethylamino) benzoic acid in 0.375 M phosphate buffer (pH 6.5), 80 mL 219 of 3-methyl-2-benzothiazoline hydrazone and 20 mL of peroxidase (0.25 unit). The 220 reaction was started by the addition of peroxidase and the increase in absorbance was