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:,
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.
5 mL of 3M sodium hydroxide, 5 mL of de-ionized water, and 15 mL of hexane were added to the reaction flask and stirred. The mixture was transferred to a separatory funnel, separated into an organic layer and water layer, and then drained. The water layer was washed twice with 10 mL of hexane. The organic layer was dried
Shake funnel and rinse off water layer ( This contains the sulfuric acid and majority of methanol). Again wash the ether with 25ml of water and then wash the organic layer with 25ml of 10% sodium bicarbonate to extract unreacted benzoic acid. Again shake separatory funnel with frequent venting of pressure and opening the stopcock. Allow the separation of layers and drain off bicarbonate layer into a beaker. Wash ether layer with saturated sodium chloride solution and retain ether layer.
The crude tetraphenylnaphthalene in a 25-ml Erlenmeyer flask and dissolved in boiling isopropyl alcohol (12 ml). The solution was cooled to room temperature and further cooled in an ice bath for 30 minutes. Crystallization of colorless crystals occurred. The product was collect in a Hirsch funnel and washed with isopropyl alcohol. The solid was left to dry over the weekend.
After the evaluation of stomach for ulcers, the gastric mucosa of glandular portion was scrapped with the help of two glass slides, weighed (100 mg) and homogenized in 1 mL of a 0.15 M, ice cold potassium chloride (KCl) solution and centrifuged at 3,000 RPM for 10 minutes (REMI centrifuge). 1 mL of suspension was taken from the above tissue homogenate in test tube and 0.5 mL of 30% w/v TCA (trichloroacetic acid) was added to it, followed by 0.5 mL of 0.8% w/v TBA (thiobarbituric acid) reagent. The tubes were then covered with aluminium foil and kept in water bath for 30 minutes at 80 °C. After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes and centrifuged at 3000 rpm for 15 minutes (R-BC DX REMI centrifuge). The absorbance of the supernatant was read in spectrophotometer (UV-1601, SHIMADZU) at 540 nm against blank.
2.4.1. Tetramethyl glucose acetylation 1gm of tetramethyl glucose was dissolved in 5ml of acetic anhydride and added to fused sodium acetate of 0.375gm and mixed for 10 minutes and allowed to cool. To this mixture 7.5ml of toluene and 5ml of dry ether were added. The whole mixture evaporated to syrup on a water bath at 50 °C. The product dissolved in the dry ether after washing with toluene.
The washings are filtered off into a 100 mL measuring flask; after cooling distilled water is added until the mark is reached (stock solution). In proportion to the expected chloride content aliquot part of this solution, which should preferably contain 50 mg – 100 mg NaCl, taken off, distilled water being added to obtain a quantity of approximately 100 mL. Subsequently 5 mL ferric alum solution, 20 mL 0.1 N AgNO3 solution and 5 mL – 10 mL ether or 1 mL nitrobenzene are added; titration is carried out by means of an ammonium thiocyanate solution 0.1 N, until the red colouring remains after
Crystal violet was then added for 60 seconds before being washed off with water. The mordant, Gram’s Iodine, was added for another 60 seconds before getting washed off with water. The heat fixed smear was then washed with 95% alcohol until the wash ran clear, leading to the final step of adding Safranin for 45 seconds before being rinsed with water. The slide was finally blot dyed with bibulous paper before it was placed under a microscope to observe the color and shape of the bacterium. 2.2 Litmus Milk Reaction A milk-based, litmus broth tube is incubated and observed after 48 hours.
The natural agar consist of Yeast Extract, Tryptone , Lactose , Manniiol, Sodium Chloride , Dip otassium Hydrogen Phosphate ,Gelatin and Agar .Then , in a conical flask 14g of nutrient agar was mixed into 500 ml of distilled water and , The mixture was stirred and dissolved until most of the agar dissolve. By non-absorbent cotton wool plug the mouth of the flask was closed. By using the autoclave the agar was sterilized for 15 minutes.The cotton wool was removed. By the flame the mouth of the flask was heated before and after pouring the agar into the Petri dishes. And, the left hand the lid of the Petri dish was lift, just enough to enter the mouth of the flask and quickly was poured in agar (about 15 cm3).
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.
Two chemical reactions are carried by adding sodium hydroxide to the acidic solution from Part I. During the first reaction is the neutralization of the excess of nitric acid in the mixture by sodium hydroxide. The second reaction takes the place after naturalization is a complete and NaOH is in excess. While the liquid inside the beaker is being stirred, with the stirring rod, 10 ml of 6 M NaOH is poured into the solution from Part I at 1 mL at a time. After each 1 mL the solution is tested for acidity with red litmus paper.