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
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:
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.
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
Rose Bengal-(bis(aminoethyl)ethylene glycol) (2) from Rose Bengal disodium salt (1) The synthesis was done following procedure from . Rose Bengal Na+ salt (915 mg, 0.90 mmol) was dissolved in DMF (2ml) and DIPEA (0.312 ml, 1.80 mmol), HATU (308 mg, 0.81 mmol) were added. After activation for 15 min, the mixture was added to O-Bis-(aminoethyl)ethylene glycol trityl resin (309 mg, 0.31 mmol) preswollen in DMF for 2 hours. The coupling reaction wrapped in aluminum foil was allowed to proceed overnight on a nitrogen bubbler apparatus.
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 ion exchange capacity is measured by the chemical titration [Hasimi A, Stavropoulou A, Papadokostaki KG, Sanopoulou M. Euro Polym J (2008) 4098-107]. In order to measure the IEC values, the dry samples were soaked in 1M aqueous NaCl solution at room temperature for 24 hours to exchange of proton by sodium ions. The ion-exchanged NaCl solution was back titrated with 0.1M NaOH solution using phenolphthalein as an indicator. The protonic conductivity measurements were made through via Nyquist plot using N4L PSM 1735 series LCZ meter with amplitude of 10 mV from 1 Hz to 1 MHz The proton conductivity values are calculated
Two drops of iodine- potassium was then added to the mixture. The procedure was repeated with the rest of the samples and isopropyl with varying drops of sodium hydroxide. Eight drops of sodium hydroxide was added to formaldehyde, 9 drops to cyclohexanone, 15 drops to benzaldehyde, and 7 drops to isopropyl. It was observed that both cyclohexanone and benzaldehyde formed a yellow layer that lasted only for a few seconds before disappearing completely while the formaldehyde mixture produce a yellow precipitate. Both acetone and isopropyl mixtures were heated since no precipitate was observed.
Firstly, 0.3 g Fe3O4 was ultrasonically dispersed into 150 ml solvent (Vmethanol/Vwater=1/1) for 20 min to obtain a uniform suspension. A 100 ml alkaline solution (0.32 g Na2CO3, 0.48 g NaOH) was added dropwise into the suspension until pH ca. 10.0 and kept for 5 min, then another 100 ml salt solution (1.02 g Mg(NO3)2⋅6H2O and 1.2 g Al(NO3)3⋅9H2O) and above alkaline solution were simultaneously added maintaining the pH in 9.5-10.0. The resultant was stirred for 5 min followed separation by using a magnet and thoroughly washing with deionized water and alcohol and dried at 60 °C overnight giving product Fe-Mg/Al
2.9. Estimation of Hydrogen peroxide (H2O2) 10 213 The concentration of H2O2 was determined by the method of Okuda et al (38). Fresh leaf 214 sample (0.5 g) was grounded in ice-cold 200 mM HClO4 and was then centrifuged at 215 1200 g for 10 min followed by neutralization of HClO4 of the supernatant with 4M KOH. 216 The insoluble KClO4 was eliminated by further centrifugation at 500g for 3 min.