3) Centrifuged at 2500 rpm for 12 mins. Upper hexane layer (supernatant) was transferred carefully into another test tube. 4) Evaporated the hexane under a stream of grade 1 nitrogen gas and added 100 µl of methanol to the residue left and vortexed for 1 min. 5) Injected 100 µl of extract in HPLC vials and closed properly. Standard curves and calculations- Retinol was quantified from standard curves peak area for each vitamin.
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.
Then it was left to boil under for 1hour. After which round bottom flask was removed from the reflux setup and held first under running room temperature water and then an ice bath until it cooled down enough to comfortably handle it. Next the cooled solution is poured into a 100ml volumetric flask and topped it off to the mark with denoised water. Subsequently, 20ml of this solution was pipetted into a conical flask. To this, 80ml of cold water and 15ml of 2M HCl was added to the conical flask.
The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min). It was suspended in minimal volume of double-distilled water. 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
The cooling curve was determined by recording the temperature at regular time intervals (every fifteen seconds) as the cyclohexane cooled, until the temperature became constant. The cyclohexane was stirred continuously. The FP tube was then removed from the ice and the cyclohexane melted. The cooling of the cyclohexane was then repeated twice and the mean of the three values was taken as the freezing point. ~0.1g of benzoic acid was accurately weighed and added to the cyclohexane and stirred vigorously until it had dissolved.
10- Transfer the ester layer to a small dry test tube and dry the ester with anhydrous CaCl2 and stir for 10 min. 11- put it in a preweighed dry round bottom flask . 14- Determine the yield, refractive index, and % yield of ester. Conditions :- 1) This reaction is catalyzed by acid, Like Fischer esterification. 2) Usage of water in step (5):So that after Estrification is completed , any excess unreacted acetic anhydride is hydrolyzed.
25 mL of a 1 M phenyl magnesium bromide in tetrahydrofuran was dispensed into the beaker by using a syringe. The resulting mixture was stirred for about 15 minutes when the purple color turned into a brown color permanently. It was then extracted first with 20 mL of dichloromethane and the bottom DCM layer containing the product was reextracted with 10 mL of dichloromethane. The final bottom layer was retained and dried with MgSO4. The drying agent was discarded when the mixture was filtered.
Then, the test media is then incubated at 37 ° C, for 18-24 hours. Rinsing reusable instruments The samples were rinsed with 40 ml of pyrogen-free water using a glass beaker that is free from pyrogens. Endotoxin testing using STV A total of 0,2mL from the water obtained from the rinsing was placed in the STV containing LAL reagent and was shaken for 20 to 30 seconds. Then STV was placed in an incubator at 37 ° C for 60 ± 2 minutes. STV was then observed by reversing the reaction tube in one smooth motion.
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
Dissolve the salt in 60 ml of tap water. Add 30 ml 6 M Hcl and stir the mixture with a glass rod. Add 12 g solid Nacl to the solution and stir the mixture for about 2 minutes. Support a 250 ml separatory funnel on a ring, making sure that the stopcock is closed and that a clean beaker is placed beneath the exit tube. Transfer the aqueous solution from the beaker to the separatory funnel.