The basic procedure was as follows: • The required amount of lime was added, as a 10% milk of lime solution (mass/mass), to melt with stirring at 80 C. • After the samples was mixed for 5 minutes, the carbonation reaction was initiated by introducing carbon dioxide in to the reaction vessel. • As soon as the pH of the liquor reached the required value, the gas supply was stopped. • The carbonated melt was stirred for a period of one minute. • The carbonated liquor was then filtered, cooled and stored in a freezer for analysis. 3.5 Control during carbonation
When the end point was reached, the pH was somewhat constant at approximately 11. The procedure was repeated for a 2nd time using a fresh sample of phosphoric acid. Now the 250 mL flask was taken and approximately 50 mL of the soft drink (sprite/mountain dew) was placed into it. The flask was shaken several times to release the carbon dioxide in the drink. The flask was then vented.
(iii) Cool with cold running tap water and add 10 ml concentrated Ammonium hydroxide. (iv) Immediately add 20 ml heptoxime reagent. (v) Add 20 ml ethyl alcohol. (vi) Dilute to volume with distilled water and mix. (vii) Measure absorbance at 445 nm, 20 min after adding reagent, using a reagent blank as
Then, 3 mL of the plasma sample was poured to a tube and 3 mL of HNO3 and 2 mL of H2O2 were added to the sample. The tube was tightly closed and centrifuged for 10 minutes at 4000 rpm. Then, the sample was placed in a hot water bath at 90°C for 100 min. Then, the digested sample was diluted with 30 mL of distilled water. Human urine sample In order to prepare human urine sample, 10 ml of urine was poured into a beaker.
2.3. Preparation procedure for DS loaded PC-SA combined beads The ionotropic gelation method was used for the preparation of DS loaded PC-SA bead. The gum (PC) was dissolved in distilled water and initially boiled for 10 minutes, then cool and keep stirring for 24 hours at 400 RPM. Then filtration
The end product was passed via sieve (no. 85) and stored in desiccators until use. 2.3. Preparation of DS loaded mucoadhesive beads PC-SA [F0], DS-SA [F1] and DS-PC-SA [F2-F6] beads were prepared by the ionotropic gelation method and the compositions are summarized in Table 1. Initially, PC gum was dissolved in distilled water and boiled for 10 min, cooled and stirred for 24 h at
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.
This diluted solution will be used in the assay as duplicate samples. Then, 1.0mL of standard glycine solution containing (7.5mg/mL) was diluted to 100mL with water using a volumetric flask. This solution contains 1.0µmole/mL of glycine. 8 tubes were set up according to the following protocol and 2.0mL of ninhydrin reagent was then added to each of the 8 tubes and were placed in a boiling water bath for 20 minutes. After 20 minutes, the tubes from the bath was carefully removed, cooled in a beaker of cold water, then 8.0mL of 50% ethanol was added and mixed well.