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Dushyant Patel Extractive Spectrophotometric Method

S.K.P.C.P.E.R., Kherva 70 M .Pharm. Thesis

c) Preparation of bromocresol green dye (0.05% w/v):

¾ Bromocresol Green reagent (50 mg) was accurately weighed and 0.1 N NaOH (0.72 ml)

was added. Ethanol (20 ml) was added and shaken well and then volume was made up to

100 ml with water.

d) Preparation of bromothymol blue dye (0.05% w/v):

¾ Bromothymol Blue reagent (50 mg) was accurately weighed and 0.02 M NaOH (4 ml)

was added. Ethanol (95 %) (20 ml) was added and shaken well and then volume was

made up to 100 ml with water.

e) Preparation of standard stock solutions:

¾ GBP (100 mg) was accurately weighed and transferred into 100 ml volumetric flask.

Distilled water (100 ml) was added to get GBP standard stock solution (1 mg/ml).

Working standard solution (200 µg/ml) was prepared by diluting GBP standard stock

solution (20 ml) to 100 ml with diluent.

5.2.4 DEVELOPMENT OF THE METHOD:

¾ In colorimetric method for optimization, different dyes like, bromocresol green,

bromothymol blue, methyl orange, thymol blue and phenolphthalein were prepared and

used. But among them only BCG and BTB gave color with drug, hence BCG (Method I)

and BTB (Method II) were used for development of the methods.

a) Optimization of conditions:

¾ Conditions under which reaction of GBP with dyes fulfill the essential requirements

were investigated. All conditions studied were optimized at room temperature (25±2

C).

b) Selection of suitable pH buffer solution:

¾ Buffer solutions of different pH (2.0 to 5.5) were prepared. Working standard solution

(5 ml) was pipette out and added to separating funnels. BCG solution (in excess) and

buffer solutions (2 ml) of different pH were added to each. Shaken well and extracted

with 10 ml of chloroform. Later the extracts were taken into 10 ml volumetric flasks,

treated with anhydrous sodium sulphate and volume was made up with chloroform, and

then absorbances were measured at 416 nm. Same procedure was applied for the BTB

and then absorbances were measured at 421 nm. It was found that drug with BCG and

BTB gave maximum absorbance at pH 4.0 (Figure 5.1 A and B)

c) Optimization of volume of buffer:

¾ Volume of buffer was optimized by changing volume of buffer and other parameters

were kept constant. Buffer solution of pH 4.0 was used for optimization. Working

standard solution (5 ml) was transferred in seven separating funnels, different volume of

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Samsung RT34M Bedienungsanleitung Seite 82