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# Experiment.

No:

Date:

## Assay of sodium benzoate and caffeine by Absorbance Ratio Method

Aim: To estimate the concentration of sodium benzoate and caffeine using Absorbance Ratio method. Requirements: Apparatus - 10ml volumetric flask, Pipettes Chemicals Sodium Benzoate, Caffeine, Distilled Water, Sample Solution
Theory: The absorbance ratio method is a modification of the simultaneous equations

procedure. It depends on the property that, for a substance, which obeys Beer's law at all wavelengths. Q-analysis is based on the relationship between absorbance ratio value of a binary mixture and relative concentrations of such a mixture. The ratio of two absorbance determined on the same solution at two different wavelengths is constant. This constant was termed as Hufners Quotient or Q-value which is independent of concentration and solution thickness e.g. two different dilutions of the same substances give the same absorbance ratio A1/ A2. In USP this ratio is referred to as a Q value. In the quantitative assay of two components in admixture by the absorbance ratio method, absorbance are measured at two wavelengths, one being the max of one of the components ( 2) and the other being a wavelength of equal absorptivity of the two components ( 1), an iso-absorptive point. At 1, A1 = aX1bcX +aY1bcY(1) At 2, A2= aX2bcX + aY2bcY(2) At 1, aX1 = aY1. Assume b = 1cm. A1 = aX1cX + aX1cY ......(3) Divide (2) by (3), A2 / A1 = aX2cX + aY2cY / aX1cX + aX1cY Divide each term by CX + CY and let FX = CX / CX + CY and FY = CY / CX + CY where FX and FY are the fractions of X and Y respectively in the mixture. A2 / A1 = aX2 FX + aY2 FY / aX1 FX + aX1 FY But , FX + FY = 1. So, FY = 1- FX A2 / A1 = FX ax2 - FX aY2 + aY2 / aX1

Observation Table: Drug/Analyte Sodium Benzoate 10 g/ml Caffeine 10 g/ml Unknown Sample Wavelength 242nm 273nm 242nm 273nm 242nm 273nm Absorbance Absorptivity =A/bc ax1= ax2= ay1= ay2= A1= A2=

Calculations:

A2 / A1 = FX ax2 /ax1 - FX ay2 /ay1 + ay2 /ay1 Let QX = ax2 /ax1 , QY = ay2 /ay1 and QM = A2 / A1 Thus QM = FX (QX QY ) + QY FX = QM QY / QX QY A1 = aX1cX + aX1cY ....... (3) A1 / aX1 = cX + cY Divide both sides by CX, A1 / cX. aX1 = cX + cY / cX = 1/ FX Thus QM QY / QX QY = cX. aX1 / A1 Hence Cx = (QM QY / QX QY) . A1/ ax1

Equation gives the concentration of X in terms of absorbance ratios, the absorbance of the mixture and the absorptivity of the compounds at the iso-absorptive wavelengths. Accurate dilutions of the sample solution and of the standard solutions of X and Y are necessary for the accurate measurement of A1 and A2 respectively.

Procedure: 1. Sodium benzoate and Caffeine injection has the following formulation: Sodium benzoate: 125mg Anhydrous Caffeine: 125mg Water q.s. : 1ml 2. Standard Solution Preparation: a) Stock solution containing 1mg/ml of sodium benzoate was prepared. From this, 1 ml of solution was pipetted and diluted to 10 ml with distilled water to give a solution of 100 g/ml. From this solution another 1 ml was pipetted and diluted to 10 ml with distilled water to give 10 g/ml sodium benzoate. b) Stock solution containing 1mg/ml of caffeine was prepared. From this, 1 ml of solution was pipetted and diluted to 10 ml with distilled water to give a solution of 100 g/ml. From this another 1 ml was pipetted and diluted to 10 ml with distilled water to give 10 g/ml caffeine. c) Determination of isobestic point: The UV spectra of both the solutions were recorded from 320 to 220nm. The spectra of both the solutions were recorded on the same chart to find the isobestic point. From the individual spectrum of caffeine the max was found to be 273nm and isobestic point at 242nm. The absorbance values of the diluted samples of the stock solutions a) and b) at 273nm and 242nm were employed for calculating the amount of Caffeine and Sodium benzoate.

Calculations continued:

3. Sample solution preparation: The given sample solution of the injection was diluted 100 times i.e. 1 ml of this solution was pipetted and diluted to 10 ml with distilled water. From this solution 1ml was pipetted and diluted to 10 ml with distilled water. The absorbance of this solution was measured at 273nm and 242nm. 4. The concentration of Sodium Benzoate and caffeine in the formulation was calculated.

Results: The concentrations of each of the component are as follows: 1. Sodium Benzoate 2. Caffeine -