Name of the experiment: Construction of calibration curve for uv-spectroscopic analysis of Paracetamol.

Principle:

In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. In spectrophotometric analysis a series of standard solutions of known concentrations are prepared and absorbance is measured using spectrophometer instrument to determine the unknown concentration of sample by Beer's Law.

A calibration is a graph where concentration is plotted against absorbance then a straight line (Beer's Law) is fit to the data that we obtained and the resulting equation is used to convert absorbance of the unknown sample into concentration.

0 2 4 6 8 10 120

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4

6

8

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f(x) = xR² = 1

Standard Calibration Curve

Concentration (µg/ml)

Abso

rban

ce

Apparatus:

UV Spectrophotometer Test tube Pipette Measuring cylinder Volumetric flask Electronic balance Filter paper

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Reagents:

Standard paracetamol powder Distilled water

Procedure:

Preparation of stock solution:

1. 5 mg paracetamol powder were weighed by electronic balance2. This powder was taken in a 50 ml volumetric flask and was filled with distilled

water upto the mark.3. The flask was shaked well until the paracetamol powder was dissolved. This

solution was the stock solution.

Preparation of working standard:

1. 9 test tubes were taken, were marked serially from 1-9 and were kept in a test tube holder.

2. Test tube marked 1 was taken and was added with 10 ml distilled water. No stock solution is added in this test tube.

3. Test tube marked 2 was taken and was added with 0.5 ml stock solution and 9.5 ml distilled water.

4. The dilution process was conducted for the rest of the test tubes.

Determination of absorbance

1. Turn on the UV spectrophotometer and wait for the calibration to be completed2. Fill 2/3rd of the cell with distilled water, set the wavelength at 249 nm and press

the auto-zero button3. Record absorbance of all the dilutions of the stock solutions4. Plot all the concentration in x-axis and absorbance in y-axis and form a standard

curve using excel in computer.5. From the standard curve find the state line equation and regression (R2) value.

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Data table:

Test Tube Number

Stock Solution

(ml)

Distilled Water (ml)

Concentration (µg/ml)

Absorbance

1 0 10 0 02 0.5 9.5 5 0.3143 1 9 10 0.5264 1.5 8.5 15 0.7595 2 8 20 0.8246 2.5 7.5 25 0.987 3 7 30 1.58 3.5 6.5 35 1.69 4 6 40 1.9

Result:

Calibration curve equation:

R2: 0.9683

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