EXPERIMENT NO.

17 Date:

PREPARATION OF STANDARD CURVE OF DICLOFENAC SODIUM

I.P. USING UV- VISIBLE SPECTROPHOTOMETER

➢ AIM:
To prepare calibration curve of Diclofenac Sodium, find out the unknown sample
concentration, slope, and regression coefficient (R) by using UV-Visible
spectrophotometer.

17.1 Figure 1 : Structure of diclofenac sodium

➢ REQUIREMENT:

• Paracetamol pure drug. Phosphate buffer pH 6.8, Distilled water.

• Volumetric flasks-1000ml, 10ml, beakers, pipettes etc.

• UV-Visible spectrophotometer [Shimadzu UV-1800] with matched quartz

cells (1 cm).

➢ PRINCIPLE:

UV-Visible spectrophotometry is one of the most frequently employed

technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible
radiation absorbed by a substance in solution. Instrument which measures the ratio, or function
of ratio, of the intensity of two beams of light in the U.V-Visible region are called Ultraviolet-
Visible spectrophotometers. In qualitative analysis, organic compounds can be identified by
use of spectrophotometer, if any recorded data is available, and quantitative spectrophotometric
analysis is used to ascertain the quantity of molecular species absorbing the radiation.
Spectrophotometric technique is simple, rapid, moderately specific, and applicable to small
quantities of compounds. The fundamental law that governs the quantitative
spectrophotometric analysis is the Beer -Lambert law.

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Diclofenac sodium is non-steroidal anti-inflammatory agent, widely used in musculoskeletal
disorders, arthritis, toothache, etc., for symptomatic relief of pain and inflammation. Diclofenac
sodium is reportedly used for topical applications. The drug undergoes substantial hepatic first-
pass metabolism and only about 50% of administered dose reaches systemic circulation. In
Rheumatoid Arthritis patients are advised to take the NSAIDs for prolong period but the side
effects such as systemic toxicity, GIT irritation, nausea, vomiting, gastric erosion, headache
are the main drawbacks of Diclofenac sodium. Because of its short biological half-life and
frequent administration, it is considered as a suitable candidate to formulate it into a sustained
release matrix type transdermal patch system.

➢ METHODOLOGY:
• Preparation of Phosphate Buffer (pH-6.8):
6.8045gm of Potassium-di-hydrogen phosphate was taken in a 1000ml volumetric
flask, then 0.896gm of Sodium hydroxide was taken and volume make up to 1000ml.

▪ Preparation of Calibration Curve:

• Preparation of stock solution
o Stock solution-1: Stock solution of drug (0.1mg/ml) is prepared by dissolving
10 mg of drug in 100 ml solution of methanol and phosphate buffer pH 6.8 in
100 ml volumetric flask (to get 100 μg/ml drug solutions) with vigorous shaking
and further sonicated for about 10 minutes.
o Stock Solution-2: 10 ml of this (stock solution 1) is diluted to 100ml with
phosphate buffer pH 6.8 to get a stock solution containing 10 μg/ml of drug.
The stock solution was filtered through Whatmann filter paper.

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❖ ABSORBANCE OF DICLOFENAC SODIUM FOR STANDARD
CURVE:

Concentration (µg/ml) Absorbance

1 0.052
1.5 0.075
2 0.093
2.5 0.114
3 0.135
3.5 0.150
4 0.187

❖ SAMPLE WITHDRAWAL OF DICLOFENAC SODIUM FOR

STANDARD CURVE:

Concentration (mg) How Much Sample Withdrawal

1 2ml
1.5 3ml
2 4ml
2.5 5ml
3 6ml
3.5 7ml
4 8ml

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 Standard Curve Of Diclofenac Sodium
0.2
0.18
0.16
ABSORBANCE

0.14
0.12
0.1
0.08 y = 0.0441x + 0.0042
0.06 R² = 0.9931
0.04
0.02
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
CONCENTRATION (µg/ml)

17.2 Fig-2: Standard Curve of Diclofenac sodium

➢ RESULTS AND DISCUSSION:

Standard Curve of Diclofenac sodium was plotted
and the concentration of unknown sample can be determined from interpolation of calibration
curve. Slope was found to be 0.0441 and regression coefficient was found to be 0.9931.
Thus, the Standard Curve of Diclofenac sodium was
plotted by using the absorbance values from the UV-Vis Spectrophotometer using various
concentration of Diclofenac sodium Solution.

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