See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/328022886

Experiment No: 4 CALIBRATION CURVE OF DICLOFENAC SODIUM

Experiment Findings · October 2018

DOI: 10.13140/RG.2.2.17262.25920

CITATIONS READS

0 1,498

1 author:

M. Venkataswamy
Vishnu Institute of Pharmaceutical Education & Research
35 PUBLICATIONS   53 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Mucoadhesive patch View project

Swine flu View project

All content following this page was uploaded by M. Venkataswamy on 02 October 2018.

The user has requested enhancement of the downloaded file.

Date: ……………… Experiment No: 4 Page no: 1
CALIBRATION CURVE OF DICLOFENAC SODIUM

M.Venkataswamy

Aim: Construct calibration curve of Diclofenac sodium, find out the unknown sample concentration,
slope and Regression coefficient (r2) by using UV-Visible spectrophotometer.
Chemicals: Diclofenac sodium pure drug, Phosphate buffer pH 6.8, Distilled water.
Instrumentation: UV-Visible spectrophotometer with matched quartz cells (1 cm)

PRINCIPLE:
Spectroscopy methods:

It is the branch of science dealing with the study of interaction between electromagnetic radiation and
matter. It is a most powerful tool available for the study of atomic and molecular structure/s and is used
in the analysis of wide range of samples. Optical spectroscopy includes the region on electromagnetic
spectrum between 100 Å and 400 μm. The regions of electromagnetic spectrum are shown in table 1.

Region Wavelength

Far (or vacuum)ultraviolet 10-200 nm

Near ultraviolet 200-400 nm

Visible 400-750 nm

Near infrared 0.75- 2.2 μm

Mid infrared 2.5-50 μm

Far infrared 50-1000 μm

Table 1: Types Regions of electromagnetic spectrum.

Ultraviolet-Visible spectrophotometry: UV-Visible spectrophotometry is one of the most frequently

employed techniques in pharmaceutical analysis. It involves measuring the amount of ultraviolet or
visible radiation absorbed by a substance in solution. Instrument which measure 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.

Beer’s law: It states that the intensity of a beam of parallel monochromatic radiation decreases
exponentially with the number of absorbing molecules. In other words, absorbance is proportional to the
concentration.
Date: ……………… Experiment No: 4 Page no: 2
Lambert’s law: It states that the intensity of a beam of parallel monochromatic radiation decreases
exponentially as it passes through a medium of homogeneous thickness. A combination of these two
laws yields the Beer-Lambert law.

Beer-Lambert law: When beam of light is passed through a transparent cell containing a solution of an
absorbing substance, reduction of the intensity of light may occur. Mathematically, Beer-Lambert law is
expressed as

A=a * b * c

Where, A=absorbance or optical density

a= absorptivity or extinction coefficient

b=path length of radiation through sample (cm)

c=concentration of solute in solution.

Both b and a are constant so a is directly proportional to the concentration c

When c is in gm/100 ml, then the constant is called A (1%, 1 cm)

Quantification of medicinal substance using spectrophotometer may carried out by preparing solution in
transparent solvent and measuring it’s absorbance at suitable wavelength. The wavelength normally
selected is wavelength of maximum absorption (λmax), where small error in setting the wavelength scale
has little effect on measured absorbance.

In calibration graph method, the absorbances of a number of standard solutions of the reference
substance at concentrations encompassing the sample concentrations are measured and a calibration
graph is constructed. The concentration of the analyte in the sample solution is read from the graph as
the concentration corresponding to the absorbance of the solution.

The single point standardization procedure involves the measurement of the absorbance of a sample
solution and of a standard solution of the reference substance. The concentration of the substances in the
sample is calculated from the proportional relationship that exists between absorbance and
concentration.

Ctest / Cstd = Atest /Astd

Where Ctest and Cstd are the concentrations in the sample and standard solutions respectively and Atest
and Astd are the absorbances of the sample and standard solutions respectively.

Diclofenac sodium is a non steroidal anti-inflammatory drug (NSAIDs) with analgesic and antipyretic
properties. It is widely used in management of mild to moderate pain particularly when inflammation is
also present as in cases of rheumatoid arthritis, osteoarthritis, musculoskeletal injuries and some post
operative conditions.
Date: ……………… Experiment No: 4 Page no: 3

Preparation Methods:

Phosphate buffer preparation:

Phosphate buffer: Place 50ml of 0.2 M Potassium di-hydrogen phosphate in a 200ml volumetric flask,
add the specified volume of 0.2 M sodium hydroxide (given in the table) and then add distilled water to
make up the volume 200ml.

Preparation of 0.2 M Potassium di-hydrogen phosphate solution: Dissolve 27.218g of potassium di-
hydrogen phosphate in sufficient distilled water containing in the 1000ml volumetric flask and to make
up to the volume 1000ml.

Preparation of solutions for Calibration curve:

Stock solution 1: Stock solution of drug (1mg/ml) is prepared by dissolving 100 mg of drug in 100 ml
phosphate buffer pH 6.8 in 100 ml volumetric flask (to get 1000 µg/ml drug solutions) with vigorous
shaking and further sonicated for about 10 minutes. (As per mentioned in the table 3).

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 100 µg/ml of drug). The stock solution was filtered through Whatmann
filter paper No.41. (As per mentioned in the table 4)

Dilutions: Take the respective samples (0.2ml, 0.4ml, 0.6ml, 0.8ml, 1ml, 1.2ml, 1.4ml, 1.6ml, 1.8ml,
2ml, 2.2ml, 2.4ml) in each test tube, add phosphate buffer pH 6.8 to make total volume of 10 ml to
produce (2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24µg/ml) respectively (As per mentioned in the table 5).

Determination of absorption maxima:

A UV absorption maximum was determined by scanning 10µg/ml solution of Diclofenac sodium in

phosphate buffer pH 6.8, in between 200-400 nm by using UV-visible spectrophotometer. Further a
representative spectrum was drawn of Diclofenac sodium in phosphate buffer pH 6.8.

Preparation of Calibration curve:

The standard solutions for the drug having concentration 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 24µg/ml
was prepared with phosphate buffer pH 6.8 from the stock solution. The absorbance of solutions of pure
Diclofenac sodium drug were measured at 276 λmax and a calibration curve was plotted between
concentration of drug (µg/ml) on x-axis v/s absorbance on y-axis to get the linearity and regression
equation which has shown in fig. 2.
Date: ……………… Experiment No: 4 Page no: 4
Calculations:
Table 2: specified volume of NaOH required preparing buffer solutions:

S.No pH Volume of 0.2M

NaOH (ml)
1 5.8 3.6
2 6 5.6
3 6.2 8.1
4 6.4 11.6
5 6.6 16.4
6 6.8 22.4
7 7 29.1
8 7.2 34.7
9 7.4 39.1
10 7.6 42.4
11 7.8 44.5
12 8 46.1

Measurement of absorbance for prepared samples:

Table 3, 4, 5:

Table 3: Preparation of Stock solution 1

Phosphate buffer
Drug (mg) Concentration of
pH 6.8 to make
Drug (µg/ml)
100 ml
100 100 1000

Table 4: Preparation of Stock solution 2

Phosphate buffer
Stock solution-1 Concentration of
pH 6.8 to make
Drug (µg/ml)
100 ml
10 100 100
Date: ……………… Experiment No: 4 Page no: 5
Table 5: Preparation of Dilutions

Volume of stock Phosphate buffer

Concentration of
solution (ml) pH 6.8 to make
Salicylic acid (µg/ml) Absorbance
10 ml
0 (Blank) 10 0 0
0.2 9.8 2 --
0.4 9.6 4 --
0.6 9.4 6 --
0.8 9.2 8 --
1 9 10 --
1.2 8.8 12 --
1.4 8.6 14 --
1.6 8.4 16 --
1.8 8.2 18 --
2.0 8 20 --
2.2 7.8 22 --
2.4 7.6 24 --
unknown - - --

UV Spectrum of Diclofenac sodium:

Fig 1: UV spectrum of Diclofenac sodium drug

Date: ……………… Experiment No: 4 Page no: 6

Draw graph according to observation values

Fig. 2: Calibration curve of Diclofenac sodium in phosphate buffer pH 6.8 at λ max 276 nm

From graph: calculate slope

Y2-Y1
Slope =……….
X2-X1

Regression coefficient (r2) determination: Formula given below

Example (enter your observation values for calculation not this values):
Table 5: Regression coefficient (r2) calculation for data given in the following table

Conc Absorb
ance = y
(ppm) n*∑x*y- {n*∑x2- n*∑y2 √{n*∑x2-(∑x)2 *
=x x*y x*x y*y ∑x*∑y (∑x)2 -(∑y)2 n*∑y2-∑y)2}

50 0.246 5*256.8- 0.602

12.3 2500 0.060516 (500*2.294) 31250 584 √18830.75= 137.225

75 0.338 25.35 5625 0.114244

100 0.456 45.6 10000 0.207936

125 0.582 72.75 15625 0.338724 18830.75

150 0.672 100.8 22500 0.451584 137.225

∑x= ∑y= ∑x2= ∑y2=

∑x*y n*∑x*y-
 Date: ……………… Experiment No: 4 Page no: 7
500 2.294 = 56250 1.173004 ∑x*∑y =

256.8 137

(∑x)2=
(∑y)2=5.
250000 262436

Where n=5

n*∑x*y-∑x*∑y 137
Therefore, r = ………………………………….. = ………………. = 0.99836
√ {n*∑x2-(∑x)2 * n*∑y2-∑y)2} 137.225

Therefore, r2 = 0.9967

REPORT: Calibration 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 …………,
Concentration of unknown sample was found to be…….....%, regression coefficient was found to
be...............

View publication stats