Volume2, Issue1, January 2011

206


Available Online at www.ijppronline.com

International Journal Of Pharma Professionals Research
Research Article

SPECTROPHOTOMETRIC METHOD OF STANDARD
CURVE PREPARATION AND CALCULATION FOR
METRONIDAZOLE.

Amit Alexander*
1
, Rashmi Chaurasia, Junaid Khan, Swarna, Sanjeev Sahu, Sandip Patel.
ISSN NO:0976-6723
1) Rungta College of Pharmaceutical Sciences and Research, Bhilai, C.G, India
Abstract
Standard curve preparation is the very basic need in field of Formulation or Research with a drug or unknown substance
before starting the further calculation for a proposed work. This work is done because most of the students are calculating
the values of slope, intercept directly from the software and find manual calculation a tedious job. Also the improper way
to handle the glassware and some minor negligence in plotting the graph can differ the results. Many of the students that
are first time preparing the standard curve faces many problems basically in searching a correct methodology and has
certain queries in regard with the preparation of the standard curve like.
Keywords: - : Standard Curve, Regression, Slope, Intercept, Metronidazole, Calibration curve, UV Spectroscopy,.
Introduction
Preparation of Standard and Regression Curve
Many laboratory tests require the outcome of a carefully
controlled chemical reaction be evaluated or read in a
photometer (colorimeter or spectrophotometer). Since these
instruments are capable of only measuring the amount of
light being allowed to pass through the cuvette, their
readout devices display % of light transmitted or
mathematically derived absorbance. One method of
obtaining concentration from % transmittance or
absorbance is through the use of a standard curve. For our
purposes, standard curves are defined as graphs with
absorption or %T plotted on the Y axis, and increasing
concentrations of standard along the X axis. If Beers Law
is followed, the resulting line representing absorbance vs.
concentration will be straight. A standard curve is
constructed after obtaining the %T/Abs readings from a
number of solutions of known concentration (standards)
used in a reaction or procedure. After the readings are
obtained each is plotted on semi-log (% transmittance) or
linear (absorbance) paper against the corresponding
concentration. If the procedure follows Beer's Law, the
points plotted will generally lie such that a straight line can
be drawn through them. The concentration of controls and
other unknowns (patient samples) can be determined by
locating their %T/Abs reading on the line, and then
Correspondence Address:
Amit Alexander
Rungta College of Pharmaceutical Sciences and Research,
Bhilai (C.G.), India.
PH: 09907333846
E-mail: [email protected]
dropping an imaginary line down from that point to
intersect the concentration axis. [1]
Linear regression is a statistical technique which is used to
estimate or predict the value of one variable, if the value of
another correlated variable is known in advance or is
assumed hypothetically.
In order to estimate the value of one of the correlated
variable from the given value of the other variable, the
regression lines are used. [2]
Once the curve is drawn, a number of things must be
considered to determine its acceptability. The majority of
the curves points should be on or close to the line. There
could be many reasons for a point not being on the line. If
the standards are formed from a series of dilutions, the
accuracy of the dilutions must be suspect. Calculations of
the dilutions and spectrophotometer errors are other
possibilities. Whether or not the curve passes through the
point of origin (the 0"), varies with the procedure. [1]
The extent of absorption of radiation by a given absorbing
system at a specific monochromatic wavelength is
governed by two classic laws of absorptiometry.
LAMBERTS (BOUGNERS) LAW: - At a given
concentration (C) of a homogeneous absorbing system, the
intensity of transmitted light decreases exponentially with
increase in path light.
BEERS LAW: - It is concerned with concentration (C) and
states that for a layer of defined path length, the intensity of
transmitted light decreases exponentially with increase in
concentration (C) of a homogeneous absorbing system. If
Beers law is followed and the procedure is linear at the
lower concentrations, the curves line generally goes
through the zero.
The combination of both these laws gives the popular law
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207

i.e. Beer Lambert Law
log Io/I = kcb.
Where k= Absorptivity of the system.
When concentration is expressed in g/100 ml., k is
described as specific absorption and is designated by the
symbol A(1%, 1cm.) i.e. absorbance of a 1%w/v solution of
a substance in a cell of 1 cm. path length. [3]
Basic Standard Curve Characteristics
1. Neatness counts. Preparing a good standard curve takes
time and practice. A sharp pencil should be used during the
early construction period.
2. Use the X axis for concentration. Determining how to
space between the individual concentrations is done by trial
and error and will also depend on the individual procedure.
3. The Y axis is labelled either %T (semi-log paper) or
Absorbance (linear paper). The amount of spacing for
absorbance readings is often times determined through trial
and error.
The following is an abbreviated list of errors or problems
encountered by students in the past. It is provided for you to
consider as pitfalls to avoid. You should keep this/these
page(s) handy when preparing your chemistry lab curves.
a. Bottom of Y axis did not start at 0.000.
b. Compressed Y or X axises.
c. Uneven spacing of Y or X axises.
d. Not labelling correctly/in the right place.
e. Drawing curve point to point.
f. Fat pencil lines/double/smeared lines.
g. Making dots on curves line for unknowns absorbance
value.
h. Drawing dotted lines on graph representing how the
concentration of unknown was determined.
i. Drawing circles around dots on the curve line.
The following Standard Curve preparation of the drug
Metronidazole will be helpful to figure out the problem
regarding procedure.
METHOD:
Solubility of Metronidazole
Reagent: 1N Hydrochloric Acid.
Procedure: Shake the preparation mechanically for 10
min. For uniform making and pipette out quantity of the
sample equivalent to 100 mg of the substance into 100 ml.
volumetric flask containing 70 ml. of 1N Hydrochloric
acid. Rinse the pipette with the acid. Shake it mechanically
for 15 min. and make the volume to 100 ml. 1 N acid.
Shake well and filter through No.1 filter paper, discard first
10-15 ml. of the filtrate. Dilute 10 ml. of the clear filtrate to
100 ml with 1N acid. [3]
Preparation of Standard Solution: Weigh accurately
100mg of pure Metronidazole & dissolve in minimum
quantity of methanol and dilute to 100.0 ml with 0.1 N
HCL. Take 10.0 ml of this stock solution & dilute further to
100.0 ml with 0.1 N HCL. Pipette out 0.2, 0.4ml, 0.6ml, .
of this solution & dilute to 10.0 ml in separate 10.0 ml
volumetric flask to make 2,4,6,8,..,20.g/ml concentration
solutions. Measure the absorbance (in UV
Spectrophotometer) at
max
277 nm (that has been found by
first scanning the sample for max as explained below).
max Determination (Spectrum Mode in UV
Spectrophotometer):
Take the sample of highest concentration for the estimation
of max from the above prepared aliquots. Fill the
reference cell and sample cell (UV Spectrophotometer)
with blank i.e 0.1N HCL and then correct the baseline.
Then take out the sample cell and fill it with the desired
sample and then proceed for scanning. The highest peak
will give the max for the drug with maximum
absorbance.
The max was found to be 277nm.
To determine the slope of the line, a graph is plotted
between Concentration vs. Absorbance, here the value of
slope is directly calculated by the use of MS-Excel.(Fig.
No. 1)


Fig. No. 1. Standard Curve Graph plotted between
Concentration and Absorbance.
For the calculation of the slope with given two points (x
1
,
y
1
) and (x
2
, y
2
) on a line , the slope of the line
is

In mathematics, the slope or gradient of a line describes its
steepness, incline, or grade. A higher slope value indicates
a steeper incline.
The slope is (in the simplest of terms) the measurement of a
line, and is defined as the ratio of the "rise" divided by the
"run" between two points on a line, or in other words, the
ratio of the altitude change to the horizontal distance
between any two points on the line. But in case of
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208

multiple points here is the method to calculate the slope
and the value of intercept
Standard curve of Metronidazole (Table. No.1): (For
Manual Calculation of the value of slope just follow the
given steps)
Equation of regressed line:
Where,




Slope =

& Intercept

J ust after getting the result Table.No.2 was
prepared for ease in the further calculations.
Table.No.1. Standard Curve Table (Readings of
absorbance against the prepared concentrations
obtained from UV Spectrophotometer Fig. No.3)
S.No Concentration Absorbance
1 2g/ml 0.141
2 4 g/ml 0.286
3 6 g/ml 0.405
4 8 g/ml 0.532
5 10 g/ml 0.677
6 12 g/ml 0.838
7 14 g/ml 0.941
8 16 g/ml 1.117
9 18 g/ml 1.189
10 20 g/ml 1.358


Fig. No. 3. Reading of the absorbance obtained from UV
Spectroscopy Schimadzu 1800.
STEPS FOR CALCULATION [4]
Step 1: Calculation of Slope i.e. the value of b.
By the formula:
b =

............................................ (1)
Where,
n= no. of observation i.e. 10
xy= 104.546
x= 110
y= 7.484

= 1540
(x)
2
= 12100

Values are obtained from Table.No.2
By putting these values in equation [1], we get
b =

b =

b =

Therefore, b = 0.06733
Step 2: Calculation of Intercept i.e. the value of a.
.................... (2)
Where,
= mean of y. (sum of observations/ no. of observations).
= mean of x. (sum of observations/ no. of observations).
b = slope.
=y/10 =x/10
= 7.484/10 = 110/10
= 0.7484 = 11
By putting the values of and in equation 2, we get
i.e.
= 0.7484 - 0.06733*11
= 0.7484 0.74063
= 0.00777
Step 3: Calculation of regressed values (Table. No.3)
(that means how much the values of y are regressed) for y-
1
, y
2
, y
3
. by putting the values of x1, x
2
,
x
3
. respectively along with a and b in the
equation of line will give the regressed values of y.
i.e.
Now for calculating the values for regressed y, we can use
the same equation of line for which the variables will be as
follows:
i.e.
Where, y = absorbance.
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209

= concentrations.
= Slope.
= intercept.
Table. No.3. Table For the calculation of Regressed
values of y (y
1
to y
10
).

If a graph is plotted between the Regressed values obtained
from Table.no.3 and the Absorbance it will be the line of
regression and the curve will be the Standard Regressed
Curve (Fig. No.2). Here the value of R
2
(Regression
Coefficient will be equal to 1, that means the line is perfect
straight.)

Fig.No.2. Standard Regression Curve Graph plotted
between Concentration and Regressed values.
Result And Discussion
Standard calibration curve of Metronidazole was prepared
in the study and a procedure in detail was explained so that
it should be ease for the beginners who are going to prepare
the standard curve the very first time. In the present study
standard curve is prepared with the slope of line as 0.067,
intercept as 0.007 and regression coefficient as 0.998 for
the same the values of regressed points of y had also
calculated and the graph is plotted for the same with
regression coefficient as 1. The above work is done
because most of the students are calculating the values of
slope intercept directly from the software and find manual
calculation a tedious job. Also the improper way to handle
the glassware and some minor negligence in plotting the
graph can differ the results.
References
1. http://www.austincc.edu/mlt/chem/chemlab3cstand
ardcurve.pdf
2. Dhall, G.D.; Chhibber, S.N.; Trivedi, Hari Om;
Chandra, Subodh; Frank mathematics for B.
Pharm, First Edition 2004, Frank Bros & Co.
(Publishers) Ltd., Pg. No. 13.25.
3. Sethi, P.D.; Quantitative Analysis of Drugs In
Pharmaceutical Formulations, Third Edition, CBS
Publishers & Distributors, New Delhi, Pg. No. 18,
226.
4. Vogel, Arthur Israel; Vogels textbook of
Quantitative Chemical Analysis, Fifth Edition,
Longman Group UK Limited, Pg.No. 144-145