Experiment no.

2
To develop a calibration curve between absorbance and known drug
concentrations for the estimation of unknown concentration graphically and
by computational values
2.1 Objectives
1. To determine unknown concentration from the graph.
2. To compute slope and y-intercept of the curve between known concentration and
absorbance on a graph.
3. To calculate the unknown concentration using slope and y-intercept of the curve.

2.2 Data
The data on concentration and absorbance is given in the Table 2.1

Table 2.1: Concentration absorbance data

Concentration (µg/ml) Absorbance

0 0
5 0.12
10 0.22
15 0.31
20 0.44
25 0.54
30 0.66
35 0.76
Unknown sample 0.36

2.3 Introduction:

The establishment of a calibration or standard curve is required for the determination of drug in
any unknown sample. UV Spectrophotometer is used for measuring the absorbance of already
prepared standard solutions of known concentrations. A graph between the absorbance and
concentration is plotted on an ordinary or a linear graph paper, where concentration being an
independent variable is taken on x-axis and absorbance being a dependent variable is taken along
y-axis.

A linear graph shows a direct relation between concentration and absorbance. The unknown
concentration is calculated through the graph and this method is referred to as graphical method.
Y-intercept and slope of the curve can also be used for the estimation of the unknown
concentration.
Variable
A variable is an object, event, time period, or any other type of category that is to be measured.
There are two types of variables-independent and dependent.
An independent variable is the variable that is changed or controlled in a scientific experiment
to test the effects on the dependent variable whereas dependent variable is the variable being
tested and measured in a scientific experiment. The dependent variable is 'dependent' on the
independent variable. As the experimenter changes the independent variable, the effect on
the dependent variable is observed and recorded.

Slope

The slope of a line is a number that measures its "steepness",

usually denoted by the letter “m”. Slope is the ratio of the change
in the y-value over the change in the x-value.
Formula for the slope is:
Slope (m) = ∆Y

∆X
Slope= from point A to B
Slope Direction

The slope of a line can be positive, negative, zero or undefined.

Positive slope

Here, y increases as x increases, so the line slopes upwards to the right.

The slope will be a positive number.

Negative slope
Here, y decreases as x increases, so the line slopes downwards to the right.
The slope will be a negative number.

Zero slope

When y does not change as x increases, so the line is exactly horizontal.

The slope of any horizontal line is always zero.

Undefined slope

When the line is exactly vertical, it does not have a defined slope. The two x coordinates are the
same, so the difference is zero.
Intercept

The point at which a curve intersects an axis is known as an Intercept. The x-intercept is the
point where the curve crosses the x-axis and the y-intercept is the point where the curve crosses
the y-axis. The equation of a line written in slope-intercept form is:

y = mx + b
Where m is the slope and b is the y-intercept

Example of intercept

In the figure above, the line cuts the x-axis at −2, 0 and y-axis at 0, −3. So, x-intercept is - 2 and
y-intercept is - 3.

Standard Curve
A standard curve, also known as a calibration curve, is a type of graph used as a quantitative
research technique. Multiple samples with known properties are measured and graphed, which
then allows the same properties to be determined for unknown samples by interpolation on the
graph.

R2 Value
R-square is a statistical measure of how close the data are to the fitted regression line.

Positive Correlation

Positive correlation is a relationship between two variables in which both variables move in
tandem—that is, in the same direction. A positive correlation exists when one variable decreases
as the other variable decreases, or one variable increases while the other increases.

Negative Correlation

Negative correlation is a relationship between two variables in which one variable increases as
the other decreases, and vice versa. In statistics, a perfect negative correlation is represented by
the value -1, a 0 indicates no correlation, and a +1 indicates a perfect positive correlation.
Graph
The collection of all points whose coordinates satisfy a given relation.

Types of Graph

The four most common are probably line graphs, bar graphs and histograms, pie charts, and
Cartesian graphs.

2.4 Procedure

i. On a linear graph adjust a suitable scale as in the figure 2.1

ii. Plot concentration on x-axis and absorbance on y-axis.
iii. Assume the curve to zero time on x-axis and read the value. It is the y-intercept(b)

0.8
0.76
0.7 y = 0.0217x + 0.0017
R² = 0.999 0.66
0.6
0.54
0.5
0.44
Absorbance

0.4
0.3 0.31
0.2 0.22
0.1 0.12 Absorbance
0 0
0 10 20 30 40

Concentration (µg/ml)

Figure 2.1: A calibration curve

iv. By using the given formula(Equation 1) calculate the value of slope(m)

m= ∆Y = Y2 – Y1 Equation 1

∆X X2 – X1
The Y2 and Y1 are the higher and lower values of the y-variable (Absorbance) whereas X2 and
X1 are the values of X (Concentration) that correlate to the values of Y2 and Y1 respectively.

v. Determine the unknown concentration corresponding to the absorbance (0.36) given in

the table 2.1 directly from the curve.
 vi. Calculate the unknown concentration by the given formula(Equation 2) and compare it
with that calculated in step 5

Unknown Concentration(x) = Absorbance of unknown – b Equation 2

Where m is the slope and b is the intercept.

Note: If the value of ‘b’ is negative, take the negative sign in consideration as -(-b) = +b

vii. Give the observation whether the curve is linear or non-linear.

viii. Put the results of the m, b and x.

2.5 Working Activity

Y-Intercept (b) = from graph

= -0.05

Slope (m) = 0.67-0.55

30-25

= 0.024

2.6 Results/Observations

Parameter Remarks/Values Unit where

applicable
The slope of the curve was (linear or Non- Linear __
linear)
Y-Intercept (b) -0.05 None
Slope of the Curve (m) 0.024 Hour-1

Unknown concentration estimated from 15µg/ml µg/ml

graph
Unknown Concentration Calculated
2.7 Learning Questions

1. What is the importance of the calibration curve?

In analytical chemistry, a calibration curve, also known as a standard 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.

2. Are the values of unknown concentration estimated graphically comparable to that

computed by equation?
Value of unknown concentration estimated from graph is 15µg/ml and value from
equation is 14.4µg/ml .So our values are comparable.

3. How is the slope a measure of sensitivity?

When the calibration curve is linear, the slope is a measure of sensitivity i.e. how much
the signal changes for a change in concentration. A steeper line with a larger slope
indicates a more sensitive measurement.

4. What does a Y-intercept of 0 mean?

A Y-intercept of 0 means that the line passes through the origin, 0,0. This means that the
line goes through the y-axis (x=0) at the 0 mark.

5. What information does R2 provide and what is its value for a perfect line?

R2 is the square of correlation coefficient(r) and provides information about how far
away the y values are from the predicted line. A perfect line would have an R 2 value of 1,
and most R2 values for calibration curves are over 0.95.

6. Why do calibration curves deviate from a straight line?

Calibration curves deviate from a straight line when A<0.1 and when A>1 because these
at these values the substances react to light differently which will cause it to not follow a
linear graph.
7. Why the regression of y on x is not the same as the regression of x on y?

This is because the procedures used in linear regression assume that all the errors are in
the y values and that the errors in the x values are insignificant.

8. What is the Residual standard deviation or standard error?

It is a statistical measure of the deviation of the data from the fitted regression line. It is
calculated using eq:

Where yi is the obsereved value of y for a given value of xi , y’ is the value of y predicted
by the equation of the calibration line for a given value of xi and n is the no. of calibration
Points.

9. What are the limitations of Beer Lambert Law?

The law tends to breakdown at very high concentrations, especially if the material is
highly scattering. Absorbance within the range of 0.2 to 0.5 is ideal to maintain the
linearity in Beer-Lambert law.

10. Why are absorbance values above 1 inaccurate?

The high conc. will have high absorbance values and in such case molecules are closer to
each other that interactions can set in which can be physical and chemical. Chemical
interactions can change the polarizability and thus absorption.

Written by: Hassan Gul, Muhammad Ilyas , Fariah Qaiser,

Rabbia Mazhar, Urooj Fatima