Lab4

PREPARATION OF
CALIBRATION CURVE
 Calibration curve definition

Calibration curve:
• It is the curve prepared from a series
of standard solution
• It used as a reference curve to obtain
the concentration of unknown sample
of the same drug
• X axis s the conc. of sample
• Y axis is the absorbance
 Stock solution

 Stock solution:
Solution of known and high conc.
from which we prepare standard
solution
 Standard solution

Standard solution:
Solution of known conc. Prepared
from stock solution using dilution
equation:
𝐶 ⋅𝑣 𝐶 ⋅𝑣
 Blank solution

 Blank solution:
Its the solution which contain all the
constituents of the sample except the
active ingredient which is required to be
measured
 Spectrophotometry
• It is a method to measure how
much a chemical substance
absorbs light
• The basic principle is that each
compound absorbs or transmits
light over a certain range of
wavelength.
• In our work usually the analysis
concerned with the absorbed
amount of light
Spectrophotometry
 Spectrophotometry
• Depending on the range of wavelength of light source, it can be
classified into two different types:
1.UV-visible spectrophotometer:
• uses light over the ultraviolet range (185 - 400 nm) and visible
range (400 - 700 nm) of electromagnetic radiation spectrum.
2.IR spectrophotometer:
uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
Methods to obtain the unknown conc.
1. Curve fitting method:

1. Curve fitting method:

To fit a straight line among
scattered points
Not reliable
Methods to obtain the unknown conc.
2. Least square fitting method:
2. Least square fitting method:
This method is based on the equation
which minimizes the sum of the squares
of deviation of the observed values from
line.

𝑦 𝑦

Where: y =observed value

ȳ = calculated value
Methods to obtain the unknown conc.
2. Least square fitting method:
X
Y (abs.) X2 X*Y
conc(mg/ml)
2
X1 Y1 X1 X1*Y1
2
X2 Y2 X2 X2*Y2
2
X3 Y3 X3 X3*Y3
2
X4 Y4 X4 X4*Y4
2
X5 Y5 X5 X5*Y5

ƩX Ʃy ƩX2 ƩX*Y
(ƩX)2
 Methods to obtain the unknown conc.
2. Least square fitting method:
X
Y (abs.) X2 X*Y ȳ
conc(mg/ml)
2
X1 Y1 X1 X1*Y1 ȳ1=c+bx1
2
X2 Y2 X2 X2*Y2 ȳ2=c+bx2
2
X3 Y3 X3 X3*Y3 ȳ3=c+bx3
2
X4 Y4 X4 X4*Y4 ȳ4=c+bx4
2
X5 Y5 X5 X5*Y5 ȳ5=c+bx5

ƩX Ʃy ƩX2 ƩX*Y
(ƩX)2
From these variables using the above equations we can obtain c & b, then by substitute
each X value we can get ȳ ( calculated value)
Calibration curve of Nitrofurantoin
Calibration curve of Nitrofurantoin
 Chemical name of drug:
1-[[[5-nitro-2-furanyl]methylene] amino]-2,4-imidazolidinedione
 Chemical structure:
Calibration curve of Nitrofurantoin

 Solubilty of drug:
It is slightly soluble in water and ethanol, and souble in DMF (dimethyl
formamide)
 Action of drug:
Nitrofurantoin is an oral antibiotic widely used either short term to treat
acute urinary tract infections or long term as chronic prophylaxis against
recurrent infections.
 Calibration curve of Nitrofurantoin
experimental procedure

1. Stock solution preparation

Preparation of 100mg/ml stock sol. Of nitrofurantoin can be done via take 10g
(10000mg) to dissolve up to 100 ml of DMF ( suitable solvent)

10000mg/100ml=100mg/ml
 Calibration curve of Nitrofurantoin
experimental procedure
2. Standard solution preparation
X
From stock sol. above prepare standard sol of deferent conc.(
conc(mg/ml)
1.5, 2,3,4,&5mg/ml),Take certain volume from the stock then
complete the volume to 100 ml by addition of DMF
1.5
To get standard sol with 1.5 mg/ml conc.:-
2
C1V1=C2V2 ---------- 100*V1=1.5*100 -------So V1 = 1.5 ml of 3
stock sol. Completed to 100ml with DMF
4
And so on for other standard conc. (2, 3, 4, & 5mg/ml)
5
 Calibration curve of Nitrofurantoin
experimental procedure
3. Absorbance
measurement X
Y (abs.)
conc(mg/ml)
Determine the absorbance of
suitable blank, 1.5,2,3,4,&5 1.5 0.02
mg /ml standard solutions by 2 0.04
spectrophotometry at 370 3 0.08
nm
4 0.12
5 0.16
 Calibration curve of Nitrofurantoin
experimental procedure
4. Apply the least squares
method: Calculate b, c, then ȳ X
conc(mg/ml)
Y (abs.) X2 X*Y ȳ

depending on the least squares method . X1 Y1 X1

2
X1*Y1 ȳ1=c+bx1
2
X2 Y2 X2 X2*Y2 ȳ2=c+bx2
∑ 𝑦 X3 Y3 X3
2
X3*Y3 ȳ3=c+bx3
∑ 𝑥⋅𝑦 ∑ 𝑥 ⋅
𝑏 𝑛 0.007 X4 Y4 X4
2
X4*Y4 ȳ4=c+bx4
∑𝑥 X5 Y5 2
X5*Y5 ȳ5=c+bx5
𝛴𝑥 X5
𝑛
∑ ƩX Ʃy ƩX2 ƩX*Y
𝑐 0.06
(ƩX)2
ȳ 𝑐 𝑏𝑥 ?
 Calibration curve of Nitrofurantoin
experimental procedure
5. Plot ȳ vs conc standard curve
X
Y (abs.) X2 X*Y ȳ
conc(mg/ml)
1.5 0.02 2.25 0.03 0.4545
2 0.04 4 0.08 0.477
3 0.08 9 0.24 0.522
4 0.12 16 0.48 0.567
5 0.16 25 0.8 0.612

15.5 0.42 56.25 1.63

240.25