7 sem/IMA practical
LAB MANUAL
INSTRUMENTAL METHODS OF ANALYSIS
PRACTICAL (PT 791) B.PHARM
7TH SEMESTER
7 sem/IMA practical
Name of Date of report:
the student:
Class Roll: Expt. No.
Title: Determination of absorption maxima of the given compound
Evaluation:
Experiment (work) planning and execution (5) ______ Signature & Date
Results and interpretation (3) ______
Understanding of the theory related to ______
experiment (2)
______
TOTAL
Evaluator
Date of Experiment:
Experiment Number: 01
Title: Determination of absorption maxima of the given compound
Determination of absorption maxima
Principle:
The wavelength at which maximum absorbance is recorded for a given sample is called Absorption
maxima or λmax. The absorption maxima is recorded by plotting a spectrum of absorbance on the Y-axis
and wavelength on X-axis. The intensity of colour in photometry and all the measurements of absorbance
in spectroscopy is carried out at absorption maxima, since for a change in the conc. at this wavelength
there is a significant change in the absorbance. This helps in increasing the resolution of the estimation
by increasing the sensitivity.
According to Beer’s law: “The intensity of monochromatic light decreases exponentially as the conc. of
the absorbing substance increases arithmetically”.
A system is said to obey Beer’s law, when a plot of conc. versus absorbance gives a linear curve. The
straight line is obtained by using line of best fit. When a non-linear curve is obtained in a plot of conc.
vs absorbance, the system is said to deviate from Beer’s law. Deviation can be positive or negative
deviation.
7 sem/IMA practical
Chemicals & Apparatus required:
100 ml Volumetric Flask, Pipette, Beaker, Uv-Spectrophotometer, Potassium Permanganate (KMnO4).
Procedure
1. 1mg/ml (1000 ug/ml) of stock solution of KMnO4 is prepared by dissolving 100 mg of the compound
in 100 ml of distilled water.
2. From the stock solution 1ml, 2ml, 3ml upto 4ml is withdrawn and diluted upto 100 ml with distilled
water. It will give (10-40) ug/ml strength.
3. Determination of λmax (absorption maxima): λmax can be determined by scanning over the range
of 380-760 nm in the Uv-visible spectrophotometer after putting the above prepared solution inside
it as we can have the absorption maxima value from instrument generated graph.
Report:
The λmax of KMnO4 was found out from the graph is ………….. nm.
Assignment Questions (Write down in your lab file under specific practical):
1. Write down a note on beer’s law in the light of spectroscopic principle.
2. Write down a short note on Absorption Maxima for a specific compound.
3. Write down the five reasons behind the deviations from Beer-Lambert’s law.
4. Write down the definitions of wavelength with proper unit.
7 sem/IMA practical
Name of Date of report:
the student:
Class Roll: Expt. No.
Title: Determination of effect of solvents on absorption maxima of the given compounds
Evaluation:
Experiment (work) planning and execution (5) ______ Signature & Date
Results and interpretation (3) ______
Understanding of the theory related to ______
experiment (2)
______
TOTAL
Evaluator
Date of Experiment:
Experiment Number: 02
Title: Determination of effect of solvents on absorption maxima of the given compounds
Principle:
Solvent effect on Absorption Spectra
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7 sem/IMA practical
Chemicals & Apparatus required:
100 ml Volumetric Flask, Pipette, Beaker, Uv-Spectrophotometer, Ibuprofen, Paracetamol, Methanol,
Chloroform and n-Hexane.
Procedure
1. Prepare 1mg/ml strength of solution of samples (Ibuprofen and Paracetamol) in Methanol, Chloroform
and n-Hexane.
2. Record the absorption spectrum of each sample solution over the range of 230-350 nm using the
appropriate solvent as blank.
Observation table
Effect of solvent on absorption maxima value (λmax)
Name of the In Chloroform In Methanol/Ethanol In n-Hexane
compound
Ibuprofen (Observed
λmax)
Paracetamol
(Observed λmax)
Report
Hence the (Observed λmax) of the Paracetamol and ibuprofen in the specific solvents are…..
Assignment Questions (Write down in your lab file under specific practical):
1. Write down a note on effect of polarity in the Absorption Maxima of the compounds.
2. Write down a short note on Red Shift and Blue Shift.
7 sem/IMA practical
Name of the Date of
student:
report:
Class Roll: Expt. No.
Title:
Evaluation:
Experiment (work) planning and execution (5) Signature & Date
Results and interpretation (3)
Understanding of the theory related
to
Experiment (2)
TOTAL
Evaluator
N.B. cut the above chart and paste above on the starting page of practical copy for each practical.
Write the remaining as mentioned below on the scheduled page.
Date of experiment:
Experiment number: 03
Title: Determination of Paracetamol content in Tablet by Spectroscopy method. Aim: To
determine the paracetamol content in tablet by Spectroscopy method. Reference:
Theory: Paracetamol the widely used analgesic and antipyretic drug which shows three λmax at 242,
243, 257 nm in neutral acidic and alkaline media. Paracetamol shows maximum absorbance at 257nm
due to presence of phenyl ring in alkaline media. The absorbance of different dilution of paracetamol is
taken at this wavelength to make the calibration curve. The absorbance of unknown sample is taken and
with the help of calibration curve the concentration is read out.
7 sem/IMA practical
Reagents required:
a. Paracetamol Reference substance and paracetamol tablet.
b. NaOH (0.1M).
Procedure:
1. Accurately weigh 100 mg of reference paracetamol powder and taken in to a 100 ml volumetric flask
and dissolved it with 50 ml of 0.1 M NaOH solution and rest of the volume with water. Shake the
content well.
2. From this stock solution pipette out 0.1, 0.2, 0.3, 0.4, 0.5 ml and mixed with rest of the volume with
0.1 M NaOH and make up to 10 ml of total volume separately.
3. Again from that 10 ml volume of solution pipette out 1, 2, 3, 4 ml and mixed with 0.1M NaOH solution
up to the volume of 10 ml separately ( If required and so on so forth).
4. Weigh accurately and powdered finely 10 tablets of paracetamol. Weigh accurately a portion of
powder (Equivalent to about 100 mg of paracetamol) into 100 ml volumetric flask where it is dissolved
with 50 ml of 0.1 M NaOH solution and rest of the volume with water and mix for 15 minutes.
5. Transfer 0.5 ml of this solution and dilute to 10 ml with 0.1M NaOH. Again from that take 5 ml of
solution and diluted up to 10 ml with 0.1M NaOH solution. If required prepare dilute solutions for the
proper absorbance values (with in Beer’s law range). In the analysis 0.1 M NaOH solution was taken
as blank solution.
Observation Table:
Serial no. Concentration Absorbance ( Working λ max is
257 nm)
Ref1
Ref2
Ref3
Ref4
Ref5
Unknown
7 sem/IMA practical
Calculation:
1. The absorbance values were plotted in the X-axis and concentrations in Y-axis. At dilute
concentrations the solutions followed Beer's-Lambert's law and the relationship between X and Y
data are found to be linear. Read out the concentration of unknown solution/tablet from the
calibration curve. Multiply the concentration with dilution factor to find out the initial
concentration.
2. Liner regression was carried out in MS Excel software and following equation was determined
in respect to as straight line equation (y= mx+(-)c
Conc. (y) (µg/ml) = (m) × Abs(x) + (– c)
Correlation coefficient, r, was found to be………………
2. Determination of drug content from tablet
Labelled claimed = Avg. Weight of tablets =
Equivalent amount of powdered drug sample to be taken as compared to pure drug = Dilution factor
(D.F) = 100
Absorbance of unknown drug solution appeared in instrument =
Actual Concentration(y) = slope (m) X Obtained Absorbance value of unknown drug solution(x) from
the instrument + (-intercept) (c). (From excel equation of pure drug solutions).
Drug content of each tablet = Actual Conc.X Average Wt. of the sample taken=
Actual content = (Drug content of each tablet / Actual drug content of each tablet) X Dilution factor X
100%
= ………………% of labelled content.
Solution concentration calculation:
Stock Solution: 100 mg/100ml = 1 mg/ml = 1000 ug/ml
Take 0.1, 0.2, 0.3, 0.4, 0.5 ml from the stock solution and dissolved upto 10 ml with NaOH.
For 0.1 ml in 10 ml the conc. will be 100 ug/10 ml = 10 ug/ml (Follow this step for the other solutions
with preparation of 20, 30, 40 and 50 ug/ml of solution)
Conclusion: Hence drug content of each tablet is mg and actual content of Drug is %.
7 sem/IMA practical
Assignments
1. Write down the concept of dilution factors.
2. Write down the definition of Assay.
3. Write down the concept of absorption maxima.
7 sem/IMA practical
Name of Date of
the report:
student:
Class Roll: Expt. No. 04
Title: Simultaneous Estimation of Ibuprofen and Paracetamol by Uv Spectroscopy
Evaluation:
Experiment (work) planning and execution (5) ______ Signature & Date
Results and interpretation (3) ______
Understanding of the theory related to ______
experiment (2)
______
TOTAL
Evaluator
Date of experiment:
7 sem/IMA practical
Significances:
7 sem/IMA practical
There are five methods for the assay techniques with Uv-spectroscopic multicomponent estimations.
They are:
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N.B. If paracetamol and ibuprofen tablets are available in any other amount rather than 325 mg and
400mg calculation should be done accordingly.
Assignment
1. Write down the applications of simultaneous method of estimation.
2. Write down a note derivative spectroscopy.
7 sem/IMA practical
LAB MANUAL/7 SEM/INSTRUMENTAL METHOD OF ANALYSIS/IMA/2025
ABG/NSCBIP
