Faculty Pharmacy

Programme B Pharm
Course Pharmaceutical Analysis III
Semester 7th Sem.
Name of the Laboratory Pharmaceutical Analysis
Laboratory Code

Suggested list of experiments

1. Colorimetric estimation of Ferrous ions using 1, 10 Phenanthroline.

2. Colorimetric estimation of sulphanilamide using N-1-Napthyl Ethylene Diamine

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dihydrochloride.
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3. Assay of Dextrose injection by colorimetry.
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4. Determination of absorption maxima for a given solution of the drug and isobestic point
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5. Estimation of Quinine sulphate by Fluorimetry.

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6. Estimation of Riboflavine by Fluorimetry.

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7. Study of quenching effect in Fluorimetry.

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8. Estimation of chlorides and sulphates by Nephelo turbidimetry

9. Simultaneous estimation of Ibuprofen and paracetamol.

10. UV Spectrometric determination of Ibuprofen tablets.

11. Estimation of Na+ ions using flame photometry.

12. Simultaneous estimation of diclofenac and paracetamol.

13, Estimation of K+ ions using flame photometry.

14. IR of samples with different functional groups (-COOH, -COOR. _ CONHR; -NH2, -NHR, OH, etc.).

15. Workshop to interpret the structure of simple organic compounds using UV, IR. NMR and

M S.

16. Estimation of Ca++ ions using flame photometry.

 Index Sheet

Name: Semester/Year:
Registration No.
Laboratory Code and Title:
Sl. Experiment Date Conducted Marks awarded Signature of
No. out of 10 Laboratory Faculty
Member
1.
2.
3.

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4.
5. ist
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6.
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7.
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8.
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9.
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10.
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11.
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12.
Total Marks scored by the Student
Total Marks ( prescribed number of Expts. x10)

Marks Scored in CE =
Total Marks Awarded X CE Marks Earmarked
Prescribed no. of Expts. X Marks allotted per experiment
 ESTIMATION OF FERROUS AMMONIUM SULPHATE BY COLORIMETRY

AIM: To estimate ferrous ammonium sulphate by colorimetry.

REQUIREMENTS: Pipette, measuring cylinder, volumetric flask, distilled water, 1,10-phenanthroline

solution , UV-spectrophotometer.

PRINCIPLE: Ferrous ions give an intense colour with 1,10-phenanthroline solution in an acidic media.
This coloured complex has maximum absorbance at 515nm. Estimation hence involves development
followed by reading the absorbance using a suitable colorimeter against a reagent blank. The
unknown concentration is found using the graph.

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PROCEDURE:

 Prepare stock solution of ferrous ammonium sulphate (100mg in 100ml water).

 Add 1ml of conc.sulphuric acid. Prepare a working standard solution(100 micro gram per ml)
 Dilute 5ml of stock solution to 50ml with water. Pipette out 1ml, 2ml, 3ml, 4ml and 5ml into
25ml volumetric flask.
 Add 1ml of 1,10-phenanthroline solution into all the volumetric flasks.
 Prepare unknown and add 1ml of 1,10-phenanthroline solution . make up the volume with
distilled water.
 Mix well and take the absorbance at 515nm.

REPORT: The absorbance of the given sample was found using UV- spectrophotometer and the
concentration was calculated. The concentration of ferrous ammonium sulphate in the given sample
was found to be _______
OBSERVATION:

Serial Working std (ml) Concentration 1,10- absorbance

no. phennthroline
solution

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Title of the Laboratory Exercise: Estimation of sulphanilamide by Colourimetry

1. Introduction and Purpose of Experiment

Sulphanilamide is the precursor for sulpha drugs. All anti - bacterial sulpha drugs contain
primary aromatic amino group with an exception of mefinide and most of them form
colourless solutions. Primary aromatic amines can be diazotised and coupled with Bratton –
Marshal reagent (BMR reagent)
The purpose of the experiment is to estimate the amount of sulphanilamide present in the
given sample.

2. Aim and Objectives

To estimate the amount of sulphanilamide present in the given sample by colourimetry
3. Experimental Set Up
Apparatus: Beakers, funnel, glass rod, Volumetric flasks, Burettes, Pippette and UV – Visible
spectrophotometer.

Chemicals and reagents: Sulphanilamide, 0.5M Hydrochloric acid, 0.1% Sodium nitrite, 0.5%

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Ammonium sulphamate, BMR reagent and distilled water
4. Experimental Procedure ist
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Construction of calibration curve:
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 Weigh accurately about 100mg of sulphanilamide and dissolve in water with the
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help of 3mlof Hydrochloric acid to make 100ml of solution in a volumetric flask.

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 Dilute the resulting solution in such a way that the concentrations of diluted
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solutions range from 0.1 to 0.25 mg/5ml (prepare 5 dilutions).

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 Pipette 5ml of each of these dilutions into separate 50ml volumetric flasks.
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 Add 5ml of 0.5M Hydrochloric acid followed by5ml of sodium nitrite to each of them
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; mix and wait for 3 minutes

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 Add 5ml of Ammonium sulphamate solution, mix and wait for three minutes
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 Add 5ml of BMR reagent

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 Measure the absorbance of the resulting coloured solutions at 540nm.

 Construct a calibration curve by taking concentration on X – axis and absorbance on
Y –axis.
Preparation of sample solution:
 Make up the given solution to the volume
 Transfer 5ml of the resulting solution to a 50ml flask
 Add 5ml of 0.5M Hydrochloric acid followed by5ml of sodium nitrite to each of them
; mix and wait for 3 minutes
 Add 5ml of Ammonium sulphamate solution, mix and wait for three minutes
 Add 5ml of BMR reagent and mix
 Measure the absorbance at 540nm

Interpolate the value on the calibration curve and obtain the concentration of the
sample. Calculate the amount of sulphanilamide present in the given sample

5. Data Collection and Tabulation

 Tabulate the data in the following format

S.No Concentration Absorbance

(mg/ml)

6. Calculations/Computations/Algorithms
Concentration of the diluted solution of the sample from the graph = mg/ml
Amount of sulphanilamide present in the given solution = mg

7. Presentation of Results

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Amount of sulphanilamide present in the given solution = mg ist
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8. Analysis and Discussions

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N N
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N
NH2
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N
HCl,NaNO2
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BMR
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O
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Cl- HN S
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O
O S O NH2
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O S O
NH2
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NH2 NH2
4-sulfamoylbenzene-1-diazonium 4-[-{4-[(2-aminoethyl)amino]
chloride naphthalen-1-yl}
diazenyl]
benzene-1-sulfonamide
H2NSO3-NH4+ + 2HNO2 → 2N2 + H2SO4+3H2O

Sulphanilamide in presence of Hydrochloric acid reacts with Sodium nitrite to form the
corresponding diazonium salt. The unreacted nitrous acid is reduced by Ammonium
sulphamate. The diazonium salt is coupled with N-1 – Naphthyl ethylene diamine di
hydrochloride solution. The colour intensity of the azo dye formed is measured using a UV-
Visible spectrophotometer in the visible region at 540 nm. The Beer– Lambert law states
that the proportion of light absorbed by a solute in a transparent solvent is proportional to
the number of absorbing molecules in the light path.
A = Log 10 (I0/I) = εbC

where: I0 = intensity of incident light, I = intensity of transmitted light, ε = molar absorptivity

C = sample concentration (in mol/L), b= cell (path) length (cm); b = 1 cm, A = absorbance
 Therefore, the absorbance is in direct relation with the concentration of the absorbing
species. The concentration can be determined by measuring the absorbance of the sample.

Conclusions

The amount of sulphanilamide present in the given sample is ________mg.

9. Comments

1. Limitations of Experiment:

a. The colour will fade on prolonged exposure to atmosphere. Therefore the absorbance has
to be measured as quickly as possible

b. Presence of excess of nitrous acid will oxidise the BMR reagent and to prevent it, the
excess nitrous acid is reduced by ammonium sulphamate solution.

c. Excess of Ammonium sulphamate will destroy the corresponding quantity of azo dye
formed. Hence stoichiometric quantities of reagents are to be used; the order of addition of

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reagents is also equally important.
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2. Limitations of Results: : The accuracy of the results depends on the technique followed to
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develop the colour and presence of primary aromatic amine impurities.

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3. Learning happened
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Learnt the colourimetric estimation of Sulphanilamide by using Bratton – Marshal reagent.

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4. Recommendations
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The method can be applied to any substance having primary aromatic amino group.
 ESTIMATION OF DEXTROSE BY CALORIMETRY

AIM - To estimate the amount of dextrose present in the given sample by colorimetry.

REQUIREMENTS - Pipette, measuring cylinder, burette , funnel , std. dextrose solution 30mg% , 1N
sodium hydroxide, DNSA solution .

PRINCIPLE -Dextrose can be estimated by calorimetric method .That is, by measuring the intensity of
colour produced when dextrose is made to react with 2,4-dinitro salicylic acid in an alkaline medium.
Then the solution is heated and the colour of varying intensity is produced depending upon the
concentration of the dextrose present. This intensity can be measured based on the absorbance of
light by colorimetry .

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PROCEDURE -
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1. PREPARATION OF DNSA SOLUTION

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 Dissolve 5g of DNSA in 100ml of 2N sodium hydroxide solution.

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 Add 250 ml of distilled water and mix wel .

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 Add 150g of roshells salt (sodiumpotassium tartarate) .

 Dissolve and make up the volume with 500ml distilled water, filter if necessary .

 Store in amber coloured bottle.

2. ESTIMATION OF DEXTROSE

 Prepare 300mg% of dextrose solution .

 Pipette 1,1.5,2,2.5,3ml dextrose solution in 15ml volumetric flask .

 Add 3ml sodium hydroxide and 3ml DNSA solution and make up the volume.

 Mix and transfer the contents in test tube and heat for 15mins in boiling water bath.

 Cool and measure the absorbance at 575 nm .

 Makeup the unknown solution and measure the absorbance.

REPORT - The concentration of dextrose in given sample was found to be ______ .

WORKING CONCENTRATION 3N NaOH DNSA DISTILLED ABSORBANCE

STD WATER
SERIAL
FORMULA
NO.

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 DETERMINATION OF λ-MAX

AIM: To determine the λ-max of KMnO4 solution.

REQUIREMENTS: Beaker, measuring cylinder, volumetric flask, cuvettes, tissue paper, KMnO4
solution.

PRINCIPLE: λ-Max is the wavelength at which maximum absorption takes place. It is a characteristic
feature of each drug.

PROCEDURE:

 Prepare KMnO4 solution (10mg%).

 Using distilled water as blank, adjust to 100% temperature.
 Switch on the colorimeter. Warm up for 15min.
 100% temperature indicates 0 absorbance.
 Add KMnO4 solution to th cuvettes and take readings from 450nm – 600nm at an interval of

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10nm.
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Select the concentration of KMnO4 to get 10% or 0.5% at the starting wavelength of 450nm.
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 Adjust to 0 absorbance using blank after change of λ every time.
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plot absorbance v|s wavelength in nm.

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REPORT: The λ-max for KMnO4 solution was found to be _____nm.

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S.NO WAVELENGTH (λ) ABSORBANCE (A)

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Title of the Laboratory Exercise: Estimation of Quinine sulphate by Fluorimetry

1. Introduction and Purpose of Experiment

Fluorescence measurements are more sensitive than colourimetric or
UV spectrophotometric methods. Fluorimetry is an example of emission spectrophotometry.
The purpose of this experiment is to estimate the amount of quinine sulphate by
fluorimetry.

2. Aim and Objectives

To estimate the amount of quinine sulphate by fluorimetry.

3. Experimental Set Up
Apparatus: Beakers, funnel, glass rod, Volumetric flasks, Burettes, Pippette and
Photofluorimeter

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Chemicals and reagents: Quinine sulphate and 0.1N sulphuric acid
4. Experimental Procedure ist
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Construction of calibration curve:
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 Weigh accurately about 100mg of Quinine sulphate and dissolve in 0.1N Sulphuric
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acid to make 100ml of solution in a volumetric flask.

 Dilute the resulting solution in such a way that the concentrations of diluted
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solutions range from 0.1 to 1ppm (prepare 5 dilutions).

 Make measurements using a photofluorometer
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 Use 360nm as excitation wavelength and 480nm as the emission wavelength.

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 Adjust the Fluorescence intensity to zero using 0.1N sulphuric acid as blank.
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 Adjust the Fluorescence intensity to 100 using the solution of highest concentration
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of the range (1ppm)

 Measure the Fluorescence intensity of the remaining four dilutions
 Construct a calibration curve by taking concentration on X – axis and Fluorescence
intensity on Y –axis.
Preparation of sample solution:
 Make up the given solution to the volume with 0.1N sulphuric acid
 Measure the Fluorescence intensity
 If the fluorescence is out of range, dilute the solution further, quantitatively.
 Note the dilution factor and measure the fluorescence intensity
 If necessary, dilute further and measure.

Interpolate the value on the calibration curve and obtain the concentration of the
sample. Calculate the amount of Quinine sulphate present in the given sample
5. Data Collection and Tabulation
Tabulate the data in the following format

S.No Concentration Fluorescence

(ppm) intensity

6. Calculations/Computations/Algorithms
Concentration of the diluted solution of the sample from the graph = ppm
Concentration of the original solution = ____ ppm

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Amount of Quinine sulphate present in the given solution = ____ mg
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7. Presentation of Results
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Amount of Quinine sulphate present in the given solution = _______ mg

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8. Analysis and Discussions

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Quinine sulphate solution in sulphuric acid is fluorescent under UV light. Though there is no
direct relationship between fluorescence and concentration, it can be shown that
concentration is proportional to Fluorescence intensity from fundamental principles of
spectroscopy. A photo- fluorometer is used to measure fluorescence. Fluorescence is an
emission phenomenon. All substances are not fluorescent. Non fluorescent substances can
be converted into fluorescent substances and estimated by fluorimetry. The fluorescent
energy is less than that of incident radiation because some energy absorbed is lost in non
radiative processes like collisions while molecules return from excited singlet state to ground
singlet state. Since excitation and emission energies are different two monochromatic
systems are to be present in a fluorometer. Fluorescent radiation is measured at right angles
to the incident light. Less expensive Photofluorometers make use of filters as
monochromators.
 Fα c

where F = Fluorescence intensity and c is concentration

Therefore, the Fluorescence is in direct relation with the concentration of the emitting
species. The concentration can be determined by measuring the Fluorescence of the sample.

Conclusions

The amount of Quinine sulphate present in the given sample is ________mg.

9. Comments

1. Limitations of Experiment:

a. The Fluorimetric determinations are for intrinsically fluorescent substances. Non

fluorescent substances can be determined by applying appropriate derivatisation
procedures to convert them into fluorescent substances

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b. Quinine sulphate is not fluorescent in hydrochloric acid solutions
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2. Limitations of Results: : The accuracy of the results depends on the accurate preparation
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of dilutions and correct calibration curve.

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3. Learning happened
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Learnt the Fluorimetric determination of Quinine sulphate.

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Learnt the construction, working and operating procedure of a photofluorometer

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4. Recommendations

Vitamins like riboflavin and thiamine chloride hydrochloride can be estimated by

fluorimetry.
Title of the Laboratory Exercise: Estimation of Riboflavin by Fluorimetry

1. Introduction and Purpose of Experiment

Fluorescence measurements are more sensitive than colourimetric or
UV spectrophotometric methods. Fluorimetry is an example of emission spectrophotometry.
The purpose of this experiment is to estimate the amount of Riboflavin by fluorimetry.

2. Aim and Objectives

To estimate the amount of Riboflavin by fluorimetry.

3. Experimental Set Up
Apparatus: Beakers, funnel, glass rod, Volumetric flasks, Burettes, Pippette and
Photofluorimeter

Chemicals and reagents: Riboflavin and 1% acetic acid

4. Experimental Procedure

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Construction of calibration curve:
 Weigh accurately about 100mg of Riboflavin and dissolve in 1% acetic acid to make
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100ml of solution in a volumetric flask.
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 Dilute the resulting solution in such a way that the concentrations of diluted
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solutions range from 0.1 to 1ppm (prepare 5 dilutions).

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 Make measurements using a photofluorometer

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 Use 440nm as excitation wavelength and 550nm as the emission wavelength.

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 Adjust the Fluorescence intensity to zero using 1% acetic acid as blank.

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 Adjust the Fluorescence intensity to 100 using the solution of highest concentration
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of the range (1ppm)

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 Measure the Fluorescence intensity of the remaining four dilutions

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 Construct a calibration curve by taking concentration on X – axis and Fluorescence

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intensity on Y –axis.
Preparation of sample solution:
 Make up the given solution to the volume with 1% acetic acid
 Measure the Fluorescence intensity
 If the fluorescence is out of range, dilute the solution further, quantitatively.
 Note the dilution factor and measure the fluorescence intensity
 If necessary, dilute further and measure.

Interpolate the value on the calibration curve and obtain the concentration of the
sample. Calculate the amount of Riboflavin present in the given sample

5. Data Collection and Tabulation

Tabulate the data in the following format
 S.No Concentration Fluorescence
(ppm) intensity

6. Calculations/Computations/Algorithms
Concentration of the diluted solution of the sample from the graph = ppm
Concentration of the original solution = ____ ppm
Amount of Riboflavin present in the given solution = ____ mg

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Title of the Laboratory Exercise: Study of Quenching of Fluorescence

Introduction and Purpose of Experiment

Quenching is decrease in intensity of fluorescence due to various factors.

Self-quenching occurs at high concentrations of the analyte due to increased collisions
among the same molecules due to overcrowding.
Certain impurities like halides will collide with excited species and reduce the intensity of
fluorescence and they are known as collisional quenchers.
The purpose of the experiment is to study the effect of quenching of fluorescence and in
turn to identify optimum concentrations of analyte and maximum permissible concentration
of quenchers for fluorescence analysis

1. Aim and Objectives

To study the pattern of quenching of fluorescence due to high concentration of analyte and
also due to increasing concentration of iodide

2. Experimental Set Up

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Apparatus: Photofluorometer, Volumetric flasks, Burette, Pipette, Beakers, funnel,
and glass rod ist
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Chemicals and reagents: Quinine sulphate, 0.1N sulphuric acid and 0.1M Potassium Iodide
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3. Experimental Procedure
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Construction of Standard curve:

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 Weigh accurately about 100mg of Quinine sulphate and dissolve in 0.1N Sulphuric
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acid to make 100ml of solution in a volumetric flask.

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 Dilute the resulting solution in such a way that the concentrations of diluted
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solutions range from 0.1 to 1ppm (prepare 5 dilutions).

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 Make measurements using a photofluorometer

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 Use 360nm as excitation wavelength and 480nm as the emission wavelength.

 Adjust the Fluorescence intensity to zero using 0.1N sulphuric acid as blank.
 Adjust the Fluorescence intensity to 100 using the solution of highest concentration
of the range (1ppm)
 Measure the Fluorescence intensity of the remaining four dilutions
 Construct a calibration curve by taking concentration on X – axis and Fluorescence
intensity on Y –axis.
Construction of curve using higher concentration range:

 Dilute the1mg/mL solution in such a way that the concentrations of diluted solutions
range from 10 to 50ppm (prepare 5 dilutions).
 Make measurements using a photo-fluorometer
 Use 360nm as excitation wavelength and 480nm as the emission wavelength.
 Adjust the Fluorescence intensity to zero using 0.1N sulphuric acid as blank.
 Adjust the Fluorescence intensity to 100 using the solution of highest concentration
of the range (50ppm)
 Measure the Fluorescence intensity of the remaining four dilutions
  Construct a curve by taking concentration on X – axis and Fluorescence intensity on
Y –axis.
Part II: Study of collisional impurity quenching:
 Prepare 1ppm solution of Quinine sulphate
 Prepare 50mL each of 0.8ppm quinine sulphate solutions containing
0, 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.005, 0.0075, 0.01 and 0.015M
Potassium iodide respectively.
 Measure fluorescence of the resulting solutions calibrating the 0.8ppm solution
free from potassium iodide for 100% intensity.
4. Data Collection and Tabulation:
Data for Standard curve :

S.No Concentration Fluorescence

(ppm) intensity

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Data for high concentration range:
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S.No Concentration Fluorescence

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(ppm) intensity
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Collisional impurity Quenching:

S.No Concentration of Fluorescence

KI(M) intensity

5. Calculations/Computations/Algorithms

A. Self- quenching:

i. Compute the quantity of 10ppm quinine sulphate solution required to prepare five
different dilutions ranging from 0.1 to 1ppm solutions
 ii. Compute the quantity of 100ppm quinine sulphate solution required to prepare five
different dilutions ranging from 10 to 50 ppm solutions

B. Collisional impurity quenching:

i. Compute the quantity of 1ppm quinine sulphate solution required to prepare
50ml of 0.8 ppm solution.
ii. Compute the quantity of 1M Potassium iodide required to prepare different dilutions
ranging from 0.0005 to 0.015M of Potassium iodide (50mL each)
( The corresponding quantities of the above two solutions to be mixed and made upto 50mL
with 0.1N sulphuric acid to study the effect of quenching by Potassium Iodide)

Presentation of Results
Plot the graphs and identify the pattern of quenching

6. Analysis and Discussions

Self - quenching: When the concentration range is between 0.1 to 1ppm,as the concentration
increases the fluorescence also increases proportionately. At higher Concentration range, the curve
gets flattened as the Concentration range is increased and at extremely high concentrations, the
fluorescence may decrease due to collisional energy loss.

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Collisional impurity quenching: As the concentration of Potassium iodide is increased the
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fluorescence intensity decreases
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7. Conclusions
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At higher concentration range, Fluorescence intensity was found to be nonlinear with

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concentration(less than expected) indicating self - quenching.

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As the concentration of KI increases, the fluorescence intensity 0.8ppm quinine sulphate

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decreases indicating collisional impurity quenching

8. Comments

1. Limitations of Experiment

With primitive instruments, adjusting to 100% at high concentration may be difficult as

the signal suppression is required

2. Limitations of Results

The results will have limited application in controlling the factors affecting fluorescence.

3. Learning happened

Learnt the operation of Photo-fluorometer and identified the effect of Concentration and
impurities on fluorescence intensity.
Title of the Laboratory Exercise: Determination of sulphates by Nephelo turbidimetry

1. Introduction and Purpose of Experiment

Nephelo turbidimetry deals with scattering of light. Nephelometry is a technique used in
immunology to determine the levels of several blood plasma proteins. For example the total
levels of antibodies isotypes or classes: Immunoglobulin M, Immunoglobulin G, and
Immunoglobulin A. It is important in quantification of free light chains in diseases such as
multiple myeloma. Dispersions can be determined by Nephelo turbidimetry. In
nephelometry scattered light intensity is measured. In turbidimetry, the intensity of
transmitted light as a function of scattered light can be measured. The study of scattering
phenomenon in general is called as nephelo turbidimetry.
The purpose of this experiment is to determine the amount of sulphate in standard and
sample turbidity in the limit test for sulphates applicable to calcium gluconate IP by
Nephelometry, compare and decide whether the sample is suitable for pharmaceutical
purposes or not. Though IP does not specify the nephelometric method, many times when
visual comparison fails Nephelometric method gives result without any ambiguity.

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2. Aim and Objectives
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To carry out the limit test for sulphates with the given sample and estimate the amount of
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sulphate present in turbidity developed in standard and sample solutions used in the limit
test and report the suitability of the sample for pharmaceutical purposes
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3. Experimental Set Up
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Apparatus: Beakers, funnel, glass rod, Volumetric flasks, Burettes, Pippette, Nessler’s
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cylinders, Cuvettes and Nephelo-turbidimeter

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Chemicals and reagents: Calcium gluconate, 25% Barium chloride solution, Standard
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sulphate solution(0.1089% aqueous solution of Potassium sulphate) , Ethanolic sulphate

solution(0.0181% alcoholic solution ), 5M acetic acid solution , Standard Formazine
suspension ( 4000NTU)
4. Experimental Procedure
Construction of calibration curve:

 Dilute Standard Formazine suspension of 4000NTU to obtain 100ml of 400NTU

suspension(stock B)
 Dilute stock B in such a way that the concentrations of diluted suspensions range
from 10 to 40NTU (prepare 5 dilutions).
 Make measurements using a Nepheloturbidimeter
 Adjust the meter reading to zero using distilled water as blank.
 Adjust the meter reading to 100 using the solution of highest concentration of the
range (40NTU)
 Measure the meter reading of the remaining four dilutions
 Construct a calibration curve by taking turbidity in NTU on X – axis and meter
reading on Y –axis.
 Preparation of standard sulphate suspension:
 To 1ml of 25% Barium chloride solution, add 1.5ml of ethanolic sulphate solution,
mix and allow to stand for 5minutes.
 Add 15ml of 0.1089%w/v solution of potassium sulphate solution and mix.
 Add 0.15ml of 5M acetic acid followed by sufficient water to produce 50ml.
 Mix and allow to stand for 5minutes.
 If necessary, dilute further quantitatively and measure.

Interpolate the value on the calibration curve and obtain the turbidity of the standard.
Calculate the amount of sulphate present in the standard.

Preparation of sample sulphate suspension

 To 10.0 g add 90 ml of boiling distilled water and boil with stirring, for not more than
10 s, until completely dissolved. Dilute to 100.0 ml with the same solvent.
 Transfer 15ml of the resulting solution which is previously filtered to a Nessler
cylinder containing 1ml of Barium chloride and 1.5ml of ethanolic sulphate solution
and mix.
 Add 0.15ml of 5M acetic acid followed by sufficient water to produce 50ml.

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 If necessary, dilute further quantitatively and measure.
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Interpolate the value on the calibration curve and obtain the turbidity of the sample.
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Calculate the amount of sulphate present in the sample.

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Compare the values and report whether the sample is suitable for Pharmaceutical
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purposes.
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5. Data Collection and Tabulation

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Tabulate the data in the following format

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S.No Turbidity (NTU) Meter reading

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6 Standard sulphate
(dilution factor = _)

7 Sample sulphate
(dilution factor_)
 6. Calculations/Computations/Algorithms
Turbidity of standard sulphate suspension
= turbidity from graph * dilution factor = X NTU
Molecular weight of Potassium sulphate = 174.259
174.259mg of Potassium sulphate ≡ 96.062mg of sulphate
108.9 mg of Potassium sulphate = 60.03mg of sulphate
100ml of Standard sulphate solution contains 60.03mg of sulphate
15ml of Standard sulphate solution contains 60.03*15/100 = 9.05mg of sulphate
X NTU = 9.08mg of sulphate
Turbidity of sample sulphate suspension = turbidity from graph * dilution factor = Y
NTU
Amount of sulphate present in the sample = 9.08*Y/X

7. Presentation of Results

Quantity of sulphate present in the sample = _______ mg

Quantity of sulphate present in the standard = _______ mg
Compare the results and report whether sample passes the limit test or not.

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8. Analysis and Discussions ist
ac
m
ar

Nephelometry and turbidimetry are methods for determining the amount of cloudiness, or
Ph

turbidity, in a solution based upon measurement of the effect of this turbidity upon the
ch

transmission and scattering of light. Turbidity in a liquid is caused by the presence of finely
en

divided suspended particles. If a beam of light is passed through a turbid sample, its intensity
tB

is reduced by scattering, and the quantity of light scattered is dependent upon the
as

concentration and size distribution of the particles. In nephelometry the intensity of the
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scattered light is measured, while, in turbidimetry, the intensity of light transmitted through
w

the sample is measured. Nephelometric and turbidimetric measurements are used in the
w

determination of suspended material in natural waters and in processing streams. The

technique is also used for determination of sulfur in coal, oil, and other organic materials;
the sulfur is precipitated as barium sulfate.
Nephelometric Turbidity Units (NTU) are the units for measurement of turbidity with
reference to the standard suspension of Formazine, a polymer formed by the action of
Hydrazine sulphate and hexamine hydrate.
5g of hydrazine sulphate and 50g of hexamine hydrate are dissolved in water separately,
mixed and allowed to stand for 48 hrs. Then the mixture is diluted to 1litre and the resulting
suspension is 4000NTU.
The limit test for sulphates principle:

Conclusions

The given sample of calcium gluconate passes/fails the limit test for sulphates
9. Comments

1. Limitations of Experiment:

a. Sulphates are common ions present in potable water. Pure distilled water has to be used.

2. Limitations of Results: : The accuracy of the results depends on the accurate preparation
of dilutions and correct calibration curve.

3. Learning happened

Learnt the Nephelometric determination of sulphate.

Learnt the construction, working and operating procedure of a Nephelo turbidimeter

4. Recommendations

Chloride limit test can also be carried out by this method. Zinc can be determined by this
method.

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Title of the Laboratory Exercise: Determination of chlorides by Nephelo turbidimetry

1. Introduction and Purpose of Experiment

Nephelo turbidimetry deals with scattering of light. Nephelometry is a technique used in
immunology to determine the levels of several blood plasma proteins. For example the total
levels of antibodies isotypes or classes: Immunoglobulin M, Immunoglobulin G, and
Immunoglobulin A. It is important in quantification of free light chains in diseases such as
multiple myeloma. Dispersions can be determined by Nephelo turbidimetry. In
nephelometry scattered light intensity is measured. In turbidimetry, the intensity of
transmitted light as a function of scattered light can be measured. The study of scattering
phenomenon in general is called as nephelo turbidimetry.
The purpose of this experiment is to determine the amount of chloride in standard and
sample turbidity in the limit test for chlorides applicable to calcium gluconate IP by
Nephelometry, compare and decide whether the sample is suitable for pharmaceutical
purposes or not. Though IP does not specify the nephelometric method, many times when
visual comparison fails Nephelometric method gives result without any ambiguity.

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2. Aim and Objectives ist
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To carry out the limit test for chlorides with the given sample and estimate the amount of
m

chloride present in turbidity developed in standard and sample solutions used in the limit
ar

test and report the suitability of the sample for pharmaceutical purposes
Ph
ch

3. Experimental Set Up
en

Apparatus: Beakers, funnel, glass rod, Volumetric flasks, Burettes, Pippette, Nessler’s
tB

cylinders, Cuvettes and Nephelo-turbidimeter

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Chemicals and reagents: Calcium gluconate, dilute nitric acid, Standard chloride
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solution(0.05845% aqueous solution of Sodium chloride) , 5% silver nitrate, Standard

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Formazine suspension ( 4000NTU)

4. Experimental Procedure
Construction of calibration curve:

 Dilute Standard Formazine suspension of 4000NTU to obtain 100ml of 400NTU

suspension(stock B)
 Dilute stock B in such a way that the concentrations of diluted suspensions range
from 10 to 40NTU (prepare 5 dilutions).
 Make measurements using a Nepheloturbidimeter
 Adjust the meter reading to zero using distilled water as blank.
 Adjust the meter reading to 100 using the solution of highest concentration of the
range (40NTU)
 Measure the meter reading of the remaining four dilutions
 Construct a calibration curve by taking turbidity in NTU on X – axis and meter
reading on Y –axis.

Preparation of standard Chloride suspension:

  To 1ml of sodium chloride solution, add 10ml of dilute nitric acid and mix
 Dilute with sufficient water to 50ml
 Add 1 ml of silver nitrate solution, mix and allow to stand for 5minutes.
 If necessary, dilute further quantitatively and measure.

Interpolate the value on the calibration curve and obtain the turbidity of the standard.
Calculate the amount of chloride present in the standard.

Preparation of sample chloride suspension

 To 10.0 g add 90 ml of boiling distilled water and boil with stirring, for not more than
10 s, until completely dissolved. Dilute to 100.0 ml with the same solvent.
 Transfer 10ml of the resulting solution which is previously filtered to a Nessler
cylinder
 Add 10ml of dilute nitric acid followed by sufficient water to produce 50ml.
 Add 1ml of silver nitrate solution and mix.
 If necessary, dilute further quantitatively and measure.

Interpolate the value on the calibration curve and obtain the turbidity of the sample.

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Calculate the amount of chloride present in the sample.
ist
Compare the values and report whether the sample is suitable for Pharmaceutical
ac
purposes.
m
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5. Data Collection and Tabulation

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Tabulate the data in the following format

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en
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S.No Turbidity (NTU) Meter reading

as
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1 40
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2
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6 Standard chloride
(dilution factor = _)

7 Sample chloride
(dilution factor_)

6. Calculations/Computations/Algorithms
Turbidity of standard chloride suspension
= turbidity from graph * dilution factor = X NTU
 Molecular weight of Sodium chloride = 58.45
58.45mg of Sodium chloride ≡ 35.45mg of chloride
100ml of Standard chloride solution contains 35.45mg of chloride
1ml of Standard chloride solution contains 35.45/100 = 0.3545mg of chloride
X NTU = 0.3545mg of chloride
Turbidity of sample chloride suspension = turbidity from graph * dilution factor = Y
NTU
Amount of chloride present in the sample = 0.3545*Y/X

7. Presentation of Results

Quantity of chloride present in the sample = _______ mg

Quantity of chloride present in the standard = _______ mg
Compare the results and report whether sample passes the limit test or not.

8. Analysis and Discussions

.in
Nephelometry and turbidimetry are methods for determining the amount of cloudiness, or
ist
turbidity, in a solution based upon measurement of the effect of this turbidity upon the
ac
transmission and scattering of light. Turbidity in a liquid is caused by the presence of finely
m

divided suspended particles. If a beam of light is passed through a turbid sample, its intensity
ar

is reduced by scattering, and the quantity of light scattered is dependent upon the
Ph

concentration and size distribution of the particles. In nephelometry the intensity of the
ch

scattered light is measured, while, in turbidimetry, the intensity of light transmitted through
en

the sample is measured. Nephelometric and turbidimetric measurements are used in the
tB

determination of suspended material in natural waters and in processing streams. The

as

technique is also used for determination of sulfur in coal, oil, and other organic materials;
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the sulfur is precipitated as barium sulfate.

w

Nephelometric Turbidity Units (NTU) are the units for measurement of turbidity with
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reference to the standard suspension of Formazine, a polymer formed by the action of

Hydrazine sulphate and hexamine hydrate.
5g of hydrazine sulphate and 50g of hexamine hydrate are dissolved in water separately,
mixed and allowed to stand for 48 hrs. Then the mixture is diluted to 1litre and the resulting
suspension is 4000NTU.
The limit test for chlorides principle:

Conclusions

The given sample of calcium gluconate passes/fails the limit test for chlorides

9. Comments

1. Limitations of Experiment:
Chlorides are common ions present in potable water. Pure distilled water has to be used.

2. Limitations of Results: : The accuracy of the results depends on the accurate preparation
of dilutions and correct calibration curve.

3. Learning happened

Learnt the Nephelometric determination of Chloride.

Learnt the construction, working and operating procedure of a Nephelo turbidimeter

4. Recommendations

Sulphate limit test can also be carried out by this method. Zinc can be determined by this
method.

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Title of the Laboratory Exercise: Simultaneous determination of ibuprofen and Paracetamol by UV
– Visible spectrophotometry

1. Introduction and Purpose of Experiment

Many pharmaceutical formulations containing two active ingredients need to be assayed in
a simple way. UV – visible spectrophotometry helps in simultaneous estimation of two
components without the necessity of separating the components under specified conditions.
The purpose of the experiment is to estimate the content of ibuprofen and paracetamol
present in tablets containing both the ingredients

2. Aim and Objectives

To determine ibuprofen and paracetamol simultaneously in tablets containing both the

ingredients.
To report the contents of tablets containing Ibuprofen and Paracetamol in combination
tablets

3. Experimental Set Up

Apparatus: Beakers, funnel, glass rod, Volumetric flasks and UV – Visible spectrophotometer.

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Chemicals and reagents: Ibuprofen, Paracetamol , Combination tablets of paracetamol and
Ibuprofen,0.1M Sodium hydroxide and distilled water ist
ac
m
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4. Experimental Procedure
Ph

Prepare solutions of 0.04 mg/mL and 0.05mg/mL concentrations of Paracetamol and

ch

Ibuprofen respectively in 0.1M solution of sodium hydroxide. Scan the solutions over the
en

wave length range 200-400nm. Over lay the spectra and identify the wave lengths λ1 and λ2
tB

such that the ratio of absorptivity of paracetamol to Ibuprofen is maximum at λ1 and

as

minimum at λ2.
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Measure the absorbance of Paracetamol and Ibuprofen at λ1 and λ2. Find the average
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weight of 20 tablets. Prepare a solution of tablet powder in 0.1N Sodium hydroxide in such a
way that the concentration of paracetamol content is about 0.04 mg/mL.

Tabulate the data as follows

5. Data Collection and Tabulation

S. No Description Absorbance

1. Absorbance of Paracetamol at λ1 (AP λ1)

2. Absorbance of Paracetamol at λ2 (AP λ2)

3. Absorbance of Ibuprofen at λ1 (AI λ1)

4. Absorbance of Ibuprofen at λ2 (AI λ2)

5. Absorbance of mixture solution at λ1 (A λ1)

6. Absorbance of mixture solution at λ2 (A λ2)

6. Calculations/Computations/Algorithm:

Calculate absorptivity of Paracetamol (aP λ1,aP λ2) and Ibuprofen (aI λ1,aI λ2)at λ1 and λ2.

From first principles of spectroscopy, the following equations can be derived.

A λ1 = aP λ1CP + aI λ1CI ……i

A λ2 = aP λ2CP + aI λ2CI ……ii where CP and CI are concentrations of Paracetamol and Ibuprofen
respectively in the mixture solution. By substituting appropriate data and solving these
simultaneous equations, CP and CI can be determined.

Calculate the amount of paracetamol and Ibuprofen in each tablet. Also calculate the
percentage content of these ingredients with respect to the amount indicated in the label.

.in
7. Presentation of Results: ist
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Amount of Paracetamol present in each tablet =
m

Amount of Ibuprofen present in each tablet =

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Percentage content of Paracetamol with reference to the label claim:

Ph

Percentage content of Ibuprofen with reference to the label claim:

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8. Analysis and Discussions

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Simultaneous equation method is one of the versatile methods for estimation of two
component pharmaceutical system. The method is useful to estimate two components
present in a formulation without the necessity of separating the components.

9. Conclusions

The given tablets contain _______mg of Paracetamol and _______mg of Ibuprofen in each
tablet. Each tablet contains ______% of stated amount of Paracetamol and __________% of
stated amount of Ibuprofen.

10. Comments

1. Limitations of the Experiment

There should be two wave lengths at which the ratio of absorptivity of component 1 to 2
should be maximum and minimum respectively
2. Limitations of Results

The concentration ratio of two components in the formulation should be in the range
over which Beer’s law is obeyed to get accurate results.

3. Learning happened

The process of simultaneous determination of two components of a formulation by UV

spectroscopy

4. Recommendations

Other combinations of drugs can also be determined by this method.

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EXPERIMENT NO.
DATE:

ESTIMATION OF IBUPROFEN I.P.RAW MATERIAL

Aim: - To estimate the amount of Ibuprofen I.P. raw material present in the given Ibuprofen I.P. raw
material by spectroscopy.

Requirements: - Ibuprofen raw material

NaOH (0.1N), volumetric flask

Principle:-

ASSAY OF MEDICINAL SUBSTANCES CAN BE DONE BY

1) Using standard absorptivity VALUE.

2) Using calibration graph
3) Single point standardization
4) Double point standardization

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Here, Ibuprofen I.P. raw material is estimated by std. calibration method. In calibration curve we plot
m

concentration on x- axis and absorbance on y- axis by the help of the graph we predict the unknown
ar

conc. of the sample with the help of regression analysis, we calculate the straight line.
Ph
ch

Y = mx + c
en

Where, m is slope
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C is y- axis intercept and x and y are the concentration and absorbance respectively.
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m = N ∑ xy – (∑x) – (∑y)

N ∑X2 – (∑X) 2

C = (∑ y) (∑x) 2 – (∑x) (∑y)

N ∑X2 – (∑X) 2

N = number of dilutions

With the help of above data we can calculate the correlation which lies between -1 and +1.

r = N (∑ xy - ∑x ∑y

N ∑x2 – (∑x) 2 N ∑y2 – (∑y) 2 ½

Procedure:-

1. PLOTTING OF CALIBRATION GRAPH FOR IBUPROFEN I.P.RAW MATERIAL

A. Preparation of 100 of Ibuprofen in 0.1N NaOH

Weigh accurately 50mg of Ibuprofen into a 50ml volumetric flask. Add about 30ml of 0.1
N NaOH dissolve well and make up the volume up to the mark to get 1 mg/ml. pipette
out 10ml of the above solution to 100 ml volumetric flask add about 50ml of 0.1N NaOH.
Shake well and make up the volume up to the mark to get 100 solution.

B. preparation of 6.8.10.12.14 concentration solution of ibuprofen

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Pipette 0.6ml, 0.8ml, 1ml, 12 ml and 1.4 ml of 100ist solution into 5 different 10ml
ac
volumetric flask. Add little of 0.1N NaOH, shake well and make up the volume up to the
m
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mark to get 6.8.10.12.14 concentration solution respectively.

Ph
ch

ʎmax of the ibuprofen in 0.1N NaOH is identified by spectral scanning of any of the
en

dilutions prepared using 0.1N NaOH as the reference. Determine the absorbance of the
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various dilutions at that nanometer using 0.1 NaOH as the reference.

as
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Plot the graph of absorbance vs. concentration and determine the correlation coff. Value
w

® of the slope (m) and the intercept (c)

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Y = mx + c

2. PREPARATION OF SAMPLE SOLUTION:-

3. Weigh about 100mg ibuprofen I.P.raw material and dissolve in 0.1N NaOH
make up to the volume with 0.1N NaOH. From this solution take 10ml and dilute it to 100ml
with 0.1N NaOH (100 from this solution pipette out 0.8ml and 1 ml of solution and
make up to 10ml (8 and 10 ) respectively.

REPORT:-
a. The percentage purity of Ibuprofen was found to be……………………..
b. The correlation coff for Ibuprofen was found to be………………..
Title of the Laboratory Exercise: Determination of Na/K by Flame Photometry

1. Introduction and Purpose of Experiment

Acidic substances can form sodium or potassium salts .The sodium/Potassium salts can be
estimated by flame Photometry. Flame photometry is also known as Flame emission
spectrophotometry. It is an atomic emission spectrophotometric method.
The purpose of this experiment is to learn the operation of a flame photometer and use it
for determination of a sodium salt.

2. Aim and Objectives

To estimate the amount of sodium ions present in the given sample by flame photometry
To learn the operation of a flame photometer
3. Experimental Set Up
Apparatus: Beakers, funnel, glass rod, volumetric flasks and flame photometer.

.in
Chemicals and reagents: Sodium Chloride and distilled water
ist
ac
4. Experimental Procedure
m
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 Connect the compressor, Fuel source with regulator and, switch on.
Ph

 Ignite the flame and aspirate distilled water

ch

 Select filter and set flame

en

 Select high concentration range

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 Select no of Standards -5
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 Save setup
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
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Calibrate using five standards between 10 to 100 ppm

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 The instrument is capable of storing the calibration data, but the values are not
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displayed.
 Then go for sample analysis
 Use the five standards used for calibration one by one to get the meter readings.
 Construct a calibration curve by plotting meter readings against concentration of
sodium in ppm
 Measure the unknown solution, interpolate the value in the graph to obtain it’s
concentration
5. Data Collection and Tabulation

S. No Concentration Meter reading

1.

3
 6. Calculations/Computations/Algorithms
2.5416g of Na Cl in one litre ≡ 1000ppm of Sodium
0.5845mg of Na Cl ≡ 0.23mg of Na

7. Presentation of Results

The given sample contains _________mg of Na = ____________mg of Na Cl

8. Analysis and
Discussions

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The working of the flame photometer involves a series of steps which is discussed in the following
sections.

Nebulisation:
The solution of the substance to be analyzed is first aspirated into the burner, which is then
dispersed into the flame as fine spray particles.
A brief overview of the process:

1. The solvent is first evaporated leaving fine divided solid particles.

2. This solid particles move towards the flame, where the gaseous atoms and ions are produced.
3. The ions absorb the energy from the flame and excited to high energy levels.
4. When the atoms return to the ground state radiation of the characteristic element is emitted.
5. The intensity of emitted light is related to the concentration of the element.
Flame photometry employs a variety of fuels mainly air, oxygen or nitrous oxide (N 2O) as oxidant. The
temperature of the flame depends on fuel-oxidant ratio.

The various processes in the flame are discussed below:

1. Desolvation: The metal particles in the flame are dehydrated by the flame and hence the
solvent is evaporated.
2. Vapourisation: The metal particles in the sample are dehydrated. This also led to the
evaporation of the solvent.
3. Atomization: Reduction of metal ions in the solvent to metal atoms by the flame heat.
4. Excitation: The electrostatic force of attraction between the electrons and nucleus of the
atom helps them to absorb a particular amount of energy. The atoms then jump to the
exited energy state.

Emission process: Since the higher energy state is unstable the atoms jump back to the stable
low energy state with the emission of energy in the form of radiation of characteristic wavelength,
which is measured by the photo detector
Conclusions

The amount of sodium chloride in the given solution =

.in
9. Comments
ist
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1. Limitations of Experiment:
m
ar

A standard solution with known molarities is required for determining the concentration of
Ph

the ions which will correspond to the emission.

ch
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The information about the molecular structure of the compound present in the sample
tB

solution cannot be determined.

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The elements such as carbon, hydrogen and halides cannot be detected due to their non-
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radiating nature.
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2. Limitations of Results: : It is difficult to obtain the accurate results of ions with higher
concentration.

3. Learning happened

Operation of flame photometer and determination of sodium by flame photometry were

learnt.

4. Recommendations

The method can be used for determination of other ions like potassium, calcium and lithium.
Experiment no. Date:

SIMULTANEOUS ESTIMATION OF DICLOFENAC AND PARACETAMOL

Aim: Simultaneous estimation of paracetamol and diclofenac in a combination formulation using U.V.
visible spectroscopy.

Procedure:

Estimation of paracetamol:-
Standard solution:- 10µg/ml of PCM in 0.1N HcL
Sample solution:- weigh accurately powder sample eq. to about 100mg of PCM. Carry out
the drug extraction with twice 25ml portion of 0.1N HCl. Filter through C 4 sintered glass
funnel. Combine the filtrate to 100ml with acid to get final concentration above 10 µg/ml,
dilute the filtrate further approximately.

Measure the extraction of both sample and standard solution at ʎmax for about 244nm.

.in
Estimation of diclofenac sodium:-
ist
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Standard solution:- 100 µg/ml
m

Sample solution:- Entire residue left after the extraction of paracetamol as described
ar

above is used for the estimation of diclofenac. Transfer the residue left in conical flask
Ph

and on top of sintered funnel quantitatively to 100ml volumetric flask with help of 0.1N
ch

NaOH. Make up the volume and carry out further dilution approx. with NaOH to get final
en

concentration of 100 µg/ml.

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Measure the absorbance of both sample and standard solution at about 276nm
w

using 0.1N NaOH as a blank.

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Report:-

The given sample of tablet contains ______________ of paracetamol and _____________ of

diclofenac sodium.
Title of the Laboratory Exercise: Comparison of IR spectra of Paracetamol and 4 –amino phenol

1. Introduction and Purpose of Experiment

4- amino phenol is a common impurity in Paracetamol as it is the raw material for synthesis
of paracetamol.
The purpose of this experiment is to distinguish between 4- aminophenol and paracetamol.

2. Aim and Objectives

To obtain the IR spectra of paracetamol and 4 – aminophenol.
To report the Characteristic IR peaks of both the compounds to distinguish them using
IRspectra

3. Experimental Set Up
Apparatus: Beakers, mortar and pestle, spatula, glass rod, China dish, Oven
and IR spectrophotometer.

Chemicals and reagents: Paracetamol, 4 – aminophenol, Xylene and Spectroscopic grade

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Potassium Bromide
ist
ac
m
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4. Experimental Procedure
Ph

Prepare 1: 100 mixture of paracetamol and KBr in a mortar and pack in the sample slot of
ch

diffuse reflectance system. Obtain the IR spectrum of paracetamol against KBr blank.
en

Similarly obtain the IR spectrum of 4 – aminophenol. Identify the characteristic IR peaks of

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both the compounds and note the distinguishing features of IR spectra of the compounds
as

under study..
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5. Data Collection and Tabulation

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S. No IR Peak Structural feature responsible

Paracetamol 4- aminophenol
6. Presentation of Results

S. No IR Peak Characteristic Paracetamol 4- aminophenol

Structural
feature

7. Analysis and Discussions

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4 – amino phenol Paracetamol

ar
Ph

The above data are useful to identify paracetamol and 4 – aminophenol respectively
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8. Conclusions
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The IR spectral data were studied for identification of paracetamol and 4 – aminophenol.
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9. Comments

1. Limitations of Experiment: Other spectra like NMR and mass are needed for identification
of compounds

2. Limitations of Results: The data may not be suitable for detecting the presence of
4 – aminophenol in Paracetamol

3. Learning happened

Learnt to operate FTIR spectrophotometer and distinguished the spectra of Paracetamol

and 4 – aminophenol

4. Recommendations

IR spectra can be obtained for other organic compounds.

Title of the Laboratory Exercise: Effect of solvent and pH on UV spectrum

1. Introduction and Purpose of Experiment

λmax is characteristic of a substance. One substance may have more than one λmax. It is the
wavelength corresponding to peak absorbance. No two compounds will have all identical
λmax values. Since it corresponds to peak absorbance values, quantitative determinations at
λmax will have highest sensitivity, least error, least interference of impurities and wide range
of concentration over which the determination will show linearity. Solvents and pH are the
factors which influence λmax values.
The purpose of this experiment is to identify the influence of solvents and pH on λmax and
also to introduce the operation of UV spectrophotometers to the students.

2. Aim and Objectives

To scan a solution of Bromophenolblue in aqueous and alcoholic solutions and in solutions

of dilute sulphuric acid and dilute sodium hydroxide in UV –Visible range and obtain the
spectra
To report the influence of solvents and pH on UV spectrum of Bromophenol blue

.in
3. Experimental Set Up ist
ac
m

Apparatus: Beakers, funnel, glass rod, Volumetric flasks and UV – Visible spectrophotometer.
ar

Chemicals and reagents: Bromophenol blue indicator solution, Dilute sulphuric acid, Dilute
Ph

sodium hydroxide, distilled water and Ethanol

ch
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4. Experimental Procedure
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Prepare four solutions of 0.04% concentration of Bromophenol blue solution in distilled

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water, ethanol, dilute sulphuric acid and dilute sodium hydroxide respectively. Measure the
w

absorbance of the resulting solutions at 5nm intervals over the range 200-800nm and plot
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the resulting values of absorbance against wave length to obtain the spectra.

Identify the influence of solvents and pH on the UV – Visible spectrum of Bromophenol blue
5. Data Collection and Tabulation

S.No Wave Absorbance

length
Distilled Ethanol Dilute Dilute
water sulphuric sodium
acid hydroxide
 6. Calculations/Computations/Algorithm

λmax of Bromophenol blue in water =

ɛmax= A/bc where c is concentration in terms of molarity and b is Pathlength in cm
Find ɛmax of the four solutions

7. Presentation of Results

S.No Parameters Water Ethanol Dilute sodium Dilute

hydroxide sulphuric acid

1 λmax

2 ɛmax

8. Analysis and Discussions

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Analyse the influence of solvents and pH on λmax and ɛmax and report any shifts in these
values.

9. Conclusions

The influence of solvents and pH on bromophenol blue were studied.

10. Comments

1. Limitations of the Experiment

Over the pH range above 4.6, there will not be much influence as Bromophenol blue is an
indicator in the pH range of 3.0 to 4.6 which changes its colour from yellow to blue.

2. Limitations of Results

Nil
3. Learning happened

Learnt the operation of UV – Visible spectrophotometer and identified the factors

influencing λmax.

4. Recommendations

Other substances can also be studied .

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