INSTRUMENTAL METHODS OF

ANALYSIS

PRACTICAL LAB MANUAL

DEPARTMENT OF PHARMACEUTICAL
CHEMISTRY
S.A.RAJA PHARMACY COLLEGE
RAJA NAGER, VADAKANGULAM-627116

1
 S.A.RAJA PHARMACY COLLEGE
RAJA NAGER, VADAKANGULAM-627116

LABORATORY CERTIFICATE

This is to certify that Smt./ Sri ……………………………………………………………… has satisfactory

completed the course of experiments in ……………………........................ practical, prescribed by the
Pharmacy Council Of India RGUHS for the ………………….. course in the laboratory of this college in the
year

Date: Signature of the Teacher In-charge

Head of the Department

Name:……………………………………………………………………………………….

Reg.No. …………………………………………………………………………………….

Examination centre …………………………………………………………………………

Date of the Practical Examination………………………………………………………......

2
 PARTICULARS OF THE EXPERIMENTS PERFORMED

Sl.No DATE NAME OF THE EXPERIMENTS P.No.

1. Introduction of U.v. spectroscopy

2. To carry out the estimation of ibuprofen in the given sample by UV

spectroscopy.

3. To Estimation Of Paracetamol by U.V. Method

4. To estimate the amount of diclofenac present in given sample.

5. To estimate the amount of ampicillin present in the given

sample.

6. To carry out the estimation of given amoxicillin capsule by

double beam spectroscopy.

7. Simultaneous estimation of aspirin and caffeine from a given

sample of combination formulation.

8. Simultaneous estimation of paracetamol and nimesulide by

UV-Visible Spectroscopy method.

9. Simultaneous estimation of paracetamol and diclofenac in a

combination formulation using UV-visible spectroscopy.

10. To carry out the simultaneous estimation of paracetamol and

ibuprofen .

11. Introduction ofchromatography.

12. To separate and identify the amino acids by paper

chromatography (ascending type).

13. To identify the given mixture of amino acid by circular [or] radial
chromatography.

14. To carry out the separation and identification of aminoacid by

thinlayer chromatography.

3
15. To carry out the separation and identification of
agivenmixtureof the aminoacids by column chromatography.

16. Introducton of H.P.L.C.

17. To determine retention time (tr) and AUC for a given sample
of diclofenac sodium by HPLC method (qualitative analysis).

18. Estimation of Diclofenac sodium from given sample by HPLC

method (quantitative analysis).

4
 Date:

Introduction To U.V –Visible Spectroscopy

The absorption of UV radiation which ranges from 200-400 nm .

The absorption of visible radiation which ranges from 400-800 nm .

In both UV and visible valence electrons absorb the energy thereby the
molecule undergoes transition from ground state to excited state.

Instruments which measure the ratio of the intensity of two beams of

light in the UV-visible region are called ultraviolet visible
spectrophotometers.

Spectroscopy is a branch of science deals with the study of interaction

of electromagnetic radiation with matter.

The energy of a EMR can be given by the following equation.

E = hν
Where , E = energy of radiation
h = plank’s constant( 6.624 × 10-34)
ν = frequency
E = hν = h.c/λ
= h.c.λ-1
While, frequency ν = c/λ

5
 Where, c = Velocity of light in vaccume
λ = Wave length
When EMR travels, a medium containing atoms, molecules or ions
where there absorption of energy, intensity of emergent light (intensity)
of incident light.

DISCUSSION:-
Spectroscopy is one of the most atomic and molecular structure
and is used in the analysis of wide range of sample. The study of
spectroscopy can be carried out under the following headings.

ATOMIC SPECTROSCOPY:-
This deals with the interaction of EMR with atoms which are most
commonly in their lowest energy state called the ground state.
MOLECULAR SPECTROSCOPY:-
This deals with the interaction of EMR with molecule. This results
in transition between rational and vibration energy level in edition to
electronic transition. Molecular spectra extends from the visible through
infra red into microwave region.
RATION (MICRO-WAVE ) SPECTRA:-
This spectra rises due to transition between the
rotational energy of gaseous molecule on the absorption of radiation
following in the microwave region. This spectra are shown by molecule
which posses a dipole movement e.g. - HCl, CO, H2O, vapours, NO, etc.
6
VIBRATIONAL – ROTATIONAL SPECTRA:-
These spectra arises due to transition included between vibrational
energy level of the molecule of the absorption at radiation belonging to
IR. IR spectra is shown by molecule when vibrational motion is
accompanied by a change in the dipole movement of molecules.
NUCLEAR MAGNETIC SPECTRA:-
It arises due to transition included between the molecule spin
energy level of a molecule in applied magnetic field.
ELECTRONIC SPECTRA:-
It arises due to electronic transition in a molecule by absorption
radiation following in visible and UV region.
ELECTRONIC SPIN RESONANCE:-
ESR results from transitions induce between electron spin energy
of molecules in an applied magnetic field.
LAW OF PHOTOMETRY:-
1) BEER’S LAW:-
It states that when monochromatic light passes through a
transparent medium containing sample. The rate of decreasing the
intensity is directly proportional to the increase in the concentration
of absorbance species.
It = I010-kc…………(1)
Where, It = intensity by travelled light
I0 = intensity of incident light

7
 k = constant
C = conc. of medium.
It states that when monochromatic light passes through at
transparent medium, the decrease in the intensity of transmitted medium
radiation is directly proportional to the increase in thickness of medium .
It = I010-kt…………..(2)
Combining (1) & (2)
It = I0.10-kct
It = I0.10-act
Above equation is called as beer’s – lambart’s law and can be
written as follows.
log I0/It = act
A = act
Where,
A = Absorbance
a = molecular absorptivity
t = thickness or pathlength of medium
c = concn of medium

8
Experiment No:1 Date:
ESTIMATION OF IBUPROFEN
AIM:-To carry out the estimation of ibuprofen in the given sample by
UV spectroscopy.
REQUIREMENT:-Beaker, volumetric flask, measuring cylinder,
pipette (10ml, 1ml)
PRINCIPLE OF UV SPECTROSCOPY:
The type of electrons present in any molecule may be
conveniently classified as
σ electron: present in saturated compounds such electrons do not
absorb near UV, but absorb vacuum UV radiation.
π electron: present in unsaturated compound example: double
bond or triple bond.
n electron: these are non bonded electrons which are not involved
in any bonding example: lone pair of electrons
Any molecule has either n electron, π electron or σ
electron or combination of these electrons. These bonding and
nonbonding electrons absorb the characteristics UV radiation and
undergoes transition from ground state to excited state by the
characteristic absorption peaks, the nature of the electron present
and hence the molecular structure can be elucidated.

9
1. LAMBART’S LAW:-
This law can be stated as follows when a beam of light as
allowed to pass through a transparent medium, the rate of decrease of
intensity with thickness of medium is directly proportional to intensity
of light.
Mathematically, the lambart’s law may be stated as follows.
-dI/dt = KI……(1)
Where, I = intensity of incident light of wave length (λ)
t = thickness of medium
k = Proportionality constant
Integrating equation & putting I= I0 & t=0
In I0/It =kt
I=I0.e-kt
It= I010-kt (changing equation to natural log)
Where, k = 1/ 2.303
2. BEER’S LAW:-
The intensity of beam of monochromatic light decreases exponentially
with increase in concentration by absorbing substance arithmetically.
It = I0e-k’c
It = I0e-kc
Combining equation,
It = I0.10-act
10
 log It/I0 = act…….(3)
Its called beer’s- lambart’s law.
Structure of Ibuprofen:

PROCEDURE:-
Preparation of standard solution:-
Weigh accurately 100 mg of ibuprofen powder, add 0.1 NaOH to
dissolve & make up volume upto 100 ml and make different
concentration 5,10,15,20,25,30 μg/ml.
Preparation of sample solution:-
Weigh accurately 10 mg of powder drug & dilute it to 100 ml to
give 100 μg/ml concentration. Take 1.5 ml of above solution & dilute to
10 ml with 0.1N NaOH to give concentration of 15 μg/ml.
Find out λmax for standard & carryout absorbance value for each sample.
REPORT:-
The λmax value for ibuprofen found to be ………….nm.
The unknown concentration was found to be……………
μg/ml.The concentration of ibuprofen in tablet was found to be ………

11
Experiment No:2 Date:
ESTIMATION OF PARACETAMOL
Aim: To Estimation Of Paracetamol by U.V. Method

References: I.P. 1996. vol-I, Organic Spectroscopy, 3rd edition by

William Kemp.

Apparatus: Volumetric flask, pipette, beaker, measuring cylinder.

Chemicals: Paracetamol (drug), 0.1 N HCl.

Structure of paracetamol:

Pharmaceutical uses of paracetamol:

analgesic for headache, musculoskeletal pain, etc

Procedure:

1) Preparation of stock solution: Dissolve 100 mg of Paracetamol

(drug) in sufficient 0.1 N HCl to produce 100 ml. From above stock
solution 1ml is taken & again diluted to 25 ml – 40 g/ml which is
the final stock solution. Take 1ml,2ml,3,4,5ml and make it upto 10ml,
To get 4, 8, 12, 16, 20g/ml. Respectively
12
 2) Preparation of sample solution: Dissolve the given sample in
100ml 0.1N HCl. Take 1ml of this solution and make the volume upto
25ml with 0.1N HCl and estimate the amount of pracetamol in the
given sample at wavelength 244 nm.

Report: The amount of pracetamol in given sample was found to

be………

13
Experiment No:3 Date:
ESTIMATION OF CAFFEINE
AIM:- To carry out the estimation of caffeine from a given sample by
U.V spectroscopy.
REQUIREMENT:- Pipette, beaker, volumetric flask, digital balance,
spatula , funnel.
REAGENT:- 7% of ammonia solution, Distilled water.
STRUCTURE OF CAFFEINE:-

PHARMACEUTICAL USES OF CAFFEINE:

Bronchial asthma and COPD
PROCEDURE:-

 ESTIMATION OF CAFFEINE:-

 Standard Solution:- Make different concentration of 4,8,12,16,20

µg/ml and measure absorbance at λmax 272 nm. Draw a standard
plot of absorbance v/s concentration.

• Sample Preparation:- Weigh powder accurately eq. to 30 mg of

caffeine in 2 ml of distilled water. Dilute 0.5 ml of the suitable to
produce final concentration (µg/ml) of caffeine citrate. Measure
absorbance at 272 nm taking water as a blank.

14
 • REPORT:- The amount of Caffine citrate in given sample was
found to be………

Experiment No:4 Date:

ESTIMATION OF DICLOFENAC SODIUM
AIM:- To estimate the amount of diclofenac present in given sample.
REQUIREMENT:- Beaker, volumetric flask, stirrer, digital balance,
funnel, NaOH, standard diclofenac sodium.
STRUCTURE OF DICLOFENAC :-

PHARMACEUTICAL USES DICLOFENAC SODIUM:-

Short term analgesics and long term anti inflammatory activity
PROCEDURE:-
Preparation of standard solution:-
Weigh accurately 100 mg of diclofenac sodium, dissolve & make
up the volume to 100 ml with 0.1 N NaOH, make different concentration
4, 8, 12, 16, 20 μg/ml. Plot the standard graph.
Sample Solution :-

15
 Weigh accurately given sample & dilute with 0.1N NaOH &
prepare 8 μg/ml concentration..
REPORT:-
The amount of diclofenac sodium in the sample was found to
be___________mg.

16
Experiment No:5 Date:
ESTIMATION OF AMPICILLIN
AIM:- To estimate the amount of ampicillin present in the given sample.
REQUIREMENT:- Beaker, volumetric flask, stirrer, digital balance,
funnel, fehling’s A& B, standard ampicillin.
STRUCTURE OF AMPICILLIN:-

PHARMACEUTICAL USES OF AMPICILLIN:-

Urinary tract infection, respiratory tract infection, meningitis,
gonorrhoea
PROCEDURE:-
Preparation of standard solution:-
Weigh 50 mg of ampicillin, dissolved in water & make up the
volume 50 ml & make up different concentration 10, 20,30, 40, 50ml.
To 2 ml of standard solution, add 3 to 5 ml of water, then add 0.5 ml of
fehling’s solution (A & B). After 1 min. measure the absorbance in
visible spectroscopy.
Preparation of sample solution:-
Accurately weigh 50 mg of powdered ampicillin & 40 ml of water,
Shake & make up the volume to 50 ml, to produce 1μg/ml.
17
Fehling’s solution:-
Prepare by stirring weighed i.e. 2 ml of fehling’s solution A & B &
diluting to 50 ml with water.
REPORT:-
The given sample found to be____________.

18
Experiment no:6 Date:
ESTIMATION OF AMOXYCILLIN
AIM:- To carry out the estimation of given amoxicillin capsule by
double beam spectroscopy.
REQUIREMENT:-
Reagents- PDAB ( P-Dimethyl- amino- benzyl, distilled water)
Apparatus- pipette, beaker, volumetric flask, measuring cylinder.
STRUCTURE OF AMOXYCILLIN:-

PHARMACEUTICAL USES OF AMOXYCILLIN:-

Urinary tract infection, respiratory tract infection, meningitis,
gonorrhoea
PROCEDURE:-
Weigh accurately 400 gm of PDBA, add 10 ml of ethyl alcohol &
2 ml of concentrated H2SO4 & dilute to 50 ml with water.
Standard solution –
1 mg/ml of amoxicillin in water.
Sample solution –
Weigh accurately equivalent 25 mg of amoxycillin & 15 ml of
water & heat on water bath to dissolve & adjust to 25 ml with water.
19
 To 2 ml of the sample & standard, add 4 ml of PDBA reagent &
heat on water bath at 60ْC for 1 hr cool to room temperature & adjust
the volume to 10 ml with water. Measure the absorbance of both sample
& standard at 410 nm. The method obeys beer’s –lambart’s law in
concentration reagent of 10-600 μg/ml.
REPORT:- The given sample was found to be__________ μg/ml.The
concentration of amoxicillin in tablet was found to be___________
μg/ml &_____________%.

20
 QUANTITATIVE ANALYSIS
1. Calibration curve method: In a single standard method, when error
is introduced in preparing the solution or measurement of absorbance,
the error in result would be greater eliminate or to minimize this error,
we can use calibration curve method. In calibration curve method, a
calibration curve is plotted using concentration Vs absorbance value of 5
or more standard solutions. A straight line is drawn either through
maximum no. of points or in such a way that there is equal magnitude of
positive and negative errors. From the absorbance of the sample solution
and using the calibration curve, the concentration of the drug ,amount
and the percentage purity can be calculated.
2. single standard or direct comparison method: In this method, the
absorbance of a standard solution of known concentration and a sample
solution is measured. The concentration of unknown can be calculated
using the formula.

A1 = €c1t

A2 = €c2t

Where,
A1, A2 – Absorbance of standard & sample

c1, c2 – Concentration of standard & sample

€ – Molecular extinction coefficient
t – Path length (1 cm)
On dividing, we get

A1/A2 = €c1t /€c2t

21
 A1/A2 = c1/c2

c2 = c1A2/A1
Since, c1, A1 and A2 are known, c2 can be calculated.
3.Using E1%1cm values: This method can be used for estimation from
formulation or raw material, when reference standard is available. E1%
1cm means the absorbance of 1% w/v solution, using a path length of 1
cm. E1% 1cm at a wavelength is a constant value for each drug and can be
seen in Pharmacopoeias and standard books on the subject.
SIMULTANEOUS SPECTROPHOTOMETRIC
DETERMINATION
when no region can be found out free from overlapping spectra of two
chromophores (groups that produce color in a compound), it is still
possible to devise a method based on measurement at two or more
wavelengths. Two dissimilar chromophores have different powers of
radiation absorption at some or several points in their absorption
spectra. If, therefore measurements are made on an unknown solution
at two wavelengths where the absorptivities of the two components
are different, it is possible to set up two independent equations and
solve them simultaneously for the two unknown concentrations.
First, it is necessary to select two points on the wavelength scale
where the ratio of the molar absorptivities is maximal. Next, it is
necessary to calculate the molar absorptivity for each component at
each wavelength selected.Thus, two simultaneous equations may be
written: C1(Є1)1 + C2(Є2) 1 = A 1

C1(Є1)2 + C2(Є2) 2 = A 2
The equations are solved for the concentration of each component
22
Experiment No:7 Date:
SIMULTANEOUS ESTIMATION OF ASPIRIN AND CAFFEINE
AIM:- Simultaneous estimation of aspirin and caffeine from a given
sample of combination formulation.
REQUIREMENT:- Pipette, beaker, volumetric flask, digital balance,
spatula , funnel.
REAGENT:- 7% of ammonia solution, Distilled water.
STRUCTURE OF ASPIRIN AND CAFFEINE:

PROCEDURE:-

 ESTIMATION OF CAFFEINE:-

• Standard Solution:- Make different concentration of 4,8,12,16,20

µg/ml and measure absorbance at λmax 272 nm. Draw a standard
plot of absorbance v/s concentration.

• Sample Preparation:- Weigh powder accurately eq. to 30 mg of

caffeine in 2 ml of distilled water by gentle shaking and filter
thorough a sintered glass funnel. Retain the residue for estimation
of aspirin. Dilute 0.5 ml of the filtrate suitable to produce final
concentration (µg/ml) of caffeine citrate. Measure absorbance at
272 nm taking water as a blank.

23
  ESTIMATION OF ASPIRIN :-

• Standard Solution:- Weigh accurately 100mg of aspirin, dissolve

and dilute to 100 ml by 7% ammonia solution, make proper
dilution to produce 4, 8, 12, 16, 20 µg/ml concentration solution ,
measure absorbance of each solution and plot standard graph.

 PREPARATION OF SAMPLE:- Dissolve entire insoluble

residue obtained above in 6 ml of 7% ammonia solution to produce
concentration of about 10 µg/ml. Measure absorbance at 240 nm
using 7% ammonia solution as a blank.
REPORT:-
The given sample contains __________of Aspirin
and______________ of caffeine.

24
Experiment No:8 Date:
SIMULTANEOUS ESTIMATION OF PARACETAMOL AND
NIMESULIDE
AIM:- Simultaneous estimation of paracetamol and nimesulide by UV-
Visible Spectroscopy method.
REQUIREMENT:- Beaker, volumetric flask, pipette, digital balance,
spatula.
REAGENTS:- 0.1N HCl,
0.1N NaOH,
Distilled water.
STRUCTURE OF PARACETAMOL AND NIMESULIDE:

PROCEDURE:-

 Preparation of standard solution of PCM & Nimesulide:-

Weigh accurately quantity of PCM & nimesulide & dilute it with
0.1 N HCl and 0.1 N NaOH respectively, to yield a final solution of
4,8,12,16,20 µg/ml. Measure absorbance for each and plot a standard
graph of absorbance v/s concentration.

25
  Preparation of sample solution:-
Take accurately weighed quantity of tablet powder eq. to 100 mg
PCM, dissolve in 0.1 N NaOH in 10 ml volumetric flask, make up the
volume to 100 ml, filtrate it. Filtrate was dissolved in 0.1 N HCl and
make up the volume up to 100 ml. Analyse both drug by UV-
spectroscopy.
REPORT:-Given sample of tablet contains __________of
PARACETAMOL and ___________ of NIMESULIDE of labeledvalue.

26
Experiment No:9 Date:
SIMULTANEOUS ESTIMATION OF DICLOFENAC AND
PARACETAMOL
AIM:- Simultaneous estimation of paracetamol and diclofenac in a
combination formulation using UV-visible spectroscopy.
STRUCTURE OF DICLOFENAC AND PARACETAMOL:

PROCEDURE:-

 ESTIMATION OF PARACETAMOL:-

• Standard Solution:- 10 µg/ml of PCM in 0.1 N HCl

• Sample Solution:- Weigh accurately powder sample eq. to about

100 mg of PCM. Carry out the drug extraction with twice 25 ml
portion of 0.1 N HCl. Filter through C4 sintered glass funnel.
Combine the filtrate to 100 ml with acid to get final concentration
of above 10 µg/ml, dilute the filtrate further approximately.
Measure the extraction of both sample and standard solution
at λmax for about 244 nm.

 ESTIMATION OF DICLOFENAC SODIUM:-

• Standard Solution:- 100 µg/ml

27
• Sample Solution:- Entire residue left after the extraction of
paracetamol as described above is used for estimation of
diclofenac. Transfer the residue left in conical flask and on top of
sintered funnel quantitatively to 100 ml volumetric flask with the
help of 0.1 N NaOH. Make up the volume and carry out further
dilution approx. with NaOH to get final concentration of 100
µg/ml.
Measure the absorbance of standard and sample at about 276 nm
using 0.1 N NaOH as a blank.
REPORT:-
The given sample of tablet contains ___________of paracetamol
and ___________ of diclofenac sodium.

28
Experiment No:10 Date:
SIMULTANEOUS ESTIMATION OF PARACETAMOL AND
IBUPROFEN
AIM:- To carry out the simultaneous estimation of paracetamol and
ibuprofen .
STRUCTURE OF PARACETAMOL AND IBUPROFEN:

PROCEDURE:-

 ESTIMATION OF PARACETAMOL:-

• Standard Solution:- 10 µg/ml of PCM in 0.1 N HCl.

• Sample Solution:- Weigh accurately exact quantity of sample eq.

to 100 mg of paracetamol. Extract the drug and filter through C4
sintered glass funnel. Measure the extinction of both solution at
λmax of about 244 nm using HCl as a blank.

 ESTIMATION OF IBUPROFEN:-

• Standard Solution:- 250 µg/ml of ibuprofen in methanol.

• Sample solution:- Dissolve the entire residue obtained in the

estimation of PCM in methanol to make 10 ml. Further dilution are
done approximately and step wise with methanol to get the final
concentration of 250 µg/ml. Measure of both sample and standard
solution at a maximum of about 267 nm using methanol as a bank.
29
REPORT:-
The given sample contains ________mg of paracetamol and
_______mg of ibuprofen.

30
 CHROMATOGRAPHY
Chromatography is a powerful separation method that finds application
in all branches of science ,chromatography was invented and named by
the Russian botanist Mikhail Tswett at the beginning of the twentieth
century .

He employed the technique to separate various plant pigment

such as chlorophyll and xanthophylls.

The name chromatography (greek chroma –colour and graphy

–writing )means colour writing.

It is used to resolve a multicomponent mixture of trace,minor or

major constituents into its individual fractions. Final identification
requires confirmation by some other analytical procedures, such as
infrared spectroscopy ,NMR or mass spectrometry qualitative analysis
can be carried out by measuring the area of the chromatographic peak.
Hence chromatography can be used for qualitative analysis.

Definition : chromatography may be regarded as an analytical

technique employed for purification and separation of organic and
inorganic subtrates.chromatography is mainly based upon then principle
that all the substances have a tendency of adhering to the substrates with
definite forces which is a combination of adsorption, partition and ion
exchange.

31
Classification in chromatography:

Based on the nature of the fixed and moving phase different types of
chromatography are as follows

1. Adsorption chromatography
2. Partition chromatography
3. Gas chromatography
4. Ion exchange chromatography
5. Exclusion chromatography
1. Adsorption chromatography;

It is a technique in which the stationary phase is solid( ex

:alumina or silica ) and the mobile phase is either a gas or liquid.
Separation takes place when one component of a two component
mixture is strongly adsorbed than the other by solid stationary phase ,the
adsorbed components are then eluted by passing suitable solvents
through the column. The commonly used adsorbents for column
adsorption chromatography are sucrose, cellulose .starch ,calcium
carbonate,calcium sulphate, silica gel,magnesium carbonate, charcoal
etc.

2. Partition chromatography:

In this technique the stationary phase is a liquid, frequently

water held on a suitable inert porous solid such as cellulose. The mobile
32
phase can be gas or liquid mixture. In this case the solute gets distributed
between the fixed liquid and the moving liquid (solvent).

Ex: paper chromatography is a special type of partition chromatography

in which the adsorbent column is a paper strip.

The commonly used solvent systems are

Stationary phase: Water

Water + acid

Water + alkali

Water + buffer

Alcohols

Formamide

Glycols.

Mobile phase:

Alcohols (n-butanol)

Isobutanol

Hydrocarbons(benzene,toluene,hexane)

Chloroform

33
 Ethyl acetate

3. Gas chromatography (Gc)

When the moving phase is a mixture of gases it is called gas

chromatography or vapor phase chromatography

The carrier gases generally used are helium and nitrogen.

Gas liquid chromatography :

The mobile phase is a gas and the stationary phase is a thin layer of
nonvolatile liquid bound to a solid support

Gas solid chromatography :

This utilizes the solid adsorbent as the stationary phase and

adsorption process takes place.

Advantages of GC :

The technique has strong separation power and even complex

mixtures can be resolved in to constituents.

The sensitivity of the method is quite high ,it is a micro method

and only a few milligram of the sample is sufficient for analysis.

4. Ion exchange chromatography

34
 Ion exchange is a process in which an interchange of ions of like
signs takes place between a solution and essentially in soluble solid (ion
exchanger) in contact with the solution .In ion exchange
chromatography a reversible exchange of ions is possible between ions
in a liquid phase (mobile phase) of a solid, insoluble substances
containing ionic sites (ion exchange resin). Ion exchange resin consists
of beads of highly polymerized cross linked organic material containing
large number of acidic or basic groups.

5. Exclusion chromatography :

It is a chromatography process in which separation of sample

component takes place according to molecular size . The two types of
exclusion chromatography techniques are

• Gel permeation

• Sieving separation

The other types of chromatograph include

• Thin layer chromatography

• High pressure or( performance) liquid chromatography

• High pressure thin layer chromatography (HPTLC)

• Counter current chromatography

35
Theories of chromatography ;

➢ Plate theory

➢ Rate theory

Plate theory

Plate theory was developed by Martin and singe , according to this

theory a chromatographic column consist of series of discrete yet
continuous horizontal layer which are termed as the theoretical plates
An equilibration of the solute between the stationary and mobile phase
takes place at each of these plates . Migration of solute occurs by a
series of stepwise transfer between one plates to the other. The
efficiency of separation in a chromatographic column gets increases as
the no of theoretical plate increases.

No of theoretical plates is given by N= L/ H

Where L = length of the column

H= height equivalent to theoretical plate

Rate theory

Rate theory is able to explain the effect of variables such as

mobile phase velocity and adsorbabilities which determine the width of
an elution band it also relates the effect of these variables on the time
taken by a solute to make its appearance at the end of the column. If a
36
molecule is attached to the stationary phase its migration down the
column is temporarily stopped , but the zone passes on that is one
molecule make it immobilized temporarily on the column while the
molecule migrate . A particle can migrate only if it is present in the
mobile phase and as a result migration down the column is highly
irregular thus some solute molecules may migrate rapidly where as
others may lags behind, the net result of all these random individual
process the width of the zone gets increased as it migrates down the
column because more time is needed for migration to take place hence
the zone width is directly related to residence or retention time on the
column and inversely proportional to the mobile phase velocity.

Development of the chromatogram

Three methods have been used to develop chromatogram , they are

frontal analysis , elution analysis ,and displacement analysis .

Frontal analysis

This method was developed by Tisellius . It consists of passing the

sample solution continuously through the adsorbent column . This
makes the active centres of the adsorbent column being occupied by the
more strongly adsorbed solutes. The less strongly adsorbed solutes are
adsorbed down the column or collect in the migrating solvent front . the
solvent issues out first from the column exit followed by the least

37
adsorbed solute . Other solutes emerge one after another depending
upon their degree of adsorbtivity

Elution analysis

This technique is most widely used to develop

chromatograms . In this technique a small quantity of sample solution is
introduced at the top of the column, pure solvent is than poured down
the column , this give rise to differential migration of solutes in the
mobile phase , each solute emerges from the column exit depending on
its partition coefficient . If the partition coffiecient for the solute in the
samples are sufficiently different the mixtures separates out in to the
bands which migrate at different rates.

Displacement analysis

In this methiod, a small quantity of a sample solution is first

introduced at the top of the column . the component of the mixtrure are
than separated by running a solution of substances which is more
strongly adsorbed than any off the component of the mixture . the
substance thus run is termed as displacing agent.
Composition of what Mann filter paper:

Alpha cellulose 98.99 %

Beta cellulose 0.3 -1%

38
 Pentosans - 0.4-0.8%

Ether soluble matter 0.015-0.02%

Ash 0.07-0.01%

Mobile phases for paper chromatography:

Isopropranol - Ammonia –water

n-butanol – acetic acid – water

Water- phenol.

Formamide – chloroform

Formamide – chloroform – benzene

Formamide - benzene – cyclo hexane

Locating or visualizing agents ;

Ninhydrin reagent – for detection of amino acids , amino sugar ,

Aniline phthalate - for detection of reducing sugars

Antimony trichloride in CHCl3 - for steroids and glycosides

Dragendroff s reagent – alkaloids

Bromocresol purple – halogen ions (except Flourine ) dicarboxylic

acids
39
Bromocresol green – carboxylic acids

40
Experiment No:11 Date:

PAPER CHROMATOGRAPHY

AIM: To separate and identify the amino acids by paper

chromatography (ascending type).

REQUIREMENTS: Whatmann filter paper grade-1, paper

chromatography chamber, capillary tube, test tubes, standard amino
acid solutions, sample amino acid solution, Ninhydrin reagent.

PRINCIPLE: Paper chromatography is based on the principle of

partition in which the substances are distributed between two liquids,
i.e., one is the stationary liquid (usually water) which is held in the
fibers of the paper and called the stationary phase, the other is the
moving liquid or developing solvent and called the moving phase /
mobile phase. The components of the mixture to be separated migrate
at different rates and appear as spots at different points on the paper.

In this technique, a drop of the test solution is applied as a small

spot on the filter paper and the spot is dried. The paper is kept in a
closed chamber and the edge of the filter paper is dipped into a
solvent called developing solvent. As soon as the liquid gets through
its capillary axis and when it reaches the spot of the test solution (a
mixture of two or more substances), the various substances are moved
by solvent system at various speeds. When the solvent moves these
41
substances to a suitable height, the paper is dried and the various
spots are visualized by suitable reagents called visualizing reagents.
The movement of the substance relative to the solvent is expressed in
terms of Rf values which are a migration parameter.

DISCUSSION: The analysis of unknown substances by the flow of

solvent on a filter paper is known as paper chromatography. This
technique proceeds by mechanism which is partly partition (distribution)
and partly adsorption. The constituents of a mixture are distributed
between the water held in the filter paper and organic solvent.

TYPES: Ascending chromatography, descending chromatography,

ascending-descending chromatography, radial chromatography, two-
dimensional chromatography.

Migration parameters: The position of migrated spots on the

chromatogram is indicated by different terms such as Rf, Rx, Rm, and Rc.
These parameters are quantitative and qualitative characteristic of a
substance.R is a function of the partition co-efficient. It is a constant for a
given substance, provided the conditions of the chromatographic system
are kept constant with respect to temperature, type of paper, duration, and
direction of development, etc.

Rf=Distance traveled by the solute from the origin line

Distance traveled by the solvent from the origin line

42
 Rx=Distance traveled by the substance from the origin line

Distance traveled by the standard substance ‘x’ from origin line

Rm=log (1/RF – 1)

In some cases, the solvent front runs off the end of filter paper, the
movement of a substance in such cases is expressed as Rx.

• Rm is used when Rf values of chemically related compounds are

very close.
Types:

(1) Partition chromatography (paper used).

(2) Paper adsorption chromatography (paper first impregnated with
an adsorbent, like silica or alumina).
PROCEDURE:

• Take a sheet of Whatmann paper draw a line 2-4cm above the

bottom boundary.
• Apply 5 micro liters of amino acid solutions at an interval of 1-
2cm.
• Dry this in a stream of hot air.
• Place this paper onto the paper chromatography chamber, saturated
with solvent system.
43
 • Allow the edge of the paper to touch the solvent.
• Run the chromatogram until the solvent has reached 3/4th of the
strip for 1hr.
• Now take out the paper and dry it in a stream of hot air.
• Visualize the spots by spraying Ninhydrin reagent on the paper and
dry it.
• Calculate the Rf value and report it.
APPLICATIONS:

(1) Purity of the sample: Purity of the sample is routinely carried

out for this direct comparison of the sample and authentic
sample is done, if the impurity present is shown as extra spots
and this can be detected very easily.
(2) Examination of the reaction: To assess whether the reaction is
complete or otherwise, this method is also used in checking
other separation process and purification process like
distillation, molecular distillation.
(3) In chromatography, it is used for the identification of natural
products like volatile oil, fixed oil, waxes, terpenes, alkaloids,
glycosides, steroids, etc.,
(4) Biochemical analysis: Separation of biochemical metabolites /
constituents from its body fluids, blood plasma serum, urine,
etc.,

44
 (5) For the detection of impurities in a pharmacopoeia drug
analysis.
(6) Various drugs like hypnotics, sedatives, anticonvulsants,
tranquilizers, local anesthetics, steroidal drugs have been tested
qualitatively.
(7) Important in multicomponent separation and formulation.
REPORT: The Rf value of sample A was found to be …….. and ……..
Therefore the sample contains

45
Experiment No:12 Date:

CIRCULAR PAPER CHROMATOGRAPHY

Aim: To identified the concentration of the given mixture of amino acid

by circular [or] radial chromatography.

Reference: 1) Instrumental method of chemical analysis by G. Chatwal

and P Anand 5th Edition page -585-590

2) Analytical chemistry by B. K. Sharma page -85-95.

Requirement: n-butanol : Glacial acetic acid : Water, 4 : 1: 5

0.2 % w/v ninhydrin in acetone and standard amino acid solution.

Apparatus: Separating funnel, petridish, sprayer, and capillary tube,

whatman filter paper.

Principle: In this technique a circular piece of paper having a wick cut

parallel to the radius from edge to center is used the center is deposited
at the paper at the upper end of the wick and flow through the paper is
placed on the edge of circular dish as a result the liquid ascend the wick
and flow through the paper the flow of mobile phase is outward from a
central part on spot

The process allowing the solvent to move along the filter paper is called
development the separation of components after the development is
46
called resolution. The amount applied as a spot to be paper is called
loading

For The Circular Paper Chromatograph

1. Choice Of Paper:

The paper is many grades of thickness and porosity in sheets. Suitable

paper for a particular can only determined by a trial and error but
whatmann No: 1 is of great importance and wide applicability for
medium flow rate

2. Temperature Control: Temperature should be kept to a minimum

either by insulating individual that with plastic foam [or] by
thermostatically controlled cabinet

3. Application of samples: Sample can be applied by a capillary tube,

platinum loop or quantitatively by a micropipette, graduated capillary or
micro syringe. Several ampoules can be spotted at once by various
automatic and semiautomatic device it is preferable to apply the sport in
small volume the position of compound can be expressed by their
retardation factor [Rf]

47
[Rf] = Distance traveled by component from original line
-------------------------------------------------------------
Distance traveled by the solvent from origin line

RF value depends upon several factors: are

1. The nature of solvent.

2. The medium used quality of the paper.
3. The nature of mixture to be separated.
4. The temperature.
5. The size of the container in which the experiment is performed.
Procedure:

1. Prepare solution of 0.1mg / 100ml each of glycine, Leucine, alanine

and mixture of glycine, Leucine,alanine.

2. The apparatus was fashioned equalized petridish and a filter disc.

3. The wick was made from a filter paper rolled into a cylinder 2-3 mm
thick at the end in the form of a brush and inserted through a small note
at the center of the paper.

48
4. The sample drop to be analyzed was placed on paper and is air dried
the developing solvent was prepared n-butanol : GL acetic acid : water
[4 : 1 : 5]

5. The was placed b/n two glass dishes the lower containing solvent the
solvent which is already saturated for 1 hr.

6. The solvent rises by capillary and run the chromatogram compleate

development and removes the paper and mark solvent front air dry the
paper and spray the ninhydrin reagent thus purple spot identify the
amino acid determine the [Rf] value.

Result: The Rf value of amino acid were found to be

Glycine-------

Leucine -------

Alanine-----------

Unknown ---------

Conclusion: The unknown Rf value 1 and 2 matches with glycine and

phenylalanine so hence the given mixture contains glycine and
phenylalanine.

49
Experiment No:13 Date:

THIN LAYER CHROMATOGRAPHY

AIM: To carry out the separation and identification of amino acid by

thin layer chromatography.

DISCUSSION: Thin layer chromatography usually employs solid layers

which adhere to the glass plate, generally by virtue of a binding agent
such as calcium, sulphate which is incorporated with this and particular
advantage is that corrosive reagent which attack paper can be used for
spraying. The prepared thin layer on glass is often called as chromates
plate.

Chromates plates prepared by applying a uniform layer of an

appropriate material in the form of an aqueous paste to clear glass
plate.The usual sorbents include silica gel, alumina, keiselghur, and
cellulose powder.

Silica gel is largely used because it can serve as a medium for the
separation of both polar compounds and non polar compounds. Spots are
placed at regular and equal distance. The mixture may be applied using a
capillary tube. Development is carried out by ascending technique in a
small glass jar.

50
PREPARATION OF SILICA GEL PLATE: Mix 2g of silica gel with
4ml of distilled water in a small conical flask and shake/mix well and
spread over glass plate.

MATERIALS

Solvent system: - n-Butanol: Acetic acid: Water (8:2:2)

Visualizing agent: - Ninhydrin reagent

Amino acids: - Alanine, Valine, Leucine, and Glycine

PROCEDURE:

Preparation of the plates

The plates are prepared by pouring slurry of adsorbent (silica gel)

in water with smooth consistency /by using a spreader.Allow the plates
to stand for 5 minutes. Then dry the plates at 120 degrees Celsius for 30
minutes. Then allow to cool and set aside for exposure to the atmosphere
for about 30 minutes.

Draw the baseline 1.5cm from the lower end of the glass plate.
Mark the points equidistant from each other and spot the sample using
capillary tube.

Preparation of the standard

51
Dissolve 5mg of each of the above amino acids separately in 0.25ml of
water. Measure the volume of water using 1ml graduated pipette. Mix a
drop of each solution to provide the test mixture dilute the remainder of
each solute separately to 1ml to give solution of the respective amino
acids. The latter will contain about 5mg/ml of each amino acid / ml.

Development of chromatography

After the application of the sample the plates are placed in previously
saturated solvent system. The solvent is allowed to pass upto 3/4 th of the
plate. Plate is then removed and air dried. Visualizing reagent
(Ninhydrin reagent) is sprayed and plates are dried in hot air oven till the
spots are developed. Rf value is calculated.

REPORT: The Rf value of the mixture of unknown sample was found

to be ………. and …………..

52
Experiment No:14 Date:

COLUMN CHROMATOGRAPHY

AIM : To carry out the separation and identification of a given

mixture of the amino
acids by column chromatography

APPARATUS : Glass column with a stop cock

Beaker

Conical flask

TLC plate

Capillary tube

CHEMICALS : Benzene , silica gel , sand , glass wool , sintered

glass

PRINCIPLE: Column chromatography is based on the principle of

selective adsorption. Mixture to be separated is dissolved in a suitable
solvent and allowed to pass through a tube containing the adsorbent. The
component which has greater absorbing power is adsorbed in the upper
part of the column. the net component is adsorbed in the lower portion
of the column which has lesser adsorbing power than the first
component, the process is continued, as a result the materials are
partially separated and adsorbed in the various part of the column, the
53
initial separation of various component can be improved by passing
either the original or some other suitable solvent slowly through the
column Two procedure are used to separate various constituents

A) The various zones are cut with a knife at boundaries and the
substance present in the zones is extracted with suitable solvent this
process of recovery of constituents is known as elution.
B) After development the column may be washed with more solvent,
now termed the eluant and each component is collected separately as it
reaches the end of the column and is released.
DISSCUSION:

Solvent is selected based on the solubility relation of substances.

The solvents commonly used as mobile phase are arranged according to
there increasing eluting power .thus elutotropic series are obtained
which vary with type of adsorbent used.

The solvents used in chromatography have three functions to perform

1. Serve to introduce the mixture to the column

2. Effect the process of development by which zones of

chromatography are separated to their fullest extent

3. Used to remove the required content of each zone from the

mechanically separated parts of the column or from the column as a

54
whole after it is partly developed. The solvents used for this purpose are
called eluants. Solvents: petroleum ether

➢ Cyclohexane

➢ Carbontetrachloride

➢ Trichloroethylene

➢ Benzene

➢ Chloroform

➢ Absolute alcohol

➢ Ethyl acetate

➢ Pyridine

PROCEDURE

1) Initially TLC was performed for the selection of solvent system

2) The sample was dissolved in suitable solvent, using a capillary tube

a small amount of sample was applied on TLC plate.

3) The TLC plate was developed in different solvent system air dried
and observed under uv light

4) The solvent which shows sufficient separation between the spots was
selected as mobile phase (benzene)
55
5) Silica gel was mixed with the mobile phase and then filled in to the
column

6) Sand was washed with the mobile phase and added in to the column
to form a layer on the top of the adsorbent

7) Sample was mixed with mobile phase and introduced in to the

column.

8) Mobile phase level was maintained above the adsorbent level

9) Eluent was collected from the bottom and presence of individual

constituents was determined by performing TLC and observing the
plates in UV

10) A series of eluent were collected and evaporated to get the pure
sample .

REPORT

56
 Date:

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Introduction

High-performance liquid chromatography (HPLC) is a form of liquid

chromatography to separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector. Compounds are separated
by injecting a plug of the sample mixture onto the column. The different
components in the mixture pass through the column at different rates due
to differences in their partitioning behavior between the mobile liquid
phase and the stationary phase.

PARAMETERS used in HPLC

RETENTION TIME (tR): is the time between sample injection (time

zero) and the appearance of the band maximum; when all conditions are
held constant, the retention time for a given peak (or compound) remains
constant.

RETENTION VOLUME: It is the volume of mobile phase required to

elute50% of the component from the column

Retention volume= retention time x flow rate

57
Separation factor: SEPARATION FACTOR (a): defined for two
adjacent bands as the ratio of k' for the second band divided by k' for the
first band; when the separation factor equals 1.00, the two bands are on
top of each other and completely unseparated. Changing the
experimental conditions can increase a and permit the two bands to be
separated. Also sometimes called

RELATIVE RETENTION

RESOLUTION (Rs ): defines how well separated two adjacent bands

are. Larger values of resolution mean better separation.

THEORIES OF HPLC

PLATE THEORY:

According to this theory, the chromatographic column consists of

continuous, narrow, horizontal plates of equal units called as

“Theoretical plates”. Though these units are hypothetical, they give rise
to a very useful method for the practical measurement of column
efficiency.

Height equivalent to theoretical plates (HETP):

It is the length of the column necessary to obtain the equilibrium of the

solute particles between the two phases & is given by

HETP = length of the column / no. of theoretical plates

58
Where L= length of the chromatographic column

N= no. of theoretical plates

HETP is given by Van Demeter equation:

HETP=A+B/u + Cu

Where,

A=eddy diffusion term or multiple path diffusion which arises due to

packing of the Column. This is unaffected by the carrier gas velocity or
flow rate. This can be minimized by uniformity in packing

LIQUID CHROMATOGRAPHY

B= longitudinal diffusion term or molecular diffusion which depends on

flow rate

C= effect of mass transfer which depends on flow rate.

u= flow rate or velocity of the mobile phase

Resolution (R):

It is defined as the distance between two adjacent peak maxima divided

by their average peak width.

N, HETP, and R are preferred as the measures of the column efficiency.

59
Retention time ( Rt ) :

It is the difference in time between the point of injection and appearance

of peak maxima.

􀂷 It is the time required for 50% of a component to be eluted from the

column.

􀂷 Measured in min or sec

RATE THEORY:

Plate theory failed to explain the ways to improve the performance of

the column, which the rate theory did. This theory explained the fact that
the mobile phase flows continuously & that the solute particles

are constantly being transported & partitioned in the column. It can be

explained by Van Deemeter equation:

TYPES OF HPLC TECHNIQUES

A) Based on modes of chromatography

1. Normal phase mode

2. Reverse phase mode

B) Based on principle of chromatography

1. Adsorption chromatography

60
2. Ion exchange chromatography

3. Size exclusion/gel permeation chromatography

4. Affinity chromatography

5. Chiral chromatography

C) Based on elution techniques

1. Isocratic separation

2. Gradient separation

D) Based on the scale of operation

1. Analytical HPLC

2. Preparative HPLC

E) Based on the type of Analysis

1. Qualitative analysis

2. Quantitative analysis

F) Based on the internal diameter of the column employed for HPLC

1. Based on modes of chromatography- Interaction or Affinity between

polar-polar and nonpolar-nonpolar

61
is more whereas the interaction affinity between the polar-no polar is
less. A. Normal phase chromatography. Separation of polar analytes by
partitioning onto a polar bonded stationary phase In this the stationary
phase is polar (e.g. silica gel) and the mobile phase is non-polar. In this
technique the non-polar compounds travel faster and are eluted first this
is because of the less affinity between the

Solute and the stationary phase. Polar compounds are retained for a
longer time in the column because of more affinity towards the

Stationary phase and take more time to be eluted from the column. This
becomes a disadvantage and the process cannot be applied

for the pharmaceutical products as most of the drug molecules are polar
in nature and takes longer time to be eluted and detected As a

result this technique is not widely used in Pharmacy

B. Reversed Phase chromatography: Separation of non-polar analytes

by partitioning onto a non-polar, bonded stationary phase.

In reverse phase chromatography, a non-polar stationary phase and polar

mobile phase is used. Hence, the polar components get

Eluted & first and the non-polar components are retained for a longer
time. Since most of the drugs and pharmaceuticals are polar

62
In nature, they arc not retained for a longer time and are eluted faster,
which is advantageous

B. Based on principle of chromatography:

1. Ion-exchange chromatography. The principle of separation is ion-

exchange, which is reversible exchange of functional group In ion
exchange chromatography, an ion exchange resin is used to separate a
mixture of similar charged ions. For cations a cation exchange resin is
used for anions, an anion exchange resin is used.

2. Affinity chromatography: uses the affinity of the sample with

specific stationary phases. This technique is mostly used in the flied

of biotechnology, microbiology, biochemistry etc.

2. Based on elution techniques:

􀂷 Isocratic elution: Here the same mobile phase combination is used

throughout. The process of separation. The same polarity or elution
strength is maintained throughout the process.

􀂷 Gradient elution: Here a mobile phase combination of lower polarity

or eluti0n strength is used followed by gradually increasing the polarity
or elution strength.

63
3. Based on the scale of operation:

Analytica1 HPLC: used only for the analytical purpose and th recovery
of the sample is not done since the qun1ity of sample used is very small
usually in micrograms. Preparative HPLC: here the individual fractions
of pure compound

Can be collected using fraction collector and the collected samples can
be reused Eg.separations of few grams of a mixture.

Based on the type of analysis:

a. Quantitave analysis: is used to identify the compound, detect

impurities and to find the number of components. It is done by the
retention time values.

b.Quantitative analysis: is done to detect the amount of individual or

several components in a mixture. It is done by comparing the peak area
of standard and sample

Principle of separation in HPLC:

When a mixture of components is introduced into a HPLC column, they

travel according to their relative affinities towards the stationary phase.
The component which has more affinity towards the adsorbent travels
slowest the component which has less affinity towards the stationary

64
phase travels faster. Since no two components have the same affinity
towards the stationary

phase, the components are separated.

Stationary Phases: Stationary phases are particles which are usually

about 1 to 20 μm average diameter (often irregularly shaped).

Polar (“Normal” Phase):

o Silica, alumina

o Cyano, amino or diol terminations on the bonded phase f Non-

Polar(“Reversed Phase”)

o, C18 to about C8 terminations on the bonded phase

o Phenyl and cyano terminations on the bonded phase

NOTE

1. Either silica or bonded silica materials should not be used above pH8
because silica itself begins to dissolve at around pH 8.

2. Chemically bonded materials also get cleaved off at pH 2.0

3. Adsorbed compounds can usually be removed from silica columns by

flushing with methanol or water and from bonded phase columns by
flushing with methanol or dichloromethane

65
4. In Adsorption chromatography, there is no additional phase on the
stationary phase particles (Silica, alumina)

5. In Partition chromatography, the stationary phase is coated on to

(often bonded) a solid support (silica, alumina, divinylbenzene resin)

The Mobile Phase in HPLC

Must do the following

1. solvate the analyte molecules and the solvent they are in

2. be suitable for the analyte to transfer “back and forth” between during
the separation process

3. Must be compatible with the instrument (pumps, seals, mixing,

detector, etc)

4. compatible with the stationary phase

5. readily available (often use liters/day)

6. of adequate purity Spectroscopic and trace-composition usually

7. Not too compressible (causes pump/flow problems)

Note:

1. Solvent purity is very important in preparative HPLC where the

sample s usually recovered by the evaporation of the solvent.

66
2. In order to achieve reproducibility in adsorption chromatography, the
Water content of the mobile phase must be controlled carefully.

3. because most part of the chromatograph in contact with the mobile

phase is constructed of stainless steel or Teflon, there are few
restrictions of the mobile phase. However, halide ions should be avoided
because these ions attack stainless steel, especially at low pH values.

4. Algal growth may result from the prolonged use of the biological
buffer solutions such as citrate and these usually have to be

discarded

5. While working with buffer solutions and other potentially corrosive

eluents, the system should be flushed out with water or a

water-methanol mixture when not in use. This is advantageous for both

the chromatograph as well as column packing material.

67
INSTUMENTATION OF HPLC

1. Degassing system

2. Pump solvent delivery system

3. Check valves

4. Pulse damper

5. Pre-columns

6. Guard column

7. Flow splitter

8. Auto sampler
68
9. Sample injection port

10.Column

11.Detector

APPLICATIONS OF HPLC

1. Quality control testing of drugs

2. In Qualitative & Quantitave analysis

3. Therapeutic monitoring of drug metabolism studies

4. Separation & control of impurities

5. In analysis of biological fluids

6. Stability studies

7. Study of metabolic pathways in basic biochemical pathways

8. Separation of positional isomers, enantiomers, Optical isomers

9. Industrial applications

a. Determination of synthetic intermediates ex: atenolol

b. In determining traces of impurity ex:Tolnafate

c. Stability studies ex Acyclovir

69
 Experiment No:15 Date:

DETERMINATION OF RETENTION TIME AND AREA UNDER

CURVE FOR A GIVEN DRUG SAMPLE BY HPLC METHOD

AIM: To determine retention time (tr) and AUC for a given sample of
diclofenac sodium by HPLC method (qualitative analysis).

PRINCIPLE: Chromatography is a physical method of separation of

different kinds of compounds from complex mixture; uses two mutually
insoluble/immiscible phases- stationary phase and mobile phase, the latter
runs over the former.

High pressure liquid chromatography (HPLC) uses a high pressure in order

to pass the mobile phase through uniformly packed stationary phase
column, at sufficient velocity and flow rates, and can be operated at various
pressures as high as 1500psi.

At such high pressure, resolution property of chromatographic system

changes; hence, also sometimes, known as high performance liquid
chromatography (as performances of stationary and mobile phases change
at high pressure).

Retention time (tr): It is the time required by the given sample to elute out
of the column with the mobile phase. The sample is injected to the
mobile phase through the injection port before the column.

70
In HPLC, tr is related parameter to, Rf (retention factor) in paper
chromatography and TLC.

INSTRUMENTATION:

(1) Mobile phase reservoir

(2) Mobile phase delivery system , consists of:
• Pumps
• Pubic damping system
• Flow regulator
• Mixing chamber
• Deaeration and filters
(3) Sample injection system
(4) Column
• Guard column
• Main column
• Oven (thermostatic chamber)
(5) Detector
(6) Data processor, recorder and display.

REPORT: Retention time for diclofenac sodium was found to

be…………… and AUC was found to be…………………
71
Experiment No:16 Date:

ESTIMATION OF DICLOFENAC SODIUM BY HPLC

AIM: Estimation of Diclofenac sodium from given sample by HPLC

method (quantitative analysis).

CHROMATOGRAPHIC CONDITION:

HPLC instrument: Water

Detector : Dual wavelength detector

Column : C18 reverse phase

(4.9mm*250mm)

Temperature : Ambient temperature

Mobile phase : Methanol

Flow rate : 1ml/min

Inlet pressure : 1824 psi

Volume injected : 30 micro liters

PROCEDURE:

Preparation of standard solution and standard curve of

diclofenac sodium:

72
 • Standard solution having concentration ranging from 5, 10,
15, 20, 25 micro grams/ml is prepared.
• Each sample is injected into column and AUC is calculated.
• A standard plot of AUC vs. concentration was prepared, from
which concentration of the unknown sample can be
determined.

REPORT: Concentration of Diclofenac sodium in the given sample is

found to be…………..

73