Paper and column and tlc

PRESENTED BY
V.SREE DEVI
(14AB1S0407)
UNDER GUIDENCE OF
A.VISWANATH
DEPARTMENT OF PHARMACEUTICAL
ANALYSISvignan pharmacy college,vadlamudi1

What is Chromatography?
 Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.
Separat
e
• Analyze
• Identify
• Purify
• Quantify
vignan pharmacy college,vadlamudi2

Uses for Chromatography
 Chromatography is used to:
• Analyze – examine a mixture, its components,
and their relations to one another
• Identify – determine the identity of a mixture or
components based on known components
• Purify – separate components in order to isolate
one of interest for further study
• Quantify – determine the amount of the a mixture
and/or the components present in the sample

Paper chromatography
 It is defined as the technique in which the analysis of
unknown substance is carried out mainly by the flow
of a solvent on specially designed filter paper.

Column chromatography
It is defined as a
separation process
involving the
uniform percolation
of a liquid solute
through a column
packed with finely
divided material.

Thin layer chromatography
•In TLC, partition, however
occurs on a layer of finely
divided adsorbent which is
supported on glass plate.
•This chromatography
using thin layer of an
adsorbent held on a plate or
other supporting medium is
known as thin layer
chromatography.

PRINCIPLES
Paper chromatography Column chromatography Thin layer
chromatography
Partition Adsorption Adsorption
Cellulose layers in filter
paper contain moisture acts
as stationary phase.
Solid material is used as
stationary phase.
Solid material is used as
stationary phase.
Organic solvents or buffers
act as mobile phase.
Liquid is used as mobile
phase.
Liquid is used as mobile
phase.
The compound which is
more soluble in stationary
phase will travel slower.
The components move
according to relative
affinities.
The components move
according to relative
affinities.
Those compounds are
eluted later.
The compound which has
more affinity towards
stationary phase travels
slower.
more affinity towards
slower.
The compound which is
more soluble in mobile
phase travels faster.
less affinity towards
faster.
less affinity towards
faster.vignan pharmacy college,vadlamudi7

STATIONARY PHASE MOBILE PHASE
water Isopropanol : ammonia : water (9:1:2)
water N-butanol : glacial acetic acid :water(4:1:5)
water Phenol saturated with water
formamide Chloroform
Formamide benzene
Formamide Benzene : cyclohexane(9:1)
DMF Cyclohexane
phenoxyethano-l Heptane
kerosene 70%isopropanol
Stationary phase and mobile phase
use in paper chromatography

Common Adsorbents for column chromatography
•Sucrose
•Cellulose
•Starch
•Calcium carbonate
•Calcium sulphate
•Calcium phosphate
•Magnesium carbonate
•Calcium oxide
•Silica gel
•Charcoal
•Magnesium oxide
•Alumina
[ The adsorbents are
given in the order of
increasing adsorption
power ]

Grouping of solvents in order of chromatographic strength is known as
elutropic series.
(Increasing eluting power)
Petroleum ether
Cyclohexane
Benzene
Chloroform
Ethyl acetate
Acetone
Ethanol
Methanol
Water
Pyridine
Organic acids
Inorganic acids
mobile phase used in column chromatography

Stationary phase and mobile phase
use in thin layer chromatography
 Stationary phase : common adsorbent for thin layer
chromatography are silica gel, alumina, kieselghur.
 Mobile phase : pure solvents or mixture of solvents are
used.
Petroleum ether>carbon tetra
chloride>cyclohexane>carbon
disulfide>ether>acetone>benzene>toluene>ethyl
Name composition
Silica gel H Silica gel without
water
Silica gel G Silica gel + caso4
Silica gel GF Silica gel + binder +
fluorescent indicator

METHODOLOGY

 Specially treated chromatographic paper
is the inert support water absorbed by
the inert support is stationary phase.
While various solvents which are
immiscible with water are mobile phase.
 A drop of mixture to be analyzed is
applied to edge of a chromatographic
paper and is dried.
 Then immersed into a cylinder with a
suitable solvent level below the appliedvignan pharmacy college,vadlamudi13

 The solvent rises by capillary action of the paper and
various components of the mixture are carried with it
at different rates.
 Here the solute distributed between the stationary
phase and mobile phase. According to partition
coefficient for each component.
 Hence the compounds are separated that is they are
distributed by zones.
 If it is colour less the chromatogram has to be
developed by applying visualizing reagents forming
coloured compounds with ions are to be detected.

 CHOICE OF THE FILTER PAPER:
 The filter paper plays an important role in the success
of paper chromatography .
 Various types of whatmann chromatography papers are
available.
 The choice of a particular whatmann
chromatography paper depends upon the type of
separation.
 Characteristics of whatmann chromatography papers
are as follows.
FAST MEDIUM SLOW
Thin paper Grade no:4 Grade no:7 Grade no:2
Grade no:54 Grade no:1 Grade no:20
Thick paper Grade no:31 Grade no:3 -
Grade no:17 Grade
no:3MM
-

Type Typical uses
Carboxyl papers Cationic separation of
protonated amines &
amino acids
Acetylated papers Rp-chromatography of
lipophilic substances like
steroids , insecticides,
Pigments & metal cations
Keiselguhr alumina,silica,
zirconia papers
Separation of low polarity
substance such as amines
, fatty acids , steroids ,
triglycerides , vitamins etc
Ion exchange papers Ion exchange paper
chromatography of various
ionic species
Hydrophilic papers Papers modified with

Rf VALUE
 The Rf value is calculated for identifying the spots.
 Rf value is the ratio of distance travelled by the solute
to the distance travelled by the solvent front.
 Rf =
Distance travelled by solute
Distance travelled by solvent
front
•The Rf value ranges from 0 to 1. but ideal values ranges
from 0.3 to 0.8.
•Rf value is constant for every compound in a particular
combination of stationary and mobile phase.

 In the column chromatography the stationary phase is
silica gel is placed as slurry, mobile phase is liquid.
 The sample is dissolved in a minimum amount of
solvent and applied to the column and passed into the
column with liquid mobile phase.
 The fundamental principle of column chromatography
is the selective adsorption of various compounds of the
mixture on the solid stationary phase.
 It is based on the fact that when a solution of complex
mixture is passed through a column of adsorbent the
various components of the mixture are adsorbed to
different extents on the adsorbent.

 The mixture to be analyzed is dissolved in a suitable
solvent and then the mobile phase is allowed to pass
through the column containing the stationary phase.
 The component which has the greatest adsorbing
power is adsorbed first, that is absorbs at the upper
part of the column.
 In this way the various components of the mixture are
separated in order of their decreasing adsorptive
powers.
 The process of separation of various components of
the mixture into different bands or zones of pure
substance, each located at different region in the
column.

FACTORS AFFECTING COLUMN EFFICIENCY
EffectFactor
Decrease of size improves separation (but very small
particles need high pressure).
Particle size of solid stationary
phase (or of support)
Efficiency increases as ratio length / width increases.Column dimensions
Non uniform packing results in irregular movement
of solutes through column & less uniform zone
formation.
Uniformity of packing
Increase in column temperature results in speed of
elution but does not improve separation .
Column temperature
Solvents should be of low viscosity (to give efficient
resolution) & h igh volatility (to get rapid recovery of
the substances).
solvent
Uniform & low flow rate gives better resolution.Solvent flow rate
Discontinuous flow disturbs resolutionContinuity of flow
Deactivation of adsorbent decreases separation.Condition of adsorbent
Substances of high concentration move slowly.Concentration of solutes

 A glass plate is coated with a loose powder or with a
slurry of an adsorbent slurries will adhere to the
surface of the glass plate, after drying as a thin layer.
 The unknown substance and reference materials are
dissolved in water or mobile phase and the solution
is applied in a row of spots, 1-2cm from the edge of
the plate with the help of capillary tube or micro
syringe.
 The chromatographic plate is placed in a jar
containing the solvent for development and the jar
with solvent vapours.vignan pharmacy college,vadlamudi23

 The jar is covered with an air tight lid.
 As the solvent ascends through the layer by capillary
action. The sample is resolved into fractions.
 The plate is carefully with drawn after the solvent front
has migrated about 75%of the length of the plate.
 The plate is then dried and sprayed with a reagent for
detection of components or more commonly exposed to
iodine vapours.
 Solute position is indicated by brown vapours.

Preparation and activation of TLC plates
 Pouring technique, the slurry prepared and poured on to a
glass plate and is spread uniformly on the glass plate. After
setting plates are dried in an oven.
 Dipping technique two plates are dipped in to the slurry and
separated after removing from slurry and dried.
 Spraying technique is like perfume spray on a cloth. the
slurry is sprayed on a glass plate using sprayer.
 Spreading technique the slurry is poured inside the reservoir
of tlc spreader. the spreader is rolled only once on the plate
after setting the plates are activated by keeping in an oven.
 Activation of tlc plates is nothing but removing moisture
from the surface of any adsorbent at high temperature.

Parameters TLC
TYPE OF
CHROMATOGRAPHIC
PLATE
HAND MADE / PRECOATED
PARTICAL SIZE
DISTRIBUTION
WIDE
PARTICAL SIZE RANGE 5 – 20 μm
SHAPE SPOT
SPOT SIZE 2 – 4 mm
LAYER THIKNESS 250 μm
SOLVENT
CONSUMPTION
50 ml
NO. OF SAMPLES MAXIMUM 12
OPTIMUM
DEVELOPMENT
DISTANCE
10 - 15 cm
SAMPLE VOLUME 1-10 μl
PARAMETERS AFFECTING TLC

Paper Column Thinlayer
Paper chromatography
used for separation of
amino acids and
oligosaccharides.
Removal of impurities
and in the purification of
compounds.
TLC has high speed of
separation.
It is also used for
structural analysis for
unknown compound.
Isolation of active
constituents.
A large number of
vitamins, antibiotics &
food products have also
been separated by using
TLC.
Separation of
carbohydrates, vitamins,
antibiotics, alkaloids,
glycosides.
Isolation of metabolites
from biological fluids.
TLC used for isolation &
characterization of
organic compounds
Identification of foreign
substance in drugs.
Estimation of drugs in
formulations or crude
extracts.
TLC is very sensitive
gives sharper zones
&better resolution.
Identification of
decomposition products.
The greatest application
of column
chromatography has
been in the separation
Purification process.
A
P
P
L
I
C
A
T
I
O
N
S

Chromatography Advantages Disadvantages
Paper It is an inexpensive but
powerful analytical tool
that requires very small
quantities of material.
They are used in many
scientific studies to identify
unknown
organic and inorganic
compounds.
Paper chromatographic
techniques can not be
used in separation of
volatile substances such
as hydrocarbons and
volatile fatty acids.
The lower limit for the
detection of most
compounds is 1-5
microgram.
Column Any type of mixture can
be separated by column
chromatography.
Any quantity of the
mixture can be separated
(g to mg).
Time consuming
method.
More amount of solvents
are required which are
expensive
Thin layer The components are
separated in very little time
as the components will
elute out very quickly.
The solvents for the TLC
plate can be changed
In this method the plate
length is limited and hence
separation takes place
only up to certain length.
The separation takes
place in an open system

Development technique in paper
chromatography
 Ascending chromatography:
 Descending chromatography:
 Ascending- descending mode:
vignan pharmacy college,vadlamudi30 Descending
chromatography

•Radial chromatography
•Two dimensional
chromatography

Development technique for column
chromatography
vignan pharmacy college,vadlamudi
 Isocratic elution technique:
 Eg. Chloroform only, pet ether:benzene=1:1 only
 Gradient elution technique:
 Eg. Intially benzene, then chloroform, then ethyl acetate, then
methanol.
 Other techniques like frontal analysis and displacement analysis
are also used.
 Frontal analysis :
 Solution of sample mixture is added continuously on the column.
no mobile phase is used for development of column.
 Displacement analysis :
 a small volume of mixture is added to the column and elution is
carried out by a solvent containing solute which has high
adsorptivity for column material.
32

Development technique for thin layer
chromatography
 One dimensional development.
 Two dimensional development.
 Multiple development technique.
 Step wise development.
33

CONCLUSION
 By using Wider choice of mobile phase any
type of mixture can be separated & any
quantity of the mixture can be separated( µg
to mg).
 separation of amino acids ,food dyes , pigments
can be done
34

REFERENCES
• Practical pharmaceutical chemistry by Beckett
& stenlake (part two).
• Instrumental methods of chemical analysis by
B .k . Sharma.……….
• PharmaceuticalAnalysis by P.PARIMO.
• PharmaceuticalAnalysis by Higuchi.
35

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