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

acetate>chloroform>alcohols>wtater>pyridine>organic

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 applied

drop.vignan 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

methanol , formamide ,

glycol etc

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.vignan pharmacy college,vadlamudi19

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

NO. OF SAMPLE PER 15 – 20

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

&identification of

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

easily.

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

there are chances that

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

paper and column and tlc

Education

stationary phase travels

mobile phase travels

isopropanolstatio phase

mobile phase use

sample vignan pharmacy

separate components

known components

mixture andor