PLANE OR PLANER

CHROMATOGRAPHY
 CHROMATOGRAPHY

 A RUSSIAN BOTANIST TSWETT IN 1906 FIRST DEVOLPED THIS TECHNIQUE.

 IT IS A TECHNIQUE FOR SEPARATING MIXTURES INTO THEIR COMPONENTS IN

ORDER TO ANALYZE,PURIFY AND IDENTIFY THE MIXTYRE OR COMPONENTS.

 THE TERM CHROMATOGRAPHY ORIGINATES FROM TWO GREEK WORDS

 CHROMA MEANS COLOUR

 GRAPHEIN MEANS WRITING

 PRINCIPLES OF CHROMATOGRAPHY

 CHROMATOGRAPHY IS A PHYSICAL PROCESS

 ANY CHROMATOGRAPHY SYSTEM COMPOSED OF THREE COMPONENTS

 STATIONARY PHASE

 MOBILE PHASE

 MIXTURE TO BE SEPARATED

 WE CAN ONLY CONTROL STATIONARY AND MOBILE PHASEAS MIXTURES ARE THE PROBLEM
WE HAVE TO DEAL WITH
 TYPES

 CHROMATOGRAPHY IS DIVIDED INTO TWO TYPES

 PLANE OR PLANER CHROMATOGRAPHY

 COLOUMN CHROMATOGRAPHY

 PLANER CHROMATOGRAPHY IS DIVIDED INTO TWO TYPES

 PAPER CHROMATOGRAPHY

 THIN LAYER CHROMATOGRAPHY

Paper
Planer
Chromatography TLC
Column
WHAT IS PAPER
CHROMATOGRAPHY?
 INTRODUCTION

PAPER CHROMATOGRAPHY FIRST INTRODUCED

GERMAN SCIENTIST,CHRISTIAN FRIEDRICH
IN 1865.

PC IS CONSIDERED TO BE THE MOST SIMPLEST AND WIDELY USED

CHROMATOGRAPHIC TECHNIQUES BECAUSE OF ITS APPLICABILITY
TO ISOLATION AND IDENTIFICATION OF ORGANIC AND INORGANIC
COMPOUNDS

DEFINATION

IT IS TYPE OF PLANER CHROMATOGRAPHY.IT IS THE

SIMPLEST AND WIDELY USEDTYPE OF CHROMATOGRAPHY
PROCEDURES WHICH RUNS ON SPECIALIZED PAPER.
 PRINCIPLES OF PAPER CHROMATOGRAPHY

 THERE ARE TWO PRINCIPLES OF PAPER CHROMATOGRAPHY

 PAPER PARTITION CHROMATOGRAPHY

PAPER IMPREGNATED WITH SILICA OR ALUMINA ACT AS

ADSORBENT(STATIONARY PHASE) AND SOLVENT(MOBILE PHASE)

 PAPER ADSORPTION CHROMATOGRAPHY

THE MOISTURE OR WATER PRESENT IN THE PORES OF CELLULOSE FIBERS PRESENT

IN THE FILTER PAPER ACT AS STATIONARY PHASE AND ANOTHER SOLVENT ACT
AS MOBILE PHASE

 IN GENERAL PAPER CHROMATOGRAPHY IS MOSTLY SAME AS

PAPER PARTITION CHROMATOGRAPHY
 PAPER CHROMATOGRAPHY

PRINCIPLE OF SEPARATION

 THE PRINCIPLE OF SEPARATION IS MAINLY PARTITION RATHER THAN ABSORPTION

 IN GENERAL PAPER CHROMATOGRAPHY IS MOSTLY SAME AS PAPER

PARTITIONCHROMATOGRAPHY
 PRACTICAL REQUIREMENTS
 1)Stationary phase & papers used
 2)Application of sample
 3)Mobile phase
 4)Development technique
 5)Detecting or Visualizing agents
STATIONARY PHASE AND PAPERS USED
WHAT MAN FILTER PAPERS OF DIFFERENT GRADES LIKE
NO.1, NO.2, NO.3, NO.4, NO.20, NO.40, NO.42 ETC. ARE
USED. IN GENERAL THIS PAPER CONTAINS 98-99% OF Α-
CELLULOSE, 0.3 – 1% Β –CELLULOSE
FACTORS THAT GOVERNS THE CHOICE OF
PAPER:
NATURE OF SAMPLE AND SOLVENTS USED.
BASED ON QUANTITATIVE OR QUALITATIVE ANALYSIS.
BASED ON THICKNESS OF THE PAPER.
 Cut the paper into
desired shape and size
depending upon work
to be carried out.
 The starting line is
marked on the paper
with an ordinary pencil
5cm from the bottom
edge.
 On the staring line
marks are made 2cm
apart from each other.
 Preparation of the solution
 Choice of suitable solvent for making solution is very
important. Pure solutions can be applied direct on the
paper but solids are always dissolved in small quantity
of a suitable solvent.
 Biological tissues are treated with suitable solvents
and their extracts obtained. Proteins can be
precipitated with alcohol and salts can be removed by
treatment with ion exchange resin.
APPLICATION OF SAMPLE
The sample to be applied is dissolved in the mobile
phase and applied as a small spot on the origin line,
using capillary tube or micropipette.
very low concentration is used to avoid larger zone

 The spot is dried on the filter paper and is placed in

developing chamber.
 MOBILE PHASE
 Pure solvents, buffer solutions or mixture of solvents
 Examples- Hydrophilic mobile phase
 Isopropanol: ammonia:water 9:1:2
 Methanol : water 4:1
 N-butanol : glacial acetic acid : water 4:1:5

Hydrophobic mobile phases

dimethyl ether: cyclohexane
kerosene : 70% isopropanol
DEVELOPMENT TECHNIQUE
 Paper is flexible when compared to glass plate used
in TLC, several types of development are possible
which increases the ease of operation.
 The paper is dipped in solvent in such a manner
that the spots will not dip completely into the
solvent.
 The solvent will rise up and it is allowed to run
2/3rd of paper height for better and efficient result.
 DETECTING / VISUALISING AGENTS
If the substance are colored they are visually
detected easily.
But for colorless substance, Physical and chemical
methods are used to detect the spot.
Non specific methods ( Physical methods)
E.g. iodine chamber method,
UV chamber for fluorescent compounds – at 254 or
at 365nm.
Rf VALUE (Retardation Factor)
In paper
chromatography the
results are represented
by Rf value which
represent the movement
or migration of solute
relative to the solvent
front.
 APPLICATIONS

 Separation of mixtures of drugs

 Separation of carbohydrates, vitamins, antibiotics,
proteins, etc.
 Identification of drugs
 Identification of impurities
 Analysis of metabolites of drugs in blood , urine ….
ADVANTAGES OF P.C
Simple ,rapid ,inexpensive ,excellent resolving
power
PRECAUTIONS IN P.C
Establishing the vapor solvent equilibrium
 WHAT IS

THIN LAYER CHROMATOGRAPHY?

 One of analysis method that is used to
identify the unknown compounds and to
determine the purity of mixture.

 This method is simple, rapid and cheap

 Widely used in pharmaceutical & food
stuff industry.
 STATIONARY PHASE

 Silica is commonly used as stationary

phase
 The separation of sample mixture will be
depent on the polarity of sample.

 Some modified silica is also used in

certain purposes.
 MOBILE PHASE

 The ability of mobile phase to move up is

depend on the polarity itself
 Volatile organic solvents is preferably used as
mobile phase.
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp
 METHOD

Solvent is transferred
into the container with
0.5-1cm in dept from the
bottom
 2. TLC Plate Preparation

 Commercialy obtained with

5cm x 20cm in size
 Prepare your size when
neccesary
 Line 1 cm from the bottom
with a pencil as a part should
be spotted.
 3.Spotting’ TLC plates

 Make sure that your sample is

liquified already.
 stick it using capillary pipe &
spott onto the line you have made

 4.‘Develop the plate’

 after spotting, put the plate inside

the chamber in the ascendant
position
 Make sure that the dept of solvent
doesn't touch the spots
 Let it develop up to the 1cm from
the top of plate
 5. Detection of spots

- The color samples are

easy to be seen and no
need to use UV lamp to
detect them
 The use of Rf as separation
parameter

- The distance taken through by the solvent to move up will be

assigned as solfent front
- The distance taken trrough by the sample to move up will be
assign as sample front
- Rf value is obtained by dividing the sample front toward solvent
front

Rf = sample front
solvent front

-
 solvent front

component B

Rf of component A = dS
dA dB

dS
component A

Rf of component B = dA
origin
dB
dS
 Advantages
 Cheap
 Simple
 The developing can be monitored visually
 Able to use various chemical as a detector