Professional Documents
Culture Documents
CHROMATOGRAPHY
DEFINITION
Thin layer chromatography (TLC) is a technique used
tseparate the components of a mixture using a thin stationary
phase supported by an inert backing.
Separation depends on competition between adsorption of
solute onto the solid surface and its desorption by the solvent
needed to elute (wash off) it .
Stationary phase: Solid
Mobile phase: Liquid
PRINCIPLE
ADSORPTION Chromatography
The component with more affinity travel slower
towards the S.P
The component with lesser affinity travel faster towards
the S.P.
In TLC separation hydrogen bonding is main
intermolecular forces involved
ANALYTICAL
PREPARATIVE TLC
NORMAL PHASE
REVERSE PHASE
Stationary phase
Polar
Non-polar
Mobile phase
Non-polar
Polar
Non-polar
Polar
Polar
Non-polar
Example of stationary
phase
Silica gel
INSTRUMENTATION
COMPONENT
PURPOSE
Developing chamber
Solid support
(chromoplates)
Stationary phase
Adsorption of material
Mobile phase
Solvent system
CHOICE OF ADSORBENT
Two properties decide the selection:
1.particle size
2. homogeniscity
Factors affecting selection:
1. Colorless
2. should have great mechanical strength
3. should not catalyze or decompose of
substance
4. should be insoluble with mobile phase & the solvent
used for elution
5. no reaction at time of separation
. Adsorbent do not adhere to glass plate
CELLULOSE
Cellulose (C6H10O5)n is a long chain polymeric
polysaccharide carbohydrate of glucose
Adsorbed water or alcohol can be retained by interaction
with hydroxyl groups
Two types of cellulose are used in planar
chromatography:
1.Polymerization b/w 400-500 glucopyranose units
2. 40 200 glucopyranose units
ALUMINIUM OXIDE
It is a chemical compound of aluminum and oxygen with
chemical formula Al2O3
Commonly referred to as alumina
Manufactured in 3 pH ranges acidic, basic and neutral
Acidic compounds phenols, sulphonic, carboxylic &
Amino acids are separated on acidic alumina
Basic compounds amines , dyes separated
STATIONARY PHASE
NAME
COMPOSITION
Silica gel H
Silica gel G
Silica gel GF
Alumina
Al203 G
Al203 + Binder
Cellulose powder
Cellulose powder
Kieselguhr G
Polyamide powder
Polyamide
Fullers earth
Magnesium Silicate
magnesol
MOBILE PHASE
2) Cyclohexane
4) Toluene
6) Benzene
8) Water
10) Pyridine
CHOICE OF SOLVENT
Selection of M.P depends upon nature of substance to be
separated
Viscosity and polarity of S.P
GLASS PLATES
Three types :
1) Full plate
: 20cm 20 cm.
2) Half plate
: 20cm 10 cm.
DEVELOPING A PLATE
TLC plate prepared , P in beaker or closed jar
Place a small amount of solvent in container.
Solvent level below the starting line of TLC, else spots dissolve
Low edge of plate dipped in solvent
Solvent travels up the matrix by capillarity
ACTIVATION OF PLATE
plate dried and activated by heating in oven for 30 minutes
at 110 C
Thickness of adsorbent layer:
A. 0.1 0.25 mm for analytical purpose
B. 1- 2 mm for preparative TLC
APPLICATION OF SAMPLE
The concentration of the sample should be 2--5l of a 1% solution
Spots should be kept atleast 2cm above the base of the plate
Go for development
ADVANTAGES
Low cost
Short analysis time
All spots can be visualized
Adaptable to most pharmaceuticals
Uses small quantities of solvents
Requires minimal training
Reliable and quick
Minimal amount of equipment is needed
Densitometers can be used to increase
accuracy of spot concentration
Development tank
The development tank
should be lined Inside
with filter paper moistened
with mobile phase to
DEVELOPMENT TECHNIQUE
Different development techniques are :
1) One dimensional development.
DETECTING AGENTS
Detecting agents are two types:
(A)Non-Specific method
1)
2)
3)
4)
Ferric chloride.
Ninhydrine in acetone.
Dregendroff reagent.
3,5 Dinitro benzoic acid.
2,4 - Dinitro phenyl hydrazine.
DETECTION
The Rf value is calculated for
DEVELOPMENT OF T L C
VISUALIZATION METHOD
Previous slide shows colored spots. Most of the time
spots wont show unless visualized.
Visualization is a method used to render TLC spots visible
A visualization method can be:
UV light
iodine vapors to stain spots
colored reagents to stain spots
reagents that selectively stain spots leaving others
unaffected
Retention
The fundamental parameter in TLC is the retardation factor, Rf:
Rf = Zs / (Zf Zo)
Zf: Distance traveled by the solvent front from the point of
application.
Zs: Distance traveled by the solute front from the point of
application.
Zo: Distance between the point of application of solvent and solute.
Zf
Zs
Zo
N = 16*(Zs / Wb)2
Where,
N: number of theoretical plates; H: plate height
s: standard deviation of the solute band (in distance units)
Wb: baseline width of the solute band (in distance units)
H = Zs /n
Zf = (xt)1/2
Where,
APPLICATIONS / USES
1) Separation
of
mixture
of
chemical,biological,plant origin.
drug
of
TLC TROUBLESHOOTING
THANK YOU