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THIN LAYER

CHROMATOGRAPHY

IN 1958, STAHL DEVELOPED STANDARD


EQUIPMENT FOR ANALYZING BY THIN LAYER
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

Polar molecules stick to plate


Non- polar molecules do not stick to plate
Non-polar molecules will spend a great amount of time
dissolved in eluent
Separation of compounds occur due to differences in
partitioning b/w liquid and S.P
More sensitive & less sample required
Spraying with corrosive agents for identification
possible

REVERSE PHASE TLC


BASED ON THE NATURE OF MOBILE
AND STATIONARY PHASE USED
NORMAL PHASE TLC
THIN LAYER
CHROMATOGRAPHY

BASED ON PURPOSE OF USE

ANALYTICAL

PREPARATIVE TLC

COMPARISON OF NORMAL PHASE &


REVERSE PHASE
PARAMETER

NORMAL PHASE

REVERSE PHASE

Stationary phase

Polar

Non-polar

Mobile phase

Non-polar

Polar

Compound eluted first

Non-polar

Polar

Compound eluted last

Polar

Non-polar

Example of stationary
phase

Silica gel

C4 ,c8 bonded phase

INSTRUMENTATION
COMPONENT

PURPOSE

Developing chamber

Create and maintain


environment for
chromatography

Solid support
(chromoplates)

Supports thin film of


stationary phase

Stationary phase

Adsorption of material

Mobile phase

Solvent system

OPERATIONAL TECHNIQUE INVOLVED


Choice of adsorbent
Preparation of plate
Preparation and application of sample
Choice of solvent
Development of chromatogram
Drying of chromatogram
Location of spot
Quantitative estimation

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

CLASSIFICATION OF ADSORBENTS USED


1. Classification according to binding strength:
A. Weak adsorbent: sucrose, starch, talc, cellulose
B. Intermediate adsorbent: silica gel, calcium carbonate, calcium
phosphate, magnesia
C. Strong adsorbent: alumina, charcoal
2. Classification according to nature:
A. Inorganic adsorbent: Silica, Silica gel, Alumina, Calcium
phosphate, Glass powder, Kieselguhr ,Magnesium silicate, Calcium
silicate, Phosphate , Ferric & Chromic oxides, Zinc carbonate &
zinc ferro cyanides, Bentonites
B. Organic adsorbent: Normal cellulose powder, Charcoal &
activated carbon, Starch, Sucrose, Manitol, Dextran gel

SILICA GEL is granular porous form of silica


Made synthetically from sodium silicate
Silica gel is solid and used in chromatography as S.P
Due to silica gel polarity non polar components tend to
elute before polar ones hence named as NPC
Hydrophobic groups (C18) attached to silica gel then polar
components elute first hence names as RPC.
Synthetic nature of silica gel enables careful control of pore
size.

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

Neutral compounds aldehydes, ketones & lactones

STATIONARY PHASE
NAME

COMPOSITION

Silica gel H

Silica gel without binder

Silica gel G

Silica gel + CaSO4

Silica gel GF

Silica gel + Binder + fluorescent indicator

Alumina

Al203 Without Binder

Al203 G

Al203 + Binder

Cellulose powder

Cellulose Without Binder

Cellulose powder

Cellulose With Binder

Kieselguhr G

Diatomaceous earth + binder

Polyamide powder

Polyamide

Fullers earth

Hydrous magnesium alumina

Magnesium Silicate

magnesol

MOBILE PHASE

1) Nature of the substance to be separated i.e whether it


is polar or non-polar.
2) Mode of Chromatography

3) Nature of Stationary phase


4) Mode Separation i.e Analytical or Preparative
technique
Examples: 1) Petroleum ether
3) Acetone
5) Ethyl acetate
7) Alcohols
9) Chloroform

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

Solvent used may be single or double phase system


e.g: n-hexane < cyclohexane< CCl4 < benzene < toluene <
CHCl3 < diethyl ether < ethyl acetate < acetone < ethanol <
Methanol < water

GLASS PLATES
Three types :
1) Full plate

: 20cm 20 cm.

2) Half plate

: 20cm 10 cm.

3) Quarter plate : 20cm 5 cm.


Microscopic slides can also be used for
monitoring the progress of a chemical reaction.

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

Moving components of samples at various rates because of their


different degrees of interaction with matrix & solubility in the
developing solvent

Non polar solvents force non polar compounds to top


of plate because the compounds dissolve well & do not
interact with polar S.P

Allow the solvent to travel up the plate until 1 cm


from top
Take the plate out and mark the solvent front
immediately.
Do not run the solvent over edge of plate

Let solvent evaporate completely.

PREPARATION AND ACTIVATION OF PLATES


The T L C plates can be prepared by following techniques :
1) Pouring
2) Dipping
3) Spraying
4) Spreading

Activation :It is nothing but removing of water/ moisture & other


adsorbed substance from the surface of any adsorbent by heating.

METHOD FOR APPLICATION OF ADSORBENT ON


THE PLATE
1. POURING- adsorbent of homogeneous particle size
made in slurry and pour on plate.
2. DIPPING- it used for small plate by dipping two plate
back to back in slurry of adsorbent in chloroform or
other volatile solvent.
3. SPRAYING- simply by spraying slurry on plate
4. SPREADING- slurry spread by using spatula or glass
rod

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

Sample is spotted using a capillary tube or micropipette

Spots can be placed at random process

Spots should be kept atleast 2cm above the base of the plate

Spotting area should not be immersed in the Mobile phase

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

TLC SUPERIOR OVER OTHER METHODS


It requires little equipment
Require little time for separation
It is more sensitive
Very small quantity of sample require for analysis
The method use for adsorption, partition, ion
exchange chromatography
Component which are separated can be recovered
easily .
Quantative separation of spot and zone are possible
For identification is permitted
Spraying of corrosive agent

Development tank
The development tank
should be lined Inside
with filter paper moistened
with mobile phase to

saturate the atmosphere


& also prevent the
EDGE EFFECT .

TLC plates are placed vertically in rectangular


chromatography tank or chamber .
Glass and stainless steel are suitable chambers.
If tank is not saturated, solvent will evaporate
and affect the Rf value.
Development should be carried out at room
temperature by covering chamber with glass plate.

DEVELOPMENT TECHNIQUE
Different development techniques are :
1) One dimensional development.

2) Two dimensional development.


3) Horizontal development.
4) Multiple development.

DETECTING AGENTS
Detecting agents are two types:
(A)Non-Specific method
1)
2)
3)
4)

Iodine chamber method.


Sulphuric acid spray method.
UV chamber for fluorescent compounds.
Using fluorescent stationary phase.

(B) Specific method


1)
2)
3)
4)
5)

Ferric chloride.
Ninhydrine in acetone.
Dregendroff reagent.
3,5 Dinitro benzoic acid.
2,4 - Dinitro phenyl hydrazine.

DETECTION
The Rf value is calculated for

identification "Rf value is the


ratio of distance travelled by
The solute to the distance
travelled by the solvent front
Distance travelled by solute
Rf =

Distance travelled by solvent front

Rf value is constant for each component only under


identical experimental condition.
Polar compounds have low Rf value
It depend on following factors Nature of adsorbent
Mobile phase
Activity
Thickness of layer
The temperature
Equilibration
Loading
Dipping zone
Chromatographic technique

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

VARIOUS TECHNIQUES TO VISUALIZE THE COMPOUNDS:

1. Sulfuric acid/ heat: destructive, leaves charred blots


behind
2. ceric stain: destructive, leaves a dark blue blot
behind polar compounds
3. Iodine: semi- destructive , iodine absorbs onto the
spots , not permanent
4. UV light: non destructive, long wavelength,
(background plate green, spots dark) short
wavelength (background plate dark, spots glow)

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

The value of Rf is related to the capacity factor (k) of the solute by


the following equation:
k = (1- Rf)/ Rf
By using the above equation, planar chromatography can be used to
obtain estimates of k for a solute on different stationary phase and
mobile phase combinations.
This can be useful in screening a number of columns or mobile
phase for use in column liquid chromatography.
EFFICIENCY
The efficiency of a separation in planar chromatography is
described in terms of plates and plate height.
N = (Zs / s)2

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

Note that the efficiency of a planar system is not constant, but


depends on the distance that the solute has traveled, or its retention
and Rf value.
The change in efficiency of a planar chromatography system with
distance and the presence of a third phase have made the derivation
of exact plate height equations for planar chromatography difficult.
These concurrently occur with another complicating factor: the flow
rate of mobile phase through a system with capillary flow is not
constant with time.
For a system with capillary flow, the change in the mobile phase
velocity with time is described by the following equation:

Zf = (xt)1/2
Where,

t = time required by the mobile phase to migrate


Zf = distance
x = the system constant

PERFORMING THE TLC ANALYSIS: CALCULATE THE RF VALUES


The Rf value is calculated by measuring the distance the
sample zone travels divided by the distance the developing
solvent travels
Values below 0.1 is considered poor: the spots are too
close to origin
Values of 0.1 to 0.8 are good and any other spots
(impurities) or other actives are resolved form each
other
Above 0.8: poor: spots may be too broad or distorted

APPLICATIONS / USES
1) Separation
of
mixture
of
chemical,biological,plant origin.

drug

of

2) Separation of Carbohydrates, vitamin, antibiotics,


proteins, etc.
3) Identification of drug. Ex :Amoxicillin, Levodopa
4) Detection of foreign substances.

5) To detect the decomposition products of drug.

6). To determine how many compounds are there in


a mixture is it real pure?
7). To determine the best solvent conditions for
separation on column
8). To identify the substances being studied
9). To monitor the compositions & appropriate
conditions of the fractions collected from Column
Chromatography
10). To monitor the progress of the reaction
11). To determine identity of two substances
12). To determine effectiveness of purification

TLC TROUBLESHOOTING

1. CAUSE: the compound runs as streak rather than a spot


REASON: the sample was overloaded
Run the TLC again after diluting your sample
Sample might contain many components
It creates many spots which run together & appear as
streak

2. CAUSE: the sample runs as a smear or a upward crescent (moon)


REASON: compounds which possess strongly acidic or basic
groups (amines or carboxylic acids) show this behavior
Add few drops of ammonium hydroxide(amines) or acetic
acid (carboxylic acids) to the eluting solvent to obtain
clear plates.

3. CAUSE: the sample runs as a downward crescent (moon)


REASON: adsorbent was disturbed during spotting caused
4. CAUSE: plate solvent front runs crookedly (curved)
REASON: adsorbent flaked of the sides of plate
Adsorbent moved towards the side of the plate
or touching the sides of the container or the
paper used to saturate the container as plate
develops.
Crookedly run plates makes it harder to measure
the Rf value accurately.
5. CAUSE: many random spots are seen on the plate
REASON: accidently check not any organic compound on
the plate or any new foreign substance touched
incidentally.

6. CAUSE: no spots seen on plate


REASON: you might have not spotted enough compound,
perhaps because the solution of the compound is too
dilute.
Try concentrating the solution or else spot it several
times in one place allowing solvents to dry b/w capillaries
Some compounds do not show under UV light
Try another method of visualization of plate
Perhaps you dont have any compounds because the
experiment did not go as well planned
If solvent level in developing jar is deeper than the origin
of the TLC plate
Solvent will dissolve the compounds into the solvent
reservoir
It allows them to move up the plate by capillary actions.
Thus you will not see the spots after the plate is developed.

THANK YOU