Chromatographic Techniques

Syllabus
Chromatographic Techniques-General concepts,
classifications, column chromatography, HPLC,
Instrumentation, Applications, Thin layer
chromatography, Experimental techniques,
Applications, advantages and disadvantages,
Gas chromatography, principles,
instrumentation and applications
 Introduction
 It is a non destructive procedure for resolving a multi-
component mixture of trace, minor, or major constituents into
its individual fractions. Invented by Russian botanist Mikhail
Tswett at the beginning of the 20th century. He used it to
separate plant pigments such as Chlorophyll and Xanthophyll
or
 Chromatography is the science which involves the study of
separation of molecules based on differences in their
composition and/or structure
• It is the collective term for a set of laboratory methods used
for the separation of mixtures.
• It is a wonderful technique to separate mixtures and involves
the preparation of a test solution, which will interact
differently with the stationary support, thereby leading to
separation of similar molecules.
 General Concepts
Chromatography may be defined as a method of separating a
mixture of components into individual components through
equilibrium distribution between two phases.
Essentially , the technique of chromatography is based on the
difference in the rate at which the components of a mixture
move through a porous medium called stationary phase
under the influence of some solvent or gas called moving
phase .
Sample is transported in a mobile phase. This mobile phase is
then forced through an immiscible stationary phase.
The chromatographic method of separation, in general,
involves the following steps:
1. Adsorption or retention of a substance or substances
on the stationary phase.
2. Separation of the adsorbed substances by the mobile
phase
3. Discovery of the separated substances by a continues
flow of the mobile phase; the method is called elution.
4. Qualitative and quantitative analysis of the eluted
substances.
 Theoretical Principles of Chromatographic
techniques
1. Analyte. It is the substance to be separated during
chromatographs.
2. Bonded phase. It is a stationary phase that is
covalently bonded to the support particles or to the
inside wall of the column tubing(Immobilized phase)
3. Chromatogram. Is the usual output of chromatograph
4. Chromatograph. Is the equipment that enables a
sophisticated separation example: Gas
chromatographic or liquid chromatographic separtion
5. Eluate. Is the mobile phase leaving the column
6. Eluent. Is the solvent that carries the analyte
7. Eluotropic series. Is a list of solvents ranked
according to their eluting power.
8. Mobile phase. Mobile phase is the phase
which moves in a definite direction. It may be a
liquid, a gas or a supercritical fluid. The mobile
phase consists of the sample being separated/
analysed and the solvent that moves the sample
through the column.
Distribution Coefficient (Equilibrium Distribution )

Definition:

Concentration of component A in stationary phase

Concentration of component A in mobile phase

Different affinity of these 2 components to stationary

phase causes the separation.
 Types of Chromatography

Based on stationary and mobile phases used we can classify the

chromatographic techniques as follows.
1. The column adsorption chromatography: A liquid mobile phase is
allowed to percolate down a solid stationary phase generally
packed in a vertical glass column. The stationary phase competes
for the solute by adsorption and the liquid mobile phase
competes for the solute by dissolution.

In this type of chromatography, sometimes both the phases

are liquids. The method is then termed as partition
chromatography. The liquid used as the stationary phase is
essentially insoluble in the liquid to be used as the mobile phase.
• Generally, partition chromatography is
performed by wetting a solid by a liquid to be
used as the stationary phase and the wet
solid is packed in a column. The liquid thus
gets immobilised in this case is due to the
difference in the solubilities of the solutes in
the two liquids. This process is also sometimes
termed as column partition chromatography.
 Principle of column chromatography
It is known that the rate of adsorption varies with a
given adsorbent for different materials. This principle of
selective adsorption is used in column chromatography.
In this method the mixture to be separated in a suitable
solvent and allowed to pass through a tube containing
the adsorbent. The component which has greater
absorbing power is adsorbed in the upper part of the
column. The next component is adsorbed in the lower
portion of the column which has lesser adsorbing
power than the first component
DI AG RAM O F SI M PLE LI Q UI D CO LUM N CH RO M ATO G RAPH Y

Sol ve nt ( m obi l e or
m ovi ng pha s e )
OOOOOOOOOOO
A + B + C Sa m pl e
OOOOOOOOOOO
( A+B+C) OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO OOOOO A OOOO
OOOOOOOOOO Col um n OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Sol i d Pa r t i c l e s OOOOOOOOOO
OOOOOOOOOOO ( pa c ki ng m a t e r i a l - OOOOO B OOOO
OOOOOOOOOO s t a t i ona r y pha s e ) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOO C OOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO

El ua nt ( e l ua t e )
GENERAL FACTORS INCREASING RESOLUTION

1. Increase column length

2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution
 Experimental Details
a) Apparatus :
 A simple straight glass tube tapered at the bottom and often
fitted with a top is still commonly used.
 The ratio of length to diameter should be greater than 20:1 ; a
ratio of 40:1 can be taken as standard.
 This tube is about 20-30 cm long and 2-3cm in diameter.
 This may hold 50-100 g of adsorbent and may retain several
grams of adsorbate.
 The adsorbent is supported on a plug of cotton or glass wool
may be employed.
 More sophisticated form of apparatus can also be used in which
detachable adapters are being used. In these, the adsorbent is
packed between two sintered glass discs and small bore teflon
tubing is used for liquid input and out put
 FLASH COLUMN CHROMATOGRAPHY

• Build column
• Apply sample
• Elute
• Collect Fractions
• Analyze by TLC
• Combine pure fractions
b) Adsorbent:
General requirements.
In order to obtain columns with good filtration properties, the adsorbents should have
following characteristics:
i) Their particle should be spherical in shape and uniform in size

ii) Their mechanical stability must be great enough to prevent the formation
of fine dust which might be deposited the channels of the packing

iii) They should not react chemically, either with the eluting solvents
or with the sample components.

iv) They should contain as small amount of soluble components as possible.

v) They should be categorically inactive and as a rule, have neutral

surface. However, exceptions are ion exchangers.
Examples.
Aluminium oxide activated by heating about 200-300ᵒC in a current of air or carbon dioxide is
the most widely used adsorbent. Other adsorbents are silica gel, activated charcoal,
magnesium carbonate, calcium carbonate, calcium sulphate, barium carb.
Typical adsorbents used in column chromatography

Alumina
Activated charcoal
Increasing adsorptive
Magnesium silicate
power for polar
molecules Silica gel
Inorganic carbonates
Starch, cellulose, sucrose

Generally, 20-30 g of adsorbent per gram of sample, packed into a column

either as a slurry or dry.
Column height should be at least ten times its diameter.
 The Adsorbent
The selective action of an adsorbent
is due to intermolecular attractions such as:
Van der Waals forces
Dipole-dipole attractions
Hydrogen-bonding
Coordination Interaction
Salt Formation
Most commonly used solids:

SILICA GEL or SILICIC ACID SiO2·xH2O

ALUMINA Al2O3·xH2O
O
- 
O O Al
- 
RO O
O Al H
R O 
C O
 
R

Dipole-dipole H-bonding
 - +
O
-O +
Al
O H
O Al -
RCOO
O -
R N H2
O
salt formation
coordination
interaction

Strength of interaction:
Salt formation > H-bonding > dipole-dipole > Van der Waals
 The Solvent
• Solvents are organic compounds
• The action of a solvent, or a series of
solvents, can be used to effect a
separation
• The polarity of the solvent should not be
changed rapidly.
Some solvents used in column chromatography

Name Structure

alkanes (petroleum ether

hexane)
benzene C 6H6
toluene C 6H5CH3
halogenated hydrocarbons CH2Cl2, CHCl3
(dichloromethane, chloroform)
diethyl ether (CH 3CH2)2O
increasing
polarity O
ethyl acetate CH 3COCH2CH3
acetone (CH 3)2C O
alcohols CH 3OH,CH3CH2OH
O
acetic acid CH 3COH
The solvents used in chromatography have three
functions to perform.
i)They serve to introduce the mixture to the columns
ii) They affect the process of development by which
the zones of chromatogram are separated to their
fullest extent. Here these are termed as developers.
iii) They are also used to remove the required content
of each zone from the mechanically separated parts of
column. Solvent used for this purpose are called
eluants
e) Successive elution:
For complex samples it may be necessary to use a
series of solvents. For example, a particular solvent
may solvent and elute only one group of compounds,
such as hydrocarbons. For the rest of the compounds
we have to use new solvent. Each change in solvent
bring about the elution from the sample of a
different set of organic functional groups. This makes
possible complete separation of the sample into
compounds with common functional groups.
The expected elution order of organic compound classes
Name of Class General Formula

fast alkanes RH
alkenes R2C CR2
ethers R2O
halogenated hydrocarbons RX
CH3

aromatic hydrocarbons , etc

increasing
polarity aldehydes and ketones RCH O and R2C O
O
esters RCOR
alcohols ROH
amines RNH 2, R2NH, R3N
O
slow carboxylic acids RCOH
f) Preparation of adsorption columns.
 The glass wool or cotton plug is used as a support for the column. It is first
kept in position in the tube. The tube is clamped vertically .

 Now the adsorbent is added in portions so that the tube is packed uniformly
with the adsorbent.

 It is pressed from the top with a flattened glass rod before the next portion is
added. This is continued till nearly two- thirds of it is filled.

 An alternate procedure involves the preparation of slurry of the adsorbent in

a suitable liquid medium like petroleum ether or sometimes as the same
solvent which is being used in the chromatographic process.

 Now the slurry is added to the tube gradually , a little at time.

 The best method is to connect the tube to a suction pump through the stop-
cock. This process is to continued till a column of desired height is obtained
g)Detectors.
These are used to monitor and determine quantitatively the dissolved
substances emerging from the column. The various detectors are as follows.
i) Optical detectors. These are old type of flow analysers which are small
cell made from glass or quartz and are used for continuous
photometric analysis with visible or UV light of appropriate wavelengths. If
the samples do not exhibit approciable absorption in the cited
regions, reagents can be added to produce color reactions which can be
measured photometrically.
ii) Differential refractometers. Claesson has employed the refraction of
emerging power for detection. This method has been improved to a
differential refractometer having a sensitivity .
iii) Detectors based on heat of adsorption. These detectors are known as
micro adsorption detectors. In this, the liquid emerging from a
separating column is passed through two cells, one located on
top of the other while the lower cell is filled with an adsorbent. In the
centre of the packing of each of the cell , the glass covered measuring
point of a small thermistor is located. The total deflection ( +ve and – ve
peak) is proportional to the concentration at least to a range of 10 2.
iii) Flame ionization detector. In these detectors, there is an
endless metal wire which is passed by the column exit. The
decomposition products of the substances, which are
transported by the wire are led to a flame ionization or
argon detectors
iv) Conductivity detectors. These are suitable for ionized
substances in aqueous solution. The effluent is passed
through the measuring cell of the detector which contains
two or three electrodes within a wheatstone bridge circuit
and is operated by alternating current.
v) Other detectors used are microwave detector, ultrasonic
detector, vapor pressure detector and mass detector.
 h) Analysis : The adsorbent may be pushed out completely
with the wooden pestle or plunger and the zones may be
separated as they leave the tube. Each zone is then
dropped immediately into the eluant and the suspension
is filtered on a sintered glass funnel to get rid of the
adsorbent.

In some cases, the column of adsorbent is not removed

from the glass tube. The developed chromatogram is
treated treated either with a single eluant or with a
succession of solvents having increasing powerful
eluants actions. The various portions of the column are thus
washed out one and collected in different receivers. If the
separation is not complete, the extracts are allowed to pass
through the adsorbent column. The series of fractions
collected in this method constituets the liquid
chromatogram.
 TLC
In thin layer chromatography, the stationary phase
is a finely divided adsorbent solid spread over a
glass plate. The mobile phase is a liquid . In gas
liquid chromatography (GLC), separation is achieved
by partition between a mobile gas phase and a
stationary liquid phase packed in a column. Paper
chromatography is essentially the same as column
chromatography, except that the packed column is
replaced by a filterpaper strip. The mobile phase is
a liquid.
 Thin Layer Chromatography
Thin layer chromatography(TLC) is a chromatography technique
used to separate mixtures. It was introduced by Izmailov and
shraiber in 1938. These workers used this technique for separating
plant extracts on 2 mm thick and firm adhesive layer of alumina set
on glass plates.

Thin layer chromatography is performed on a sheet of glass, plastic,

or aluminum foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide, or cellulose (
blotter paper). This layer of adsorbent is known as the stationary
phase.

TLC is often named by other names such as drop, strip, spread

layer, surface chromatography and open column chromatography
 A small amount of an appropriate solvent (elutant) is
poured into a glass beaker or any other suitable
transparent container (separation chamber) to a depth of
less than 1 centimeter.

 A strip of filter paper (aka "wick") is put into the chamber,

so that its bottom touches the solvent, and the paper lies
on the chamber wall and reaches almost to the top of the
container.

 The container is closed with a cover glass or any other lid

and is left for a few minutes to let the solvent vapors
ascend the filter paper and saturate the air in the chamber.
(Failure to saturate the chamber will result in poor
separation and non-reproducible results).
• After the sample has been applied on the plate,
a solvent or solvent mixture (known as the
mobile phase) is drawn up the plate via
capillary action. Because different analytes
ascend the TLC plate at different rates,
separation is achieved.
• The solvent is allowed to completely evaporate
off, otherwise a very poor or no separation will
be achieved. If a non-volatile solvent was used
to apply the sample, the plate needs to be
dried in a vacuum chamber.
Thin layer chromatography can be used to monitor
the progress of a reaction, identify compounds
present in a given mixture, and determine the purity
of a substance. Specific examples of these
applications include: analyzing ceramides and
fatty acids, detection of pesticides or insecticides in
food and water, analyzing the dye composition of
fibers in forensics , assaying the radiochemical purity
of radiopharmaceuticals, or identification of
medicinal plants and their constituents
 Experimental technique
1. Coating materials. TLC plates are usually commercially
available, with standard particle size ranges to improve
reproducibility. They are prepared by mixing the adsorbent,
such as silica gel, with a small amount of inert binder like
calcium sulfate (gypsum) and water. This mixture is spread
as a thick slurry on an unreactive carrier sheet, usually
glass, thick aluminum foil, or plastic. The resultant plate is
dried and activated by heating in an oven for thirty
minutes at 110 °C. The thickness of the adsorbent layer is
typically around 0.1 – 0.25 mm for analytical purposes and
around 0.5 – 2.0 mm for preparative TLC
2. Preparation of Thin layers in Plates.

Various methods of preparing thin layers in plates are as follows:

i) Pouring. A measured amount of the slurry is put on a given size plate which is
kept on a level surface. The plate is then tipped back and forth to spread the
slurry for uniform surface. Although this method is simple, yet it is not used to a
great extent.
ii) Dipping. In this method plates are prepared by dipping them at a time, back to
back in chloroform or chloroform – methanol slurries of the adsorbent. This
method also not of much use.
iii) Spraying. In this method a small pointer is used to spray the slurry on the plate
but it is difficult to obtain uniform layers on a single plate and also there will be
variation from plate to plate.
iv) Spreading. The slurry is placed in an applicator. This is either moved over the
stationary plate or it is held stationary and the plate is pushed or pulled through.
This spreading method is the most important nowadays to obtain thin and
uniform layers.
v) Precoated plates. Ready to use thin layers of the common adsorbents are now
available precoated on glass or plastic sheets. These plates are quite expensive .
However, these avoid the purchase of fairly expensive coating equipment. The
thickness of precoated plastic sheets usually varies from 0.1 to 0.2 mm
 3. Activation of adsorbent
• Mix the absorbent, water and a binder such as
calcium sulfate, Silica gel, paper and alumina
• Spread a thin layer of absorbent on an unreactive
hard surface
– Glass, plastic, thick aluminum
• Heat in oven at 110°C for 30 mins. to activate and
dry the plate. In order to obtain very active
layers, silica gel and alumina plates can be heated
to 150ᵒ C for about 4 hours.
 4. Purification of Silica Gel ‘G’ layers
Silica gel contains iron as an impurity which causes a considerable
distortion of the chromatography. Therefore, it becomes essential to
purify the adsorbent.
Iron free layers have been obtained by giving the coated and air-dried
plates a preliminary development of the plate with methanol-Con. HCl
(9:1 v/v). The iron gets migrated with the solvent front to the upper
edge of the plate.
The plates are again dried and activated at 110ᵒ C.

Generally, calcium sulphate originally present as binder dissolves out

during the cleaning process. The silica gel purified by this method will
be reused to obtain plates with any other suitable binder such as
gypsum, starch or agar.
 5. Sample application
Agla microsyringe is generally used for
transferring the sample solution to the thin
layers for quantitative work. However, capillary
tubes may be used for qualitative work. The
range of sample volume and concentration
possible is far greater than with paper
chromatography. Solvents used for sample
solutions should be volatile and as non-polar as
possible.
 6. Development Tank

 The development tank used in paper chromatography is also used in TLC.

 Ascending chromatography is the most common technique in TLC.
 In TLC, the plane is placed in a development chamber at an angle of 45ᵒ
C.
 The bottom of the chamber is covered upto 1 mm by the solvent.
 Three sides of the tank are lined with solvent impregnates paper while
top is covered tightly with the lid.
 It is important in TLC that the development chamber is perfectly
saturated with solvent vapors.
 It is essential so as to avoid unequal solvent evaporation losses from the
development plate which can lead to various types of random behavior
usually resulting in generally lack of reproducibility in Rf values and edge
effect.
 Descending TLC also possible. It has no advantages over the ascending
technique in terms of efficiency of separation and speed of analysis.
Apparatus also more complicated.
 TLC Procedure
• Place a small amount of
solvent in a beaker

• In pencil, draw a straight

line across the plate about
1 cm from the end of the
plate

• Place a drop of sample

solution on the line
 TLC procedure

• Add filter paper

• Place in solvent

• Sealed container
 6. Solvents

• Choose a solvent depending on the polarity of the

compound
Petroleum ether
Cyclohexane
• Least Polar
Toluene
Chloroform
Acetone
Ethanol
Methanol

• More polar
– The solvent can be a mixture of compounds but
the polar solvent properties will over take the
non-polar one.
• 10-30% Methly tert-butyl ether, MTBE, in hexane, C6H14,
works well
• 10-30% Methylene chloride, CH2Cl2, in hexane, C6H14,
for a less polar mixture
• 10-30% Acetone, CH3COCH3, in Methylene chloride,
CH2Cl2, for a more polar mixture
– Trial and error is the best way to approach which
solvent to use.
 7. Visualization
• Destructive visualization
– Spray plate with H2SO4, and then bake in the oven at 110ºC for
15-20 minutes. Compound is destroyed but all spots will be
visible
• Nondestructive visualization – because of the use of a
UV light the sample will not be destroyed. Although,
not all of the spots on the plate will be visible.
– Long wave UV
– Short wave UV
– Semi-destructive visualization
 Visualization
A plate under a UV light to display the compounds
after they were developed
 Interpretation
Calculation of Rf Value
Distance that the Spot Traveled
 R f Value
Solvent Front Distance
 Rf Value

• What affects the Rf value?

– Temperature
– Solvent
– Thickness and amount of spot
– Other compounds
 Rf Value
– The Rf value needs to be between 0.0 and 1.0
• If the value is over 1.0 or less than 0.0, the calculation
is wrong (you goofed)
– If the Rf value is greater than 0.8 or lower than
0.2 the values are hard to interpret, thus
creating a larger error
– The best Rf values are 0.3 to 0.6
 Results
• Multiple spots from one sample can be achieved.
 Pros for TLC
• Sensitivity
• Speed
• Inexpensive
 Cons for TLC
• Too little of sample
• Too much of sample
• Subjective
 Forensic Analysis using Thin Layer
Chromatography
• Ink analysis
– Determines the specific chemicals
– Uses organic solvents
– Results are compared to a database of pen ink
 Forensic Analysis using Thin Layer
Chromatography
• Dye analysis
– Fibers
• Significant evidence
• Use thin layer
chromatography to
determine the
different dyes in the
fiber
– See how the colors elute
 Forensic Analysis using Thin Layer
Chromatography
• Organic acid analysis
– Separation of carboxylic acids
– Organic acids are in textile, food
preservatives, and medical agents
 Forensic Analysis using Thin Layer
Chromatography
• Pesticide analysis
– Pesticides are a hazard to the environment
– Many deaths are the results of poisoning
from pesticides
– Pesticides are classified by their use or
chemical type
– Determination of organophosphorus
compounds in pesticide
 Calibration/Standards TLC
• No calibration
• Standards
– Compare to other known substances
– Rf value
 How TLC works
• Sample solution is dissolved by solvent
• The solution sample will travel at different
distances based on solubility, polarization, size
• Silica gel
– Polar substances do not move far
– Non polar substances move farther up the plate

HO OH OH

R Si Si Si R
O O O O
R R R
Separation Process
Different compounds in the sample mixture travel at
different rates due to the differences in their
attraction to the stationary phase, and because of
differences in solubility in the solvent. By changing the
solvent, or perhaps using a mixture, the separation of
components (measured by the Rf value) can be
adjusted. Also, the separation achieved with a TLC
plate can be used to estimate the separation of a
flash chromatography column
Separation of compounds is based on the competition of the
solute and the mobile phase for binding places on the stationary
phase. For instance, if normal phase silica gel is used as the
stationary phase it can be considered polar. Given two
compounds which differ in polarity, the more polar compound
has a stronger interaction with the silica and is therefore more
capable to dispel the mobile phase from the binding places.
Consequently, the less polar compound moves higher up the
plate (resulting in a higher Rf value). If the mobile phase is
changed to a more polar solvent or mixture of solvents, it is more
capable of dispelling solutes from the silica binding places and all
compounds on the TLC plate will move higher up the plate
It is commonly said that "strong" solvents (elutants) push the analyzed
compounds up the plate, while "weak" elutants barely move them. The order of
strength/weakness depends on the coating (stationary phase) of the TLC plate.
For silica gel coated TLC plates, the elutant strength increases in the following
order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride,
Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile,
Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid,
Formic acid (strongest). For C18 coated plates the order is reverse. Practically
this means that if you use a mixture of ethyl acetate and hexane as the mobile
phase, adding more ethyl acetate results in higher Rf values for all compounds
on the TLC plate. Changing the polarity of the mobile phase will normally not
result in reversed order of running of the compounds on the TLC plate. An
eluotropic series can be used as a guide in selecting a mobile phase. If a
reversed order of running of the compounds is desired, an apolar stationary
phase should be used, such as C18-functionalized silica.
• Analysis
• As the chemicals being separated may be colorless, several methods exist to visualize the spots:
• Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added
to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer
will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
• Iodine vapors are a general unspecific color reagent
• Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate[7]
– Potassium permanganate - oxidation
– Iodine
– Bromine
• In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected
to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.
• Once visible, the Rf value , or retardation factor, of each spot can be determined by dividing the
distance the product traveled by the distance the solvent front traveled using the initial spotting site
as reference. These values depend on the solvent used and the type of TLC plate and are not physical
constants.
Applications
In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary
tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a "co-
spot" with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to run. The analysis is
qualitative, and it will show if the starting material has disappeared, i.e. the reaction is complete, if
any product has appeared, and how many products are generated (although this might be under-
estimated due to co-elution). Unfortunately, TLCs from low-temperature reactions may give
misleading results, because the sample is warmed to room temperature in the capillary, which can
alter the reaction—the warmed sample analyzed by TLC is not the same as what is in the low-
temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde.
As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of
development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway
in the final step and lutein the first compound staining yellow.

In one study TLC has been applied in the screening of organic reactions [8] for example in the fine-
tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for
instance iron(III) chloride) are placed separately on the base line, then reacted and then instantly
analyzed.
 Superiority of TLC over other Chromatographic Technique

The main advantages of TLC are listed below.

i) Simple equipment. The TLC requires simple equipment.

ii) Short development time. In TLC, development time is only about 1 hour or so for
good separation on inorganic adsorbent layers. However, column and paper
chromatography require several hours or days.

iii) Wide choice of stationary phase. The method may be employed for adsorption,
partition or ion exchange phenomena.

iv) Early recovery of separated components. It is possible to remove the powdery

coating of plates by scraping with a knife. This means that a spot or zone may be
removed quantitatively, and the required compound dissolved in a suitable solvent
is determined by calorimetric or spectrophotometric analysis.

v) Separation effects. The separation effects achieved by thin layer chromatography

are usually superior to those obtained paper chromatography.
vi) Easy visualization of separated compounds.
Detection of fluorescence compounds under UV light is easier than on
paper because the inorganic background does not fluoresce.

vii) Sensitivity. Extremely sharp delineated spots are obtained in TLC. Even
the usual spray reagents will able to detect much smaller quantities than
with the more diffused spots obtained in paper chromatography. This
increase in sensitivity is of the order of 10 to 100 folds as compared as that
of paper chromatography

viii) Variable thickness of thin layers. The method used in TLC analysis may
also be applied for the preparative separation with the aid of thicker
layers as to most separation by column chromatography

ix) Chemically inert stationary phase. The greatest merit of an inert

stationary phase as used in TLC lies in affording vigorous means of
detection including the application of strong heat or of corrosive reagent
such as conc.H2SO4
 HPLC-High performance liquid
chromatography
HPLC is a chromatographic technique that can separate a mixture and
is used in biochemistry and analytical chemistry to identify, qualify and
purify the individual components of the mixture with the following
features.
i) Where solvent is forced to through under high pressures upto
400atm. That makes the technique too much faster.
ii) We can use very much smaller particle size for the column packing
material which gives a much greater surface area for interactions
between the stationary phase and the molecules flowing with
solvents.
iii) It allows much better separation of the components of the
mixture
iv) Detection methods are highly automated and extremely sensitive