Separation techniques in analytical chemistry

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Dr.G.R.Bhagure S.Y.B.Sc. Sem-IV Study material
Paper-III Sem-IV
Separation Techniques in Analytical Chemistry
Separation Techniques in Analytical Chemistry (02 L)
1.1. An Introduction to Analytical Separations and its importance in analysis.
1.2. Estimation of an analyte without effecting separation.
1.3. Types of separation methods
1.3.1. Based on Solubilities (Precipitation, Filtration Crystallisation)
1.3.2. Based on Gravity- Centrifugation
1.3.3. Based on volatility-Distillation ;
1.3.4. Based on Electrical effects-Electrophoresis
1.3.5. Based on retention capacity of a Stationary Phase -Chromatography;
1.3.6. Based on distribution in two immiscible phases-Solvent Extraction;
1.3.7. Based on capacity to exchange with a resin-Ion Exchange;
• Why separation techniques are essential?
Matrix are interfering in the determination of analyte so to remove matrix separation techniques are
essential.
• Based on solubilities: filtration, precipitation, crystallization
i) Filtration: It is a physical method of separation technique in which undissolved solid present
in liquid can be separated by passing through sieve or filter paper.
ii) Precipitation: It is a chemical method of separation technique in which dissolved solid
present in liquid can be separated by precipitation reaction followed by passing through sieve or
filter paper.
iii) Crystallization: This method of separation can be used for purification of solids ( by
removing impurities) or can be used to separate two or more solids present in a mixture.
Basic principle involved in the crystallization is; less soluble salt /compound crystallize out
first and more soluble salt/ compound crystallizes later.

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If salt differ in solubility to greater extent simple crystallization is used where as salt differ in
solubility to lesser
extent fractional crystallization is used.
iv) Centrifugation: (Based on Gravity) Centrifugation is of particular importance as a separation
technique in biochemistry. Cellular components can be separated by centrifugation. For example,
lysosomes can be separated from other cellular components by repeated differential centrifugation.
In this technique the ppt particle thrown away from centre of gravity, because of which it get settle to
bottom of the test tube, which can be easily separated by decantation
v) Based on Volatility:
Distillation: It is a method of purification of liquid or it can used to separate two or more liquid
present in a mixture.
In distillation liquid is heated to boiling stage ,vapours so formed are condensed and converted
into liquid. (by passing vapours through water condenser)
If a solvent differ in boiling point to greater extent simple distillation is used where as solvent
differ in boiling point to lesser extent fractional distillation is used.
vi) Based on Electrical effect::
Electrophoresis: It’s a method of separation technique for charged species.
In this method charged species migrate towards oppositely charged electrode under the applied
electric field.
vii) Based on Retention capacity of stationary phase:
Chromatography: It’s a physical method of separation in which sample to be separated is
allowed to pass through stationary phase, separation occurs by passing mobile phase.
Chromatography involves two phases i.e. stationary and mobile phase
stationary phase: The phase which remain stable ( not moving ,non reactive, thermally stable)
can be called as
stationary phase.( stationary phase may be solid or liquid )
Mobile Phase: The phase which is in moving stage (dynamic) can be called as Mobile phase
(Mobile phase may be liquid or gas)

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viii) Based on Capacity to exchange with resin: Ion exchange Chromatography: In this
chromatographic technique separation of ions based on reversible exchange of ions between like
sign.
ix) Size-Exclusion Chromatography: Separation of molecules takes according to molecular
size.
In this separation technique mixture passes through a bed of porous particles, bigger particles
separated first while smaller particles separated later as they take longer to pass through the
bed.
1.5. Solvent extraction (06 L)
1.5.1. Introduction, Nernst distribution Law, Distribution Ratio, Partition Coefficient.
1.5.2. Conditions of extraction: Equilibration time, Solvent volumes, temperature, pH.
1.5.3. Single step and multi step extraction, Percentage extraction for single step and
multistep extraction. Separation factor.
1.5.4. Batch and continuous extraction
SOLVENT EXTRACTION
Solvent extraction is based on the principle that when a solute is brought in contact with
two immiscible solvents, one of which is invariably water and the other organic, the solute
distributes itself in a fixed ratio in the two solvents. In certain cases, at some optimum

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conditions, the solute can be more or less completely transferred from one phase to another.
Thus, the technique finds application in separation, purification and enrichment. There is another
important aspect of solvent extraction in the form of extractive Spectrophotometry. The
absorbance of coloured metal complexes, particularly metal chelates, extracted in the organic
phase is measured. In favorable circumstances, it may be possible to concentrate the metal,
separate it from interferences and develop the absorbing system in a single step. Thus, the
extractive spectrophotometry invariably scores over normal spectrophotometry in terms of
selectivity and sensitivity.
Nernst Distribution Law (1891):
The law states that, a solute will distribute itself between two immiscible solvents in such a
manner that, at equilibrium, the ratio of concentration of the solute in the two phases at a
particular temperature will be a constant.
K=
𝐶1
𝐶2
Where C1= concentration of solute in solvent 1
and C2= concentration of solute in solvent 2
The law is valid only if the solute remains in same molecular state.
If solute undergoes association or dissociation in that case Nernst distribution law is modified
and stated into another form known as distribution ratio.
Distribution ratio =
𝑻𝒐𝒕𝒂𝒍 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 𝑨 𝒊𝒏 𝒂𝒍𝒍 𝒇𝒐𝒓𝒎𝒔 𝒊𝒏 𝒔𝒐𝒍𝒗𝒆𝒏𝒕𝟏
𝑻𝒐𝒕𝒂𝒍 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 𝑨 𝒊𝒏 𝒂𝒍𝒍 𝒇𝒐𝒓𝒎𝒔 𝒊𝒏 𝒔𝒐𝒍𝒗𝒆𝒏𝒕𝟐
Characteristics of distribution ratio:
i) D is dimensionless or has no units
ii) At given temperature D is constant.
iii) D is independent of the total amount of solute
iv) D and K are related with each other.
Choice of solvents:
i) Organic liquid should be good solvent for solute to be extracted i.e. K or D value must be
in favour of solvent.
ii) Organic liquid must be quickly settle out as separate layer.

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iii) Specific gravity should be greater or considerably lesser than 1.
Efficiency of Extractionswhen ‘n’ number of extraction is carried out
Let V dm3 of aqueous solution contain W gm of solute, in order to extract it this solution is
extorted with V0 dm3 of organic solvent.
After extraction let W1 gm of solute unextracted at the end of first extraction.
The expression for W1 can be given as;
Distribution ratio =
𝑻𝒐𝒕𝒂𝒍 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 𝑨 𝒊𝒏 𝒐𝒓𝒈𝒂𝒏𝒊𝒄 𝒑𝒉𝒂𝒔𝒆
𝑻𝒐𝒕𝒂𝒍 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 𝑨 𝒊𝒏 𝒂𝒒𝒖𝒆𝒐𝒖𝒔 𝒑𝒉𝒂𝒔𝒆
=
𝑪𝒐
𝑪𝒂𝒒.
Amount of solute extracted in Vo dm3 of organic solvent will be = (W-W1)
D=
𝑊−𝑊1
𝑉𝑜
𝑊1
𝑉
…………………………(1)
D=
(𝑊−𝑊1) 𝑉
𝑊1 𝑉𝑜
…………………(2)
W1DVo= (W-W1) V…………….(3)
W1DVo=WV-W1 V……………..(4)
W1DVo+ W1V=W V …………….(5)
W1(DVo+V)=W V…………(6)
W1= (
𝑽
𝑫𝑽𝒐+𝑽
) 𝒙 𝑾 ………………..(7)
If extraction is continued with the remaining solute namely W1 gm in V dm3 of aqueous layer
with the same volume of Organic solvent Vo dm3 and the amount of solute unextracted is now
W2 gm then;
D=
𝑊1−𝑊2
𝑉𝑜
𝑊2
𝑉
………………………… (8)
W2= (
𝑽
𝑫𝑽𝒐+𝑽
) 𝒙 𝑾𝟏 …………………………(9)
After n extraction with Vo dm3 of organic solvent every time, the amount of solute remaining
unextracted will be given by the relation

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Wn= (
𝑽
𝑫𝑽𝒐+𝑽
) 𝒏𝒙 𝑾 ………………..(10)
Where ;Wn represents the amount of solute in gms unextracted after ‘n’ extraction,
Thus lesser is Wn ,greater is efficiency of extraction .
Efficiency of Extractionswhen single extraction is carried out:
For single extraction using entire volume of organic solvent i.e. nVo,
Let the amount of solute unextracted be Wn, then;
D=
𝑊−𝑊′𝑛
𝑛𝑉𝑜
𝑊′𝑛
𝑉
………………………… (11)
D=
(𝑊−𝑊,𝑛) 𝑉
𝑛𝑉𝑜𝑊′𝑛
……………………………….(12)
W’n= (
𝑽
𝒏𝑽𝒐𝑫+𝑽
) 𝒙 𝑾 …………………………..(13)
Percentage Extraction:
For single extraction the percentage extraction can be given as
From equation no.7
W1= (
𝑉
𝐷𝑉𝑜+𝑉
) 𝑥 𝑊
And the amount of extraction (W-W1) will corresponds to
W-W1 = W- (
𝑉
𝐷𝑉𝑜+𝑉
) 𝑥 𝑊 ……………..(14)
The fraction extracted (x) will corresponds to X=
𝑊−𝑊1
𝑊
X=1- (
𝑉
𝐷𝑉𝑜+𝑉
) ……………..(15)
X= (
𝐷𝑉𝑜
𝐷𝑉𝑜+𝑉
) ………………..(16)

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(𝑋 =
𝐷
𝐷+
𝑉𝑎𝑞.
𝑉𝑜
) …………………(17)
The Percentage Extraction E=100 x X
E= (=
100 𝑥 𝐷
𝐷+
𝑉𝑎𝑞.
𝑉𝑜
)…………………(18)
The Percentage Extraction depends on distribution ratio as well as volume ratio
𝑽𝒂𝒒.
𝑽𝒐
of two
phases.
For n extraction the fraction ‘x’ that is extracted is given by the expression
X=1- (
𝑉
𝐷𝑉𝑜+𝑉
)
n
……………..(19)
E=𝒙 𝑿 𝟏𝟎𝟎 = 𝟏𝟎𝟎 [1- (
𝑉
𝐷𝑉𝑜+𝑉
)
n
] ……………….(20)
Where:
V= Volume of aqueous phase
Vo= Volume of organic phase
Separation Factor: It is related to two different solutes.
Let A and B are two different solutes present in Organic phase and aqueous phase.
𝜷 =
[𝑨]𝒐
[𝑩]𝒐
[𝑨]𝒘
[𝑩]𝒘
=
[𝑨]𝒐
[𝑨]𝒘
[𝑩]𝒐
[𝑩]𝒘
=
𝑫𝑨
𝑫𝑩
1.6. Chromatography : (05L)
1.6.1. Introduction to Chromatography
1.6.2. Classification of chromatographic methods based on stationary and mobile phase

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1.6.3. Paper Chromatography: Principle, techniques and applications of Paper
Chromatography in separation of cations.
CLASSIFICATION OF CHROMATOGRAPHY
Introduction:
The chromatographic techniques were introduced to the scientific world in a very modest
way by M. Tswett in 1906. He employed the technique to separate various pigments such as
chlorophylls and xanthophylls by passing a solution of these compounds through a glass column
packed with finely divided calcium carbonate. Later on this technique was named as liquid-solid
adsorption chromatography. About the same time, Thompson and Way had realized the ion
exchange properties of soils. Almost after three decades, in 1935, Adams and Holmes observed
ion exchange character in crushed phonograph. This particular observation opened up the field
for synthetic organic resin exchangers. Thereafter, the subject of ion exchange Chromatography
started expanding in its utility. The concept of gas-liquid chromatography was first introduced
by Martin and Synge in 1941. They were also responsible for the developments in liquid-liquid
partition chromatography. Moreover, in 1944, from Martin’s laboratory, the separation of amino
acids by paper chromatography was reported. This made people to realize the importance of
such a simple technique for tedious separations. Simultaneously the efforts were being made to
understand the theoretical aspects of chromatography. The high point of all these activities
reached in 1952 when the contributions of Martin and Synge were recognized and they were
awarded the Nobel Prize for their work in chromatographic science.
Definition
Chromatography is a physical method of separation technique in which the components
to be separated are distributed between two phases, one of these is called a stationary phase and
the other a mobile phase which moves on the stationary phase in a definite direction. The
components of a mixture redistribute themselves between two phases by a process which may be
adsorption, partition, ion exchange or size exclusion.
The stationary phase may be solid or liquid where as mobile phase may be liquid or gas.

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Classification of chromatographic techniques:
Depending on the physical nature of stationary phase and mobile phase chromatographic
techniques are classified into two types.
1) Adsorption Chromatography: Stationary phase is solid and mobile phase is liquid or Gas
2) Partition Chromatography: Stationary phase is Liquid and mobile phase is liquid or Gas
Types of Adsorption Chromatography:
a) Thin Layer Chromatography: Stationary phase ( Alumina or Silica gel) is solid and mobile
phase is liquid
b) Column Chromatography: Stationary phase ( Alumina or Silica gel) is solid and mobile
phase is liquid
c) Ion Exchange chromatography : Stationary phase ( Ion Exchange resin) is solid and mobile
phase is liquid
d) Gas Solid Chromatography : Stationary phase ( Alumina or Silica gel) is solid and mobile
phase is Gas (Inert Gas: He, Ne, Ar)
e) Gel Chromatography: (Size exclusion Chromatography): Stationary phase (Sephadex gel)
is solid and mobile phase is liquid
Types of Partition Chromatography:
a) Paper Chromatography: Stationary phase is Liquid and mobile phase is liquid .
b) High Performance Liquid Chromatography: Stationary phase is Liquid and mobile phase
is liquid .
c) Gas Liquid Chromatography: Stationary phase is Liquid and mobile phase is Gas (Inert
Gas: He, Ne, Ar).
Expected questions:
Q. Describe the classification of chromatographic technique? …………(2)
Q. State the stationary and mobile phase in TLC and Paper Chromatography…………(2)
Q. State the stationary and mobile phase in any two Chromatographies? …………(2)
Q. Define adsorption chromatography and partition chromatography…………(2)

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PAPER CHROMATOGRAPHY
1.0.Principle: (Question: Explain the principle of paper chromatography)
In paper chromatography stationary phase is liquid as well as mobile phase is also
liquid. In paper chromatography solute undergoes partition between the two liquid phases. The
rate of transfer of solute and its effective separation on paper will depend on partition coefficient
of the solute between the two phases. The solutes from the original mixture will have migrated
along paper at different rates, forming a series of separated spots. For identification purposes
spots are characterized by Rf values.
Rf values can be calculated as:
Rf value=
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒂𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒖𝒕𝒆 𝒇𝒓𝒐𝒏𝒕
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒇𝒓𝒐𝒏𝒕
(Question: Name the stationary phases and mobile phase for paper chromatography)
2.0.Experimental Procedure:
(Question: Describe the Experimental Procedure for Paper chromatography)
1) Preparation of the Paper.
2) Solvent system used
3) Preparation of the sample.
4) Application of the sample.
5) Development of the Chromatograms.
6) Identifying the Spots
2.1.Preparation of the paper:
The paper used for chromatography is short fibred cellulose papers with high purity and
certain special properties. Whatman no.1 is strong, medium fast, pure cellulose paper that is
widely used.
For the separation of polar substances special ion exchange paper (containing ion
exchanging groups) can be used.

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For the separation of the component which is hydrophilic in nature, esters of cellulose can
be used.
Cut a square piece of high-quality filter paper to fit into your development chamber. The
dimension of the paper should be 3- 5 cm. in width and 18-20 cm. in length. With a pencil,
draw a straight line about 2 cm from the bottom edge of the paper. Make a circle on this line in
middle position for application of sample.
2.2) Solvent system:
In paper chromatography the liquid stationary and mobile phase is used.
The solvents should have following characteristics;
i) The solvents should not react with any component during separation.
ii) The chemical compositions of the solvents should not change with time.
iii) The Rf value for the component should be anywhere between 0.06 to 0.95.
iv) The distribution ratio of the component should be independent of its concentration.
v) The solvents used may be miscible or immiscible but one of the solvent should be polar that
can work as stationary phase.
The paper shows affinity with polar solvent that can work as stationary phase.
If water is used as stationary phase then no special saturation is necessary. If polar solvent other
than water is used then it is necessary to remove the water from the paper.
Ex. Water and Ethanol.
2.3. Preparation of the sample.
The solid sample is dissolved in organic solvent having low boiling point. The percentage
of the sample in the solution should be 0.1—1%. About 10 micro liter of the sample are
transferred to the paper by using capillary or micro syringe. If the sample is of biological
origin, then proteins, lipids and inorganic ions present in excess are to removed for better
separation.

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2.4. Application of the sample
The point of the application of the sample or the origin is marked with pencil on the
paper. The sample should be applied by micro pipette or capillary. 10-20 micro liter samples is
to be applied. After application of sample on the marked spot solvents associated with sample
solution is evaporated by using hair drier or current of hot air.
2.5. Development of the Chromatograms.
(Question: Describe various method of development of Chromatogram)
In paper chromatography chromatogram is developed by using following ways.
i) Ascending
ii) Descending
iii) Horizontal
iv) Radial
v) Multiple
i) Ascending Development:-
In this solvents are placed in a jar or in a flask or in cylinder at the bottom. A small
concentrated spot of solution that contains the sample of the solute is applied to a strip of
chromatography paper about two centimeters away from the base of the plate, usually using a
capillary tube for maximum precision. The paper is suspended vertically by means of thread. The
solvents run up through the fiber of the paper by capillary action. The paper is then dipped into a
suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the
solvent, and placed in a sealed container. Different compounds in the sample mixture travel at
different rates due to competition between the paper fibers and solvent for the solutes. Paper
chromatography takes anywhere from several minutes to several hours.
Ascending Paper
Chromatography

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2.6. Location of the Separated component: The substances separated by paper
chromatography are colorless. These separated substances are detected by using visualizing
agent. The paper is dried at temperature 100O
c. The spots are detected by means of physical or
chemical method
2.6.1. Physical Method
Question: ( How separated components are identified by physical method?)
In this method paper is exposed to ultraviolet light in the wave length range 240-260 nm.
The compound produces florescent spots. Compounds which do not produce florescent spots are
exposed to high pressure mercury vapour lamp.
2.6.2. Chemical Method
(How separated components are identified by Chemical method?)
In this method the separated compound is allowed to react with with another reagent to produce
florescent spots.
Example:
i) H2S gas is passed over the paper for the visualization of metal ions.

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ii) Iodine vapors are used for the detection of unsaturated organic compounds.
iii) Acid –base indicators are used to detect carboxylic acids.
iv) Ninhydrin are used to detect amino acids.
2.7. Qualitative Analysis Or Interpreting the Data:-
The Rf value for each spot should be calculated. Rf value is characteristic for any given
compound. For identification known Rf values can be compared to those of unknown substances
for the identifications.
𝑹𝒇 𝑽𝒂𝒍𝒖𝒆 =
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒂𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒖𝒕𝒆 𝒇𝒓𝒐𝒏𝒕
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒇𝒓𝒐𝒏𝒕
Note: Rf values often depend on the temperature, solvent, and type of paper used in the
experiment; the most effective way to identify a compound is to spot known substances next to
unknown substances on the same chromatogram.)
Application of Paper Chromatography:
(Q.Give application of Paper chromatography
i) Paper chromatography is used for the separation of organic, inorganic, biochemical
and natural products.
ii) Metal ions having same chemical properties or belonging to the same group can be
detected and separated.
iii) The technique is useful for the separation of metal ligand chelate or complexes, it’s
also useful for the separation of isomer.
iv) The purity of a sample can be tested. In this case pure sample can produce only one
spot where as impure sample can produce two or more.
v) It is useful in the field of biochemistry.

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vi) It is very useful technique in the field of food, drugs and cosmetics.
1.6.4. Thin layer Chromatography Principle, technique and Applications in determining the
purity of a given solute; Following progress of a given reaction .
THIN LAYER CHROMATOGRAPHY - TLC
1.0.Principle

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Thin Layer Chromatography is adsorption chromatography in which stationary phase is solid and
mobile phase is liquid. In TLC, plate used is a sheet of glass, metal, or plastic which is coated with a thin
layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is
spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a
developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is
the mobile phase, and it slowly rises up the TLC plate by capillary action. As the solvent moves past the
spot that was applied, equilibrium is established for each component of the mixture between the
molecules of that component which are adsorbed on the solid and the molecules which are in solution. In
principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent
and some components will be carried farther up the plate than others. When the solvent has reached the
top of the plate, the plate is removed from the developing chamber, dried, and the separated components
of the mixture are visualized. Series of separated spots are characterized by Rfvalues for identification
purposes.
2.0.Experimental Procedure:-
i) Adsorbent
ii) Preparation of plate
iii) Mobile phase
iv) Development of chromatogram
v) Qualitative and quantitative analysis.
i) TLC Adsorbent :-
Silica gel is the most commonly used material for the formation of thin layer on glass plate or a
plastic plate. Alumina (Al203) can also used. The adsorbent material should have fine particles.
ii) Preparation of plate:
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
thick slurry on an uncreative 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.
iii) TLC Solvents or Solvent Systems.

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A single solvent or mixture of two solvents can work as mobile phase in TLC .Ex.
petroleum ether, carbon tetrachloride, chloroform, ethyl acetate, hexane can used as mobile
phase.
iv) Development of chromatogram:
The chromatogram is developed by ascending process. The solvent or mixture of
solvents to be used as mobile phase is placed in tall jar lined with filter paper and covered by
glass lids. This ensures that the space in the jar is saturated with the solvent vapors. A small spot
of solution containing the sample is applied to a plate, about one centimeter from the base. The
plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a
sealed container. . The solvent moves up the plate by capillary action and meets the sample
mixture, which is dissolved and is carried up the plate by the solvent. 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 most 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). The solvent is allowed to run till it covers most of the thin layer. Then plate
is taken out the chamber and subjected for the detection/ location of separated component.

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Application of sample and development of
Chromatogram by Ascending way
High
Pola
r
25
Les
s
Pola
r
Identification of Component:
a) Physical Method
Question: ( How separated components are identified by physical method?)
In this method plate is exposed to ultraviolet light in the wave length range 240-260 nm. The
compound produces florescent spots. Compounds which do not produce florescent spots are exposed to
high pressure mercury vapour lamp.
b) Chemical Method
(How separated components are identified by Chemical method?)
In this method the separated compound is allowed to react withwith another reagent to produce florescent
spots.
Example:
v) H2S gas is passed over the paper for the visualization of metal ions.
vi) Iodine vapors are used for the detection of unsaturated organic compounds.
vii) Acid –base indicators are used to detect carboxylic acids.
viii) Ninhydrin are used to detect amino acids.

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Application of TLC:
i) Qualitative analysis: -
If the separated components are colored then identification is very easy. All the
visualizing agents used in paper chromatography (Detecting agents or indicators) can be used in
TLC.
Ex.1) H2S gas is passed over the plate for the visualization of metal ions.2) Iodine vapors are
used for the detection of unsaturated organic compounds.3) Acid –base indicators are used to
detect carboxylic acids. 4) Ninhydrin is used to detect amino acids.
Plate is dried using warm air. The indicator is spread over the plate. The Rf value of the
separated component is find out and it is compared with the literature value for qualitative
analysis.
For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75)
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
ii) Quantitative analysis:
i) The size of the spot increases with the amount. Square root of the spot area is find out from
that amount of solute can be found out.
ii) Potentiodensitometry of the plate is carried out.
iii) Flurometry or emission Spectroscopy is also used.
iv) Separated spot is removed by knife edge .Its dissolved in proper solvent and its amount is
finding out by volumetric analysis.
Other Applications of TLC:-
Thin layer chromatography finds many applications, including
TLC can is applicable in the field of medicinal preparations, pharmaceutical preparations, natural
product extract and related compounds.
i) Assaying the radiochemical purity of radiopharmaceuticals
ii) Determination of the pigments in plants.
iii) In forensic science Laboratory detection of pesticides or insecticides in food, poison etc.

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iv) Analyzing the dye composition of fibers in forensics, orIdentifying compounds present in a given
substance
v) Monitoring organic reactions.
vi) To carry out clinical study to carry out qualitative and quantitative analysis of biological and
metabolic samples to detect disease.
vii)Semi quantitative analysis can also performed by extracting the spot in suitable solvent and it’s
determined by volumetric analysis or any other instrumental technique.
Advantages of TLC over Paper Chromatography:
i) Separation is sharper in TLC than paper Chromatography.
ii) TLC is much more rapid.
iii) The use of inorganic layer eliminates background organic effects in spectroscopic
analysis.
iv) More reactive reagents like Sulphuric acid can be used.
The separated spots are more distinct hence detection methods are more
sensitive
1.4. Electrophoresis: Principles, Basic Instrumentation, Working and Application in
separation of biomolecules like enzymes and DNA. (02L)

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Separation techniques in analytical chemistry

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