The document discusses different types of chromatography techniques used to separate mixtures. It describes chromatography as a collective term for laboratory methods that separate mixtures by dissolving them in a mobile phase that carries the mixture through a stationary phase. The key types discussed are paper chromatography, thin layer chromatography, gas chromatography, column chromatography, and high performance liquid chromatography. HPLC uses very small column packing and high pressure to improve separation speed over other chromatography methods.

1. Miss Shazia Ghafoor
Biological techniques

2. Presented by
Mahpara nabeel
Chromatography and its types

3. chromatography
 Chromatography(from Greek chroma= color;
graphein= to write)
 Collective term of laboratory techniques for the
separation of mixtures.
 Mixtures is dissolved in a fluid called mobile
phase, which carries it through a structure holding
another material called stationary phase
 The various constituents of mixture travel at
different speeds, causing them to separate.
 Separation is based on differential partioning
between mobile and stationary phase

4. History
 Chromatography literally “color writing” was first
employed by Russian scientist MIKHAIL TSVET
in 1900.
 He continued his work primarily for separation of
plant pigments such as chlorophyll, carotenes,
and xanthophylls.
 Since these components have different
colors(orange, green and yellow) they gave the
technique its name

5. principle
 Chromatography usually consist of mobile and
stationary phase. The mobile phase refers to the
mixture of substance to be separated dissolved in
a liquid or gas. The stationary phase is a porous
solid matrix through which the sample contained
in the mobile phase percolates. The intraction
between mbile phase and stationary phase
results in the separtion of compound from
mixture.

6. Types of chromatography
 Paper chromatography
 Thin layer chromatography
 Gel chromatography
 Column chromatography
 Ion exchange chromatography
 Gel filtration chromatography
 Gas liquid chromatography
 Affinity chromatography

7. Paper chromatography
 It is a technique that involves placing a small dot
on line of sample solution onto a strip of
chromatography paper. Paper is placed in a jar
containing a shallow layer of solvent & sealed.
The solvent rises through a paper, it meets the
sample mixture, which starts to travel up paper
with solvent.
 Rf= distance travelled by substance
distance travelled by solvent front
 Rf value help us for identification of unknown

8.  sometimes it is rather difficult to
separate mixture of substance by a
single run with one solvent system.
In such a case, a second run is
carried by different solvent system,
in a direction perpendicular to a first
run. This is called as TWO DIMESIONAL
chromatography

10. Thin layer chromatography
 Thin layer chromatography(TLC) is a widely
employed laboratory technique & is similar to
paper chromatography. However instead of using
a stationary phase of paper, it involves stationary
phase of of a thin layer of adsorbent like silica
gel, alumina, or cellulose.
 It has advantage of faster run, better separation,
and choice between diffeent adsorbents.

12. Gas chromatography
 Also called as gas-liquid chromatography
 A separation technique in which mobile phase is
a gas. It is method of choice for separation of
volatile substance or volatile derivatives of certain
non volatile substances.
 Stationary phase is an inert solid liquid
impregnated with a non motile liquid.
 In gas chromatography, a sample is rapidly
heated and vaporaized at the injection port.
Sample is transported through the column by a
mobile phase consisting of an inert gas. Sample
components are separated based on their B.P &
relative affinity for stationary phase, which is most
often a viscous liquid(wax) within column. .

13.  The higher a component’s affinity for the
stationary phase, the slower it comes off the
column.
 Components are then detected and represented
as peaks on a chromatogram
 The mixture of volatile material is injected into the
column along with the mobile phase.
 The separation of volatile mixture is based on the
partition of components between mobile
phase(gas) & stationary phase(liq.) hence name
GAS-LIQUID CHROMATOGRAPHY

14.  It is well suited for use in
 Petrochemical, environmental monitoring &
industrial chemical field
 Sensitive, rapid and reliable

16. Column chromatography
 It is a separation technique in which the
stationary bed is within a tube. Absorbents are
packed into a column in a glass tube. This serves
as a stationary phase, leaving an open
unrestricted path for the mobile phase in the
middle of tube.
 Absorbents such as silica gel ,alumina, charcoal
powder, and calcium hydroxyapatite are used.

17.  The sample mixture in solvent is loaded on this
column. The individual compounds get
differentially adsorbed on to the adsorbent.
 The elution is carried out by the buffer system
which is mobile phase.
 The individual compounds come out of column at
different rates which may be collected separately
and identified.

19. High performance liquid chromatography(HPLC)
 Liquid chromatography is a separation technique
in which the mobile phase is a liquid. Liquid
chromatography can be carried out either in a
column or plane. Present day Liquid
chromatography that generally utilize very small
packing and relatively high pressure is referred to
as high performance Liquid
chromatography(HPLC)
 Since the chromatic techniques are slow and time
consuming, hence the separation can be greatly
improved by using high pressure in a range of
5000-10000 psi; hence this technique is also
referred to as high pressure Liquid

20.  In HPLC the sample is forced by a liquid at high
pressure(the mobile phase) through a column
that is packed with a stationary phase composed
of irregularly or spherically haped particles.
 The interaction between mobile and stationary
phase leads to the separation of mixture

22. Thanks for your attention