This document provides an overview of different chromatographic techniques classified based on their mechanism of separation, physical state, shape of the chromatographic bed, and physical or chemical methods. It describes key techniques including adsorption chromatography, partition chromatography, gas-liquid chromatography, ion-exchange chromatography, size exclusion chromatography, thin layer chromatography, paper chromatography, normal phase chromatography, reverse phase chromatography, and high performance liquid chromatography. The document serves to introduce common chromatographic concepts and provide examples of different techniques.

1. CLASSIFICATION OF
CHROMATOGRAPHIC TECHNIQUES
Advanced Separation Techniques

2. ADVANCED SEPARATION
TECHNIQUES
Prepared by: Ayesha Rasty
Submitted to: Dr. Farhat Nosheen
University of Education, Jauharabad Campus

3. CONTENTS
 Introduction
 Common steps in Chromatography
 Chromatographic terms
 Based on Mechanism of Separation
 Adsorption Chromatography
 Partition Chromatography
 Based on physical state
 Gas-liquid Chromatography
 Gas-solid Chromatography
 Liquid-solid Chromatography
 Liquid-liquid Chromatography
 Based on shape of chromatographic bed
 Planar Chromatography
 Column Chromatography
 Based on physical or chemical method
 Column chromatography
 Size exclusion Chromatography
 Size exclusion Chromatography
 Extraction Chromatography
 Normal Phase Chromatography
 Reverse Phase Chromatography,
 Ion – exchange chromatography
 Thin layer Chromatography
 Paper chromatography
 Super critical fluid Chromatography
 HPLC
 Affinity Chromatography

4. INTRODUCTION
 In 1906, Mikhail Tswett, the Russian botanist discover chromatography.
 Chromatography is a separation technique in which a sample is equilibrated
between a mobile and a stationary phase.
 Chromatography, a group of methods for separating very small quantities of
complex mixtures, with very high resolution, is one of the most important
techniques in environmental analysis.
 Widely used technique for separation, isolation and identification of
compounds. Such as organic matter such as amino acids, starch, pigments
for vitamins and plants, etc.

5. THE CHROMATOGRAPHIC METHODS OF SEPARATION,
INVOLVE IN THE FOLLOWING STEPS:
• Adsorption or retention of a substance.
• Separation of the adsorbed substances by the mobile phase.
• Recovery of the separated substance by continuous flow of mobile phase.
This method is known as elution.
• Qualitative and quantitative analysis of the eluted substances.

6. CHROMATOGRAPHIC TERMS
 The analyte is the substance to be separated during chromatography.
 A chromatogram is the visual output of the chromatograph.
 The eluate is the mobile phase leaving the column.
 The eluant is the solvent that carries the analyte.
 The detector refers to the instrument used for quantitative detection of
analytes after separation.

7. BASIS OF CLASSIFICATION OF CHROMATOGRAPHY
 Based on mechanism of separation
 Based on physical state
 Based on shape of chromatographic bed
 Based on physical or chemical method

8. BASED ON MECHANISM OF SEPARATION
Adsorption Chromatography
Partition Chromatography

9. Adsorption Chromatography
• Adsorption Chromatography is process of separation of
components in a mixture introduced into chromatography system
based on the relative difference in adsorption of components of
stationary phase present in chromatographic column.
• Adsorption Chromatography is one of the oldest type of
chromatography.
• The equilibration by the mobile & stationary phase accounts for
the separation of different solutes.
• It utilizes a mobile liquid or gaseous phase that is adsorbed onto
the surface of a stationary solid phase.

10. Partition Chromatography
• Chromatography in which separation is based mainly on differences
between the solubility of the sample components in the stationary phase
or on differences between the solubility of the components in the mobile
and stationary phases.
• This form of chromatography is based on a thin film formed on the
surface of a solid support by a liquid stationary phase.
• Solute equilibrates between the mobile phase and stationary liquid.

11. BASED ON PHYSICAL STATE
Gas-liquid Chromatography
Gas-solid Chromatography
Liquid-solid Chromatography
Liquid-liquid Chromatography

12. Gas- Liquid Chromatography
• Mobile phase is an unreactive gas such as nitrogen(the carrier
gas)
• The stationary phase comprises of a small amount of liquid held
on a finely divided inert solid support.
• Gas- liquid chromatography is very sensitive and can be used to
detect small quantities of substances.
• It is often used in forensics.
Stationary phase used
Dimethyl Poly Siloxane(350oC)
• Hydrocarbons, Polynuclear aromatics
Poly(phenyl methyl) Siloxane(250oC)
• Steroids, Pesticides, Glycols

13. Gas- Solid Chromatography
• Gas chromatography employs an inert gas as the mobile
phase.
• The mobile phase is a gas, often nitrogen, but sometimes
helium, hydrogen or occasionally another gas. It is called
the “carrier gas” .
• Common solids are charcoal, a synthetic zeolite called
“molecular sieve”, or a combination of the two.
• Separation depends on the relative partial pressures of the
sample components above the stationary phase.
• Gas solid chromatography is relatively rare, but it is used to
separate atmospheric gases.

14. Liquid- Solid Chromatography
• Mobile phase is liquid
• Preferred mobile phase is non-polar or slightly polar.
• Liquid chromatography can be carried out either in a column
or a plane.
• The porous adsorbent is polar and separation is based on the
properties of classes of compounds-e.g., amines(alkaline)
from alcohols(neutral) and esters(neutral) from acids.
• Popular adsorbents are Silica and Alumina

15. Liquid- liquid Chromatography
• The first liquid-liquid system was reported by A. J. P. Martin
who used water supported on silica gel as the stationary phase
and n-heptane as the mobile phase.
• Mobile phase is a liquid(usually solvent or a simple binary
solvent mixture) and the stationary phase is also a liquid(which
must be immiscible and insoluble in the liquid mobile phase).
• The system is inherently unstable, as the stationary phase will
always have some solubility in mobile phase

16. BASED ON SHAPE OF CHROMATOGRAPHIC BED
• Planner Chromatography
• Paper Chromatography
• Thin layer Chromatography
• Column Chromatography
• Packed column Chromatography
• Open tubular column Chromatography

17. Planner Chromatography
• Stationary phase is present on a plane.
• Plane can be a paper, serving as such or impregnated by a
substance as the stationary bed(paper chromatography) or a
layer of solid particles spread on a support such as a glass
plate(Thin layer chromatography).
• Different compounds in the sample mixture travel different
distances according to how strongly they interact with the
stationary phase as compared to the mobile phase.

18. Column Chromatography
• Stationary bed is within a tube.
• The particles of the solid stationary phase or the support coated
with a liquid stationary phase may fill the whole inside volume of
the tube (packed column) or be concentrated on or along the
inside tube wall leaving an open, unrestricted path for the mobile
phase in the middle part of the tube(open tubular Column).
• Differences in rates of movement through the medium are
calculated to different retention times of the sample.

19. BASED ON PHYSICAL OR CHEMICAL METHOD
Column chromatography, Size exclusion Chromatography,
Size exclusion Chromatography, Extraction Chromatography,
Normal Phase Chromatography, Reverse Phase Chromatography,
Ion – exchange chromatography, Thin layer Chromatography,
Paper chromatography, Super critical fluid Chromatography,
HPLC, Affinity Chromatography

20. Ion Exchange Chromatography
• In this type of chromatography, the use of a resin (the stationary solid
phase) is used to covalently attach anions or cations onto it.
• Solute ions of the opposite charge in the mobile liquid phase are
attracted to the resin by electrostatic forces.

21. Molecular Exclusion Chromatography
• Also known as gel permeation or gel filtration,
• This type of chromatography lacks an attractive interaction between
the stationary phase and solute.
• The liquid or gaseous phase passes through a porous gel which
separates the molecules according to its size.
• The pores are normally small and exclude the larger solute
molecules, but allows smaller molecules to enter the gel, causing
them to flow through a larger volume.
• This causes the larger molecules to pass through the column at a
faster rate than the smaller ones.

22. Affinity Chromatography
• This is the most selective type of chromatography employed.
• It utilizes the specific interaction between one kind of solute
molecule and a second molecule that is immobilized on a
stationary phase. For example, the immobilized molecule may be
an antibody to some specific protein.
• When solute containing a mixture of proteins are passed by this
molecule, only the specific protein is reacted to this antibody,
binding it to the stationary phase.
• This protein is later extracted by changing the ionic strength or pH.

23. Size Exclusion Chromatography
• A mixture of molecules dissolved in liquid (the mobile phase) is
applied to a chromatography column which contains a solid
support in the form of microscopic spheres, or “beads” (the
stationary phase).
• The mass of beads within the column is often referred to as the
column bed.
• The beads act as “traps” or “sieves” and function to filter small
molecules which become temporarily trapped within the pores.
• Larger molecules are “excluded” from the beads .
• Large sample molecules cannot or can only partially penetrate the
pores, whereas smaller molecules can access most or all pores.
• Thus, large molecules elute first, smaller molecules elute later,
while molecules that can access all the pores elute last from the
column.
• Particles of different sizes will elute(filter) through a stationary
phase at different rates.

24. Thin Layer Chromatography
• The separation depends on the relative affinity of compounds
towards stationary and mobile phase.
• The compounds under the influence of mobile phase (driven by
capillary action) travel over the surface of stationary phase.
• During this movement the compounds with higher affinity to
stationary phase travel slowly while the others travel faster.
• Thus separation of components in the mixture is achieved.
• Once separation occurs individual components are visualized as
spots at respective level of travel on the plate.
• Their nature or character are identified by means of suitable
detection techniques.

25. Paper Chromatography
• Paper chromatography is an analytical method that is used to
separate coloured chemicals or substances, especially pigments.
• This can also be used in secondary or primary colours in ink
experiments.
• This method has been largely replaced by thin layer chromatography,
but is still a powerful teaching tool.
• Double-way paper chromatography, also called two-dimensional
chromatography, involves using two solvents and rotating the paper
90° in between.
• This is useful for separating complex mixtures of compounds having
similar polarity, for example, amino acids.
• If a filter paper is used, it should be of a high quality paper.
• The mobile phase is developing solutions that can travel up to the
stationary phase carrying the sample along with it.

26. Normal Phase Chromatography
• In normal phase chromatography, the stationary phase is polar, and
so the more polar solutes being separated will adhere more to the
stationary adsorbent phase.
• When the solvent or gradient of solvents is passed through the
column, the less polar components will be eluted faster than the more
polar ones.
• The components can then be collected separately, assuming adequate
separation was achieved, in order of increasing polarity.

27. Reverse Phase Chromatography
• In reverse phase chromatography, the polarities of the mobile and
stationary phases are opposite to what they were when performing
normal phase chromatography.
• Instead of choosing a non-polar mobile phase solvent, a polar
solvent and non-polar stationary phase will be chosen.

28. Extraction Chromatography
• An important modification in terms of stationary phase is
introduced by loading the extractant used for solvent extraction
on a hydrophobized inert support and irrigating the support
with aqueous solvent. This is known as extraction
chromatography.

29. High Performance Liquid Chromatography
• High-performance liquid chromatography (HPLC; formerly referred
to as high-pressure liquid chromatography), is a technique in
analytic chemistry used to separate the components in a mixture, to
identify each component, and to quantify each component.
• It relies on pumps to pass a pressurized liquid solvent containing
the sample mixture through a column filled with a solid adsorbent
material.
• Each component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as
they flow out the column.

30. Supercritical Fluid Chromatography
• Supercritical Fluid Chromatography (SFC) is a form of normal phase
chromatography, that is used for the analysis and purification of low to
moderate molecular weight, thermally labile molecules.
• It can also be used for the separation of chiral compounds.
• Principles are similar to those of high performance liquid
chromatography (HPLC), however SFC typically utilizes carbon dioxide
as the mobile phase; therefore the entire chromatographic flow path
must be pressurized.
• The supercritical phase represents a state in which liquid and gas
properties converge, supercritical fluid chromatography is sometimes
called "convergence chromatography."

31. REFERENCES
 http://www.waters.com/webassets/cms/category/media/other_images/primer_T_Ion_Exchange
_CHromatography.jpg
 http://xray.bmc.uu.se/Courses/MPC/students_files/ION_EXCHANGE_CHROMATOGRAPH Y.pptx
 http://www.thefreedictionary.com/chromatography
 http://www.britannica.com/EBchecked/topic/115917/chromatography/80518/Liquid- chromatography
 http://www.ltt.com.au/simulab/5/Laboratory/StudyNotes/snClassifChromatoMeth.htm
 http://en.wikipedia.org/wiki/Paper_chromatography
 http://en.wikipedia.org/wiki/Aqueous_normal-phase_chromatography
 http://s3.amazonaws.com/ppt-download/principlesandapplicationofchromatography- 130121011302-
phpapp02.pptx?response-content
disposition=attachment&Signature=lvlIqsf0i6utymK3xLIDRIThCG8%3D&Expires=1426432
486&AWSAccessKeyId=AKIAIA7QTBOH2LDUZRTQ
 http://s3.amazonaws.com/ppt-download/hplc-120622032932-phpapp01.pptx?response- content-
disposition=attachment&Signature=of%2FKpbRF%2BdvtUfOS0nOATt4kMIg%3D&Expires=
1426433000&AWSAccessKeyId=AKIAIA7QTBOH2LDUZRTQ

32. THANK YOU