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CH ROM ATO GRA PHY. Jigarkumar B. Patel K.B.I.P.E.R. INTRODUCTION. History of chromatography. Chromatography was first developed and defined by the russian botanist Mikhail S. Tswett in 1903.

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ch rom ato gra phy


Jigarkumar B. Patel



History of chromatography

Chromatography was first developed and defined by the russian botanist Mikhail S. Tswett in 1903.

He produced a colorful separation of plant pigments using a column of calcium carbonate (chalk).

The word chromatography comes from the Greek words for color "chroma" and write "graphein". So chromatography means \'to write with color\'

1938 Paper and TLC

1952 Gas-liquid chromatography GLC

1968 High pressure liquid chromatography HPLC

1980s Super critical fluid chromatography SFC


How it works?

In all chromatography there is a mobile phase and a stationary phase.

The stationary phase is the phase that doesn\'t move and the mobile phase is the phase that does move.

The mobile phase moves through the stationary phase picking up the compounds to be tested.

As the mobile phase continues to travel through the stationary phase it takes the compounds with it.

At different points in the stationary phase the different components of the compound are going to be absorbed and are going move with the mobile phase.

This is how the separate from the other components is achieved.


Is a technique used to separate and identify the components of a mixture.

  • Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium.
  • Molecules that spend most of their time in the mobile phase are carried along faster.

USP 2000: “Chromatography is procedure by which solutes are separated by dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in given direction and in which the individual substance exhibits different mobilities by reasons of differences in adsorption, partition, solubility, vapor pressure, molecular size or ionic charge density. The individual substance thus separated can be identified or quantitatively measured by suitable analytical method.”

usp 2009
USP 2009
  • Chromatography is defined as a procedure by which solute are separated by a dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular size, or ionic charge density. The individual substances thus separated can be identified or determined by analytical procedures.
iupac 1993
IUPAC 1993:
  • “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.”

mobile phase: solvent

  • may be a liquid or a gas.
  • stationary phase : column packing material
  • may be a solid, liquid supported on a solid or a gel.
  • This definition suggests that chromatographic separations have three distinct features:
  • They are physical methods of separation;
  • Two distinct phases are involved, one of which is stationary while the other is mobile; and
  • Separation results from differences in the distribution constants of the individual sample components between the two phases.
purpose of chromatography
Purpose of Chromatography
  • Analytical - determine chemical composition of a sample
  • Preparative - purify and collect one or more components of a sample
uses for chromatography
Uses for Chromatography

Chromatography is used by scientists to:

Analyze – examine a mixture, its components, and their relations to one another

Identify – determine the identity of a mixture or components based on known components

Purify – separate components in order to isolate one of interest for further study

Quantify – determine the amount of the a mixture and/or the components present in the sample

uses for chromatography1
Uses for Chromatography

Real-life examples of uses for chromatography:

Pharmaceutical Company – determine amount of each chemical found in new product

Hospital – detect blood or alcohol levels in a patient’s blood stream

Law Enforcement – to compare a sample found at a crime scene to samples from suspects

Environmental Agency – determine the level of pollutants in the water supply

Manufacturing Plant – to purify a chemical needed to make a product


Types of Chromatography

Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)

Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)


Basically, molecules can be separated from one another if there are differences in one or more of the following:

      • 1. Volatility
      • 2. Polarity
      • 3. Molecular size
      • 4. Charge

Separation of closely related components of a complex mixture. Mobile phase forced through a stationary Phase (column or a planar surface).Components of a mixture distribute between mobile and stationery phase to varying degree. More solubility in Mobile Phase – fast progress- low retention Time (Rt). More solubility in stationery phase – slow progress – long retention time(Rt) 


In the animation below the red molecules have more affinity towards the stationary phase than the green molecules.


For example, suppose we have a mixture of two solutes, A and B, dissolved in a suitable buffer solution.

  • A column is filled with beads of an insoluble substance that is expected to bind differentially and reversibly to A and B.
  • The column is initially equilibrated by running a certain volume of the buffer through it, and the mixture applied to the top of the column as a narrow layer.
  • If the stopcock of the column is carefully opened, the layer of solutes will pass into the column, and the solutes can be washed out slowly by pouring more buffer into the column.
  • This process is called elution, and the solution emerging at the bottom of the column is called the eluate.

Chromatogram –

Detector signal vs. retention time or volume


Let us assume, for the purpose of this illustration, that solute B binds more strongly to the matrix than solute A.

  • Then, as the mixture travels down the column, the molecules of B will be retarded with respect to the molecules of A.
  • In time, they will separate into bands, and be eluted at different times.

Classification of chromatography

  • Depending on depth of separation(Geometry):
  • 1) Column Chromatography:
  • The stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity.
  • 2) Planar Chromatography:
  • The stationary phase is supported on a flat plate or the interstices of a paper and the mobile phase moves through the stationary phase by capillary action or by gravity.

Classification of chromatography

Nature of mobile phase :

On the basis of types of Mobile Phase and Stationary Phase and the kinds of equilibrium involved in the transfer of solutes between the two phases. Liquid Chromatography Gas Chromatography Supercritical Chromatography



Based on instrument/ apparatus

Based on working principle/

Mode of chromatography

Nature of Stationary Phase

Column Chromatography

Paper Chromatography


Gas Chromatography


SCF Chromatography

Electrophoretic Chromatography

1-3: Gives separation basically

4-7: give separation and separated compound is analyzed quantitatively and qualitatively.

Adsorption Chromatography

Partition Chromatography

Ion exchange Chromatography

Size exclusion Chromatography




  • Column Chromatography:
  • used for the separation of large quantity of drug.
  • a) Adsorption Column Chromatography
  • b) Partition Column Chromatography
  • 2) Paper Chromatography:
  • Separation and identification of very small quantity of drug (µg) e.g. amino acids.
  • a) Ascending Paper Chromatography
  • b) Descending Paper Chromatography
  • c) Circular Paper Chromatography
  • d) Two Dimensional Paper Chromatography


3) Thin Layer Chromatography:

Separation and identification of very small quantity of drug (µg), limit test, assay.

Advantage: fast as compared to PC.

a) Two Dimensional Thin Layer Chromatography

b) Continuous development TLC

c) High Performance Thin Layer Chromatography (HPTLC)

4) Gas Chromatography:

For gaseous and volatile substance.

a) Gas-Solid Chromatography (GSC)

b) Gas-liquid Chromatography (GLC)



5) High Performance/Pressure Liquid Chromatography:

a) Normal Phase HPLC

b) Reverse Phase HPLC

6) Super Critical Fluid Chromatography

7) Electro Chromatography/ Capillary Electrophoretic Chromatography



  • Adsorption (liquid-solid) Chromatography:
  • Substance of different chemical type.
  • S.P. : Adsorbent
  • Adsorb different solute to different extent.
adsorption chromatography

Adsorption Chromatography

Adsorption chromatography is probably one of the oldest types of chromatography around.

It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.

The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.



2) Partition (liquid-liquid) Chromatography:

Substance having similar chemical type.

Both the phases must be liquid.

one having greater partition co-efficient in m.p. than s.p., it will move fast and vice versa.

Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography)

partition chromatography

Partition Chromatography

This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase.

Solute equilibriates between the mobile phase and the stationary liquid



3) Ion-exchange Chromatography:

Ionic and inorganic Substance.

Separation is based upon ionic charge density.

S.P.: ion exchangers which has capacity to exchange ions.

Support chemically bonded to anionic or cationic group

Separation on the basis of charge on the solute on the principle of opposite charges attracting each other.

Resolution influenced by pH & ionic strength of buffer.

Cationic Sulfonate exchanger strongly acidic

Carboxylate exchanger weak

Anionic Quaternary ammonium strongly basic

Tertiary amine weak

ion exchange chromatography

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.



4) Size exclusion(MolecularExclusion/gel permeation) chromatography:

Size exclusion chromatography is a type of chromatography in which the stationary phase is a molecular sieve (Different gel material).

Therefore, the molecules are separated according to differences in their molecular size.

Non interactive mode of separation.

The particles of column packing have various size pores and pore networks

Solute molecules are retained or excluded on the basis of their hydrodynamic volume.

Column packing:

- semi rigid cross linked - polymer gels

- rigid controlled pore size glasses or silicas

molecular exclusion chromatography

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.



  • 3) Miscellaneous Chromatography:
  • a) Ion pair Chromatography:
  • Control of hydrophobicity and hydrophilicity counter ion is added.
  • Production of ion pair comprising a sample ion and oppositely charged ion component of mobile phase.
  • Non polar interaction of ion-pair with stationary phase.
  • Ion pair agent first absorbs onto stationary phase via non polar interaction.
      • Sample ion interacts with the agent.


  • 3) Miscellaneous Chromatography:
  • b) Chiral Chromatography:
  • S.P.: Optically active
  • Thus, entiomer can be separated.
  • c) Affinity Chromatography:
  • Specific interactions between stationary phase and solute molecule.
  • Antigen - antibody
  • enzyme - substrate or inhibitor
  • Hormone - binding protein
  • S.P.: Enzyme (highly selective in interaction with substrate)
  • OR
  • Substrate
affinity chromatography

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.


Two types of partition chromatography:1. Liquid-liquid --- liquid stationary phase is held on support particles through physical adsorption, easy to lose during the mobile phase running.2. Bonded-phase --- the stationary phase is chemical bonded to support surfaces.In early partition chromatography, liquid-liquid type dominated, but nowadays bonded phase has become predominant.Partition chromatography, now normally referred as to Bonded-phase chromatography, has two types:

Partition Chromatography: a Typical LC


Normal-phase LC:The stationary phase is polar (silica or chemically modified surface) and the mobile phase is non-polar (hexane, ether or other non-polar organic solvents). Good for separation of non-polar solutes.Reversed-phase LC:column packing is made of chemically modified silica (normally with non-polar surface) and the mobile phase is polar (water, or polar organic solvents like alcohols, acetonitrile). Excellent for solubilizing and separating polar solutes. This type of LC is very useful for biological or medical research which normally deal with water solutions.LC plays with balancing average concentration in mobile phase versusconcentration in the stationary phase.Keq = Cstat/Cmobile


Adsorption chromatography (HPLC)

Nature of stationary phase

i) Normal phase

Stationary phase - silica, alumina

Mobile phase - CH2Cl2, IPA, MeOH, Hexane

ii) Reverse phase

Stationary phase - non polar

Mobile phase - MeOH, ACN, Aqueous buffers


Normal phase retention by two possible interactions

1.Displacementofsolvent molecules which are adsorbed on the stationary phase.

2. Sorption of solute molecule into a layer of solvent molecule without their displacement from stationary phase.

Retention of solutes decreases with decreasing polarity

The relationship between polarity and elution times for normal-phase and reversed-phase chromatography
comparison of reversed phase media of different chain length
Comparison of Reversed-Phase Media of Different Chain Length

Peak ID

1 – Uracil

2 – Phenol

3 – Acetophenone

4 – Nitrobenzene

5 – Methyl Benzoate

6 – Toluene



Stationary phase selection criterion is highly dependent on the particular application and separation aims.

In general:

1. C18 (Octyldecylsilane – ODS) Bonded Ligand

The most popular and functional of the available reversed-phase stationary phases.

Particularly useful for the separation of non-polar analyte species.

2. C8 Bonded Ligand

While less non-polar than the C18, it provides similar selectivity while giving appreciably shorter run times.

3. C4 / C3 Bonded Ligand

These shorter-chain hydrocarbons are not as stable or as retentive as their longer chain C18 and C8 counterparts, but provide good separations for a variety of protein and polypeptide analytes.

4. CN (Cyano) Bonded Ligand

Interacts with polar functional groups, allowing its use in both reversed-phase and normal phase chromatography.

5. NH2 (Amino) Bonded Ligand

Interacts with polar functional groups. Commonly used for the separation of sugars and polysaccharides.

6. Phenyl Bonded Ligand

Exhibits a more polar nature than either the C18 or C8, the π electron clouds providing sites of interaction for a variety of aromatic (ring) analytes.


Partition (Reversed-phase) Chromatography

Analyte retention increases with increasing stationary phase carbon chain length.

Some typical bonded phases used in reversed-phase HPLC are described below:



In normal-phase HPLC, the stationary phase is polar, often the silica itself, and the mobile phase is relatively non-polar (such as hexane, iso-propylether, toluene,etc.).

The least polar component elutes first.

Increasing the polarity of the mobile phase decreases the elution time.

In reversed-phase HPLC, the stationary phase is non-polar, often a hydrocarbon, and the mobile phase is relatively polar (such as water, methanol or acetonitrile).

The most polar component elutes first.

Increasing the polarity of the mobile phase increases the elution time.



Method Application

1 Normal phase

CN rugged, fairly polar, general utility

OH more polar than cyano

NH2 highly polar, less stable

Silica rugged, cheap, less convenient to operate,used in prep LC


Method Application

2 Reversed phase

C18 rugged, highly retentive

C8 slightly less retentive than C18

C3, C4 less retentive, mostly for peptides & proteins

C1 least retentive, least stable

Phenyl moderately retentive, some selectivity difference

CN moderately retentive, both normal &reverse phase

NH2 weak retention, less stable, carbohydrates

Polystyrene pH 1 - 13, better shape & longer life for some separations

s p silica gel
S.P.-Silica gel:
  • Wide range of solventsto be available as the mobile phase and this increases both the complexity of the analytical procedure and the solvent disposal costs.
  • Water de-activates silica rapidly to such an extent that it exhibits very weak interactions with most substances during the separation process and, as a consequence they are only slightly retained and thus, poorly resolved.
  • Another serious problem with silica as a stationary phase is that it is significantly soluble in water and, thus, if aqueous solvents are used as the mobile phase, the column performance will gradually deteriorate due to the dissolution of the silica.
  • This will be particularly serious if the pH of the mobile phase is outside the range of 4.0 - 8.0.

Today there is a large number of bonded phases available with an extensive range of polarities but, of these, the reverse phases are by far the most commonly used in modern LC.

  • The term reversed phase originated from the work of Martin and Synge when they were examining different phase systems.
  • Martin and Synge replaced a phase system of water as the stationary phase and a hydrocarbon as the mobile phase by a hydrocarbon as the stationary phase and water as the mobile phase.
  • In fact this can be called a phase reversal and the alternative stationary phase was called the reverse phase.

In liquid chromatography, liquid-liquid systems are unstable as, however small the solubility of the stationary phase may be in the mobile phase, the stationary liquid phase will be eventually stripped from the column.

  • It was therefore found necessary to chemically attach the stationary phase to the support to ensure a stable system and these materials were called bonded phases.
  • The early bonded phases were silica based and prepared by reacting the hydroxyl groups on the surface of the silica with organic silyl chlorides or silyl esters, any remaining unreactedsilanolgroups being blocked by methylation with hexamethyldisilazane (capping reagent).

A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

A stationary phase chemically bonded to a support that is used for the separation.

It is the most commonly used LC mode.

The most popular support used is microparticulate silica gel.

An organosilane, such as octadecyl (for reversed-phase chromatography), is the most accepted type of bonded phase.

Approximately 70 percent of all HPLC is carried out on chemically bonded phases.

characteristics of bonded phases
Characteristics of Bonded Phases
  • Attach aliphatic hydrocarbon chains to the surfaceby means of the silicon-oxygen-carbon linkage.
  • The silicon-oxygen-carbon linkage is very weak and the bonded phase readily hydrolyzed from the surface, thus, regenerating the original hydroxyl groups of the silica gel.
  • An alternative bonding method that involved the use of chlorsilane reagents.
  • When a chlorsilane reacts with a hydroxyl group of the silica gel surface, the hydrocarbon chain is attached by the much stronger and stable silicon-carbon link.
the different classes of bonded phase
The Different Classes of Bonded Phase

Some Commonly Used Silane Reagents Used in Bonded Phase Synthesis

brush type bonded phases
"Brush" Type Bonded Phases
  • By employing mono-substituted silanes single layers of organic moieties could be bonded to the silica surface and these materials were called brush phases.
  • The brush type phase was the first class of bonded phase to be prepared and the basic methodology for its production, employing a mono-chlorsilane reagent.
  • The product is made of a surface that is covered with dimethyloctyl chains, attached like the bristles of a brush, hence the term \'brush\' phase.
oligomeric type bonded phases
"Oligomeric" Type Bonded Phases
  • By alternately treating silica with di-substituted silanes and water in a heated fluidized-bed system, oligomeric bonded phases can be synthesized whereby the oligomer is only attached to the silica at one position.
  • In the synthesis of oligomeric phases, a di-substituted silane, (i.e. dichlorsilanes) must be employed as the silanizing reagent.
  • As one chlorine atom of the dichlor reagent reacts with a surface hydroxyl group, a mono-chlor silane is bonded to the silica accompanied by the evolution of hydrochloric acid.

Alternately treating the bonded phase with water and a dichlorsilane,a series of methyloctylsilyl residues can be attached to each other that builds up the oligomeric reverse phase.


When the last oligomeric synthesis has been completed and the final oligomer attached, the remaining silanol groups are capped with trimethyl chlorsilane or hexamethyltrisilazane.

  • This reaction terminates the oligomeric chain with a trimethyl silyl group.
bulk type bonded phases
"Bulk" Type Bonded Phases
  • Employing tri-substituted silanes in the presence of water the organic moieties could be cross linked with ether groups and form a type of a polymer.
  • These polymeric phases were strongly held to the silica matrix and were very stable and were given the name bulk phases.
  • The bulk type phase consists of a somewhat open polymeric film of stationary phase covering the silica surface that is formed by the use of trichlorsilyl reagents.

In the second stage it is presumed that the \'octyldichloro\' chain reacts with some of the excess water to from a \'octyldihydroxy\' chain.

  • The polymer is cross-linked by water that is added to the silica prior to reaction.

When the silica surface is saturated with water and is treated with octyltrichlorsilanea reaction occurs with both the hydroxyls of the silica surface and the adsorbed water, causing a cross-linking reaction and the octylsilanyl polymer is built up on the surface.

As a result of the polymerization process, the stationary phase assumes a multi-layer character and that is why it is termed a \'bulk\' phase.

The \'octyldihydroxy chain can, in turn react, with two more octyltrichlorsilane molecules and start building the polymer on the surface.

the use of alkoxysilane reagents in bonded phase synthesis
The Use of Alkoxysilane Reagents in Bonded Phase Synthesis
  • If the alternative alkoxysilane reagents are employed for bonding, the method of synthesis remains very similar. The same solventscan be used but, as no hydrochloric acid is generated.
  • The most reactive alkoxy reagents are the methoxy and ethoxysilanes and their reaction with a hydroxyl group is accompanied by the release of methanol or ethanol.
sorption interaction mono layer adsorption

The Distribution of Solvents A and B as a Mono-layer on a Silica Gel Surface

bilayer adsorption of solvent

The Distribution of Solvents A and B as a Bi-layer on a Silica Gel Surface

Different types of solute interaction that can occur on silica surfaces covered with a solvent bi-layer

Mechanisms of Separation:

  • Partitioning
  • Adsorption
  • Exclusion
  • Ion Exchange
  • Affinity
separation mechanism classification based on attractive forces
Separation mechanism/Classification based on Attractive Forces
  • Adsorption - for polar non-ionic compounds
  • Ion Exchange - for ionic compounds
    • Anion - analyte is anion; bonded phase has positive charge
    • Cation – analyte is cation; bonded phase has negative charge
  • Partition - based on the relative solubility of analyte in mobile and stationary phases
    • Normal – analyte is nonpolar organic; stationary phase MORE polar than the mobile phase
    • Reverse – analyte is polar organic; stationary phase LESS polar than the mobile phase
  • Size Exclusion - stationary phase is a porous matrix; sieving