Introduction to chromatography


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Definition of chromatography:

Definition of chromatography IUPAC definition ( International Union of pure and applied Chemistry) (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 while the other moves in a definite direction. The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.

Chromatography :

Chromatography Russian scientist Tswett in 1906 used a glass columns packed with finely divided CaCO 3 to separate plant pigments extracted by hexane. The pigments after separation appeared as colour bands that can come out of the column one by one. Tswett was the first to use the term "chromatography" derived from two Greek words " Chroma " meaning color and " graphein " meaning to write.

Classification Of Chromatographic Techniques:

Classification Of Chromatographic Techniques Different methods were attempted for classification of chromatography: According to mechanism of separation As per the types of mobile phase Based on Physical means (Methods holding the stationary phase)

According to mechanism of separation :

According to mechanism of separation Adsorption chromatography Partition chromatography Size exclusion chromatography Affinity chromatography Ion exchange chromatography Ion pair chromatography

a) Adsorption Chromatography::

a) Adsorption Chromatography : Here The stationary phase is a solid with adsorption power and mobile phase may be liquid or gas. The Mixture components will be adsorbed on the surface of the stationary phase by weak ionic forces. Different component have diff. tendency to adsorb on stationary phase. The forces involved are relatively weak and effective only over short distance. Mobile phase effectively counter act this attractive force

b) Partition Chromatography: :

b) Partition Chromatography: Here the stationary phase is a liquid coated or bonded on solid surface and mobile phase may be liquid or gas. Cellulose powder and wet silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. Sample mixtures are separated according to relative tendency of the sample components to partition between a mobile phase and stationary phase based on the solubility in two phases.

c) Size exclusion chromatography:

c) Size exclusion chromatography It is also called gel permeation chromatography Here stationary phase used are polymer which have been cross linked to yield an open network with numerous pores of consistent size. Here separation of sample mixture is based on their effective size in solution Very large molecule, which are not fit in pores are excluded and eluted first


Cont. Small molecule are free to diffuse in and out of the pores so that their path is longer and they eluted out later.

d) Ion Exchange Chromatography::

d) Ion Exchange Chromatography: It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded . Ions of opposite charges(counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin . Mixture of Alkaloids(compounds with positive charges)can be separated on anionic exchanger, while mixture of organic acids (negative charges) can be separated using cationic exchanger.

e) Ion pair chromatography:

e) Ion pair chromatography In this method a reagent which dissociates to give ions opposite in charge to those of sample solute is added to the mobile phase. The added ion combine with the charged solute to form ion pairs which is non polar and will partition more readily into the stationary phase.

f ) Affinity chromatography:

f ) Affinity chromatography Here the stationary phase is a specific ligand which is immobilized by chemically binding it to an insoluble matrix. Various enzymes, protines , antibodies can be effectively separated using this affinity chromatography.


ACCORDING TO MOBILE PHASE(classification) Liquid chromatography Gas chromatography Supercritical fluid chromatography(SFC)

a) Liquid chromatography (LC):

a) Liquid chromatography (LC) The mobile phase is liquid. In case of separation by adsorption the stationary phase is solid so it is called: Liquid-Solid Chromatography (LSC). If separation occurs through partition the stationary phase is liquid so it is called: Liquid -Liquid Chromatography (LLC).

b) Gas chromatography (GC):

b) Gas chromatography (GC) Here the mobile phase is inert gas nitrogen or helium. If the stationary phase is solid it is called: Gas–Solid Chromatography (GSC) . When stationary phase is liquid it is called: Gas-Liquid Chromatography (GLC ).

Supercritical Fluid Chromatography (SFC):

Supercritical Fluid Chromatography (SFC) In Supercritical Fluid Chromatography (SFC), the sample is carried through a separating column by a supercritical fluid (typically carbon dioxide) where the mixture is divided into unique bands. Advantage of SFC is that, it provides rapid separation. It also not require any organic solvent, which we have to require in HPLC.

Based on physical means(methods holding the stationary phase):

Based on physical means(methods holding the stationary phase) Planar or Plane Chromatography: Columnar or Column Chromatography (CC):

a) Planar or Plane Chromatography: :

a) Planar or Plane Chromatography: In this type of chromatography the stationary phase is used in the form of layer. Plane chromatography is further classified into: a- Thin Layer Chromatography (TLC): b- Paper Chromatography (PC):

b)Column Chromatography (CC): :

b)Column Chromatography (CC): The stationary phase is held in to a tube made of glass or metal. Here,the mixture to be analyzed is applied to the top of the column. The liquid solvent (the eluent ) is passed through the column by gravity or by the application of air pressure.

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If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography . If the solvent is forced down the column by positive air pressure, it is called flash chromatography.

Elution Chromatography on Columns :

Elution Chromatography on Columns Elution involves washing a species through a column by continuous addition of fresh solvent. The sample is introduced at the head of a column, components of the sample distribute themselves between the two phases. Introduction of additional mobile phase (the eluent ) forces the solvent containing a part of the sample down the column, where further partition between the mobile phase and fresh portions of the stationary phase occurs.


Chromatogram If a detector that responds to solute concentration is placed at the end of the column and its signal is plotted as function of time (or of volume of the added mobile phase), a series of peaks is obtained. Such a plot, called a chromatogram It is useful for both qualitative and quantitative analysis.

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Chromatogram chromatogram - concentration versus elution time W h W b Where: t R = retention time t M = void time W b = baseline width of the peak in time units W h = half-height width of the peak in time units Inject

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Retention time (t r ): It is the time required to elute a sample component from the stationary phase. Retention Vol.(V r ) : It is the vol. of mobile phase required to elute the sample component from the stationary phase. Relation between t r and v r : V r = f * t r Here, f= flow rate Dead time: The time t M for the unretained species to reach the detector is called the dead time.

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Distribution coefficient : It is the ratio of the conc. Of the species in the stationary phase to the mobile phase. K= c s /c m Retention factor or capacity factor: It is used to describe the migration rates of solutes on columns. For a solute A, the retention factor k` A is defined as k` A = K A V S /V M where K A is the distribution constant for the species A.

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When k' is <1.0, separation is poor When k' is > 30, separation is slow When k' is = 2-10, separation is optimum

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Relative Migration Rates: The selectivity Factor: The selectivity factor  of a column for the two species A and B is defined as  = K B /K A where K B is the distribution constant for species B and K A is the distribution constant for species A.  is always greater than unity. A relationship between the selectivity factor and retention factors:  = k` B / k` A Where k` B and k` A are the retention factors. A value of a >1.1 is usually indicative of a good separation

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Resolution: It is a quantitative measure of column which provides its ability to separate two analytes . For a symmetrucal peak with gaussian shape, resolution is represented by eqn., R=2(t 2 -t 1 ) / (w 1 +w 2 ) where t 2 and t 1 are retention time of adjacent peaks and w1 and w2 are the peak widths. Resolution of 1.5 indicates complete separation between adjacent peaks.

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If one of the peak is assymetrical , resolution is given by, R= t 2 -t 1 /(w 1R +w 2L ) Here, w 1R represents peak width to right of t 1 and w 2L represents peak width left to t 2.

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