Chromatography


Chromatography (from Greek Chroma "color and graphy “writing") is the techniques for the separation of mixtures.
The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.
The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile & stationary phases. 

History of Chromatography


Chromatography, literally "color writing", was first employed by Russian scientist Mikhail Tswvett in 1906.

He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls.
Since these components have different colors (green, orange, and ,   Yellow            respectively) they gave the technique its name.


Principles of Chromatography

It consists of mobile phase and stationary phase.


The mobile phase refers to the mixture of substances to be separated dissolved in a liquid or a gas.

The stationary phase is a porous solid matrix through which the sample contained in the mobile phase percolates.

The interaction between the mobile phase and the stationary phase results in the separation of the compound from the mixture.

Components of chromatography
•Stationary phase
–Liquid
–Solid
•Mobile phase
–Liquid  
–Gas
•Process condition
–Temperature
               Others

The chromatographic method of separation, in general, involves following steps

Adsorption or retention of substances on the stationary phase
Separation of the adsorption of subs. by the mobile phase
Recovery of the separated substances by a continuous flow of the mobile phase; the method being called elution.
Qualitative and Qantitative analysis of the eluted substances

Chromatographic terms
Stationary phase or adsorbent substance that stays fixed inside the column 
Analyte is the substance to be separated during chromatography.
Chromatogram is the visual output of the chromatograph.
 Eluate is the mobile phase leaving the column.
 Eluent is the solvent that carries the analyte.
 Detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.
Elution  the process of washing out a compound through a column using a suitable solvent.

Classification of chromatography

BASED ON MECHANISM OF SEPARATION

I.Adsorption chromatography
II.Partition chromatography
III.Ion exchange
IV.Size exclusion
The most of chromatography based on first two principals  


BASED ON SHAPE OF CHROMATOGRAPHIC BED

Planner chromatography
I.Paper chromatography
II.Thin layer chromatography
Column chromatography
I.  Packed column chromatography
II. Open tubular column chromatography

BASED ON PHASES
Solid phase chromatography
i.Solid-liquid chromatography
ii.Solid-gas chromatography
Liquid phase chromatography
i.Liquid-liquid chromatography
ii.Liquid –gas chromatography


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 to stationary phase present in chromatography column

Adsorption chromatography is one of the oldest types of chromatography.

The equilibration between the mobile and 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


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 & the stationary liquid.

Ion Exchange Chromatography
Ion exchange chromatography (IEC) uses an ion exchange mechanism to separate analytes based on their respective charges. 
It is usually performed in columns but can also be useful in planar mode. 

IEC uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins.

In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain.

There are two types of ion exchange chromatography: 
Cation-Exchange and Anion-Exchange. 
In the Cation-Exchange Chromatography the stationary phase has negative charge and the exchangeable ion is a cation, whereas, in the Anion-Exchange Chromatography the stationary phase has positive charge and the exchangeable ion is an anion.  
IEC is commonly used to purify proteins using FPLC (Fast protein liquid chromatography) 


Size-exclusion chromatography

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). 

Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. 

The average residence time in the pores depends upon the effective size of the analyte molecules.
However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. 
It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. 

It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.


BASED ON SHAPE OF CHROMATOGRAPHIC BED

Planner chromatography


Planar chromatography is a separation technique in which the stationary phase is present on a plane.

The 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.

The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance


 


Column Chromatography

Column chromatography is a separation technique in which the 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



Gas-Solid chromatography(G.S.C.)

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.

Solid-Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid.
The preferred mobile phase is a nonpolar or slightly polar...
Liquid chromatography can be carried out either in a column or a plane.

In liquid-solid chromatography the porous adsorbent is polar and separation is based on the properties of classes of compounds—e.g., amines (alkaline) from alcohols (neutral) & esters (neutral) from acids Popular adsorbents are Silica and Alumina. 

Liquid-Gas Chromatography

The 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 forensic tests
Stationary phase used in (LGC)

Liquid-Liquid Chromatography (LLC)

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.

LLC is a chromatography separation technique in which the mobile phase is a liquid (usually a 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


Applications of chromatography

Separation of mix of drugs of chemical, biologic and plant origin.
Separation of carbohydrate (sugars), vitamins, antibiotics, proteins, alkaloids, glycosides and amino acids. 
Identification of drug.
Identification of impurities.
Identification of related compound.
Identification of decomposition product.
Analysis of metabolite of drug in blood, urine etc.