a field of analytical chemistry that investigates analytes using scientific instruments
Block diagram of an analytical instrument showing the stimulus and measurement of response.
Chromatography
is the separation of the components of a mixture based on the different degrees to which they interact with two separate material phases
CHROMATOGRAPHY
Mobile phase – moving phase
Stationary phase – does not move
CHROMATOGRAPHY
The mixture (sample) is usually introduced into the mobile phase, which is then made to movethrough the stationary phase.
The components are attracted to and slowed by the stationary phase to varying degrees, and as a result, they move along with the mobile phase at varying rates, and are thus separated
CHROMATOGRAPHY
The mobile phase can be either a gas or a liquid,
while the stationary phase can be either a liquid or solid
CHROMATOGRAPHY
All techniques which utilize a gas for the mobile phase come under the heading of gas chromatography.
All techniques that utilize a liquid mobile phase come under the heading of liquid chromatography
Partition Chromatography
The mobile phase is a liquid that moves through a liquidstationary phase as the mixture components partition or distribute themselves between the two phases and become separated.
PARTITION CHROMOTOGRAPHY
The separation mechanism is thus one of the dissolving of themixture components to different degrees in the two phasesaccording to their individual solubilities in each.
PartitionChromotography
This form of chromatography is based on a thin fim formed on the surface of a solid support by a liquid stationary phase.
Solute equilibriates between the mobile phase and the stationary liquid.
Partition Chromatography
The stationary phase actually consists of a thinliquidfilm chemically bonded to the surface of finely divided solid particles
Such a stationary phase cannot be removed from the solid substrate by heat, reaction, or dissolving in the mobile phase.
Partition Chromatography
The mobile phase can also be a gas
Partitionchromatography
a type of chromatography in which the stationary phase is a liquid chemically bonded to the surface of a solid substrate, while the mobile phase is either a liquid or gas.
Partition Chromatography
The mixture components dissolve in and out of the mobile and stationary phases as the mobile phase movesthrough the stationary phase, and separation occurs as a result
Adsorption Chromatography
The separation mechanism is adsorption
AdsorptionChromatography
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.
Adsorption Chromatography
The stationary phase consists of finely divided solidparticles packed inside a tube, and the mixture components adsorb or stick to the surface of the solid
Adsorption Chromatography
The nature of the adsorption involves the interaction of polar molecules, or molecules with polar groups, with a very polar solid stationary phase.
The mobile phase can be either a liquid or a gas
IonExchangeChromatography
It is a method for separating mixtures of ions, both inorganic and organic.
Ion Exchange Chromatography
The stationary phase consists of very small polymer resin beads that have many ionic bonding sites on their surfaces. These sites selectively exchange ions with certain mobile phase compositions as the mobile phase moves.
Ions that bond to the charged site on the resin beads are thus separated from ions that do not bond
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 electrostaticforces.
Ion Exchange Chromatography
The stationary phase material can be either:
an anionexchangeresin, which possesses positively charged sites to exchange negative ions,
or a cationexchangeresin, which possesses negatively charged sites to exchange positive ions.
Ion Exchange Chromatography
The mobile phase can only be a liquid
Ion Exchange Chromatography
Positively charged protein binds to negatively charged bead
Negatively charged protein flows through
Ion Exchange for Deionization of Water
Wide columns packed with a mixture of:
an anion exchange resin that exchanges dissolved anions for hydroxide ions,
a cation exchange resin that exchanges dissolved cations for hydrogen ions
... are used because water that is passed through such a column becomes free of ions (deionized) since the hydrogen and hydroxide ions combine to form more water.
Partition Chromatography (Paper & TLC)
Thin film of liquid formed of a solid inert support
Liquid or Gas
Solutes equilibrate between the 2 phases according to their partition coefficients
Adsorption Chromatography
Solid that attracts solutes
Liquid or Gas
Solutes move at different rates according to the forces of attraction to the stationary phase
IonExchangeChromatography
Solid resin that carries fixed ions & mobile counterions of opposite charge attached by covalent bonds
Liquid containing electrolytes
Solute ions of chage opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions
Size Exclusion Chromatography
It is a technique for separating dissolved species on the basis of their size
Size Exclusion Chromatography
The stationary phase consists of porouspolymerresin particles. The components to be separated can enter the pores of these particles and be slowed from progressing through this stationary phase as a result.
The mobile phase for this type can only be a liquid
Chromatography Configurations
Configurations can be broadly classified into 2 categories:
Planar methods
Column methods
PLANAR METHOD
utilize a thin sheet of stationary phase material
the mobile phase moves across this sheet, either upward (ascending chromatography), downward (descending chromatography), or horizontally (radial chromatography).
PLANAR METHODS:
Paper Chromatography
Thin Layer Chromatography
COLUMN METHODS:
Open Column Chromatography
Instrumental Chromatography
Paper chromatography
makes use of a sheet of paper having the consistency of filter paper (cellulose) for the stationary phase
Paper Chromatography
Since filter paper is hydrophilic, the stationary phase is actually a thin film of water unintentionally adsorbed on the surface of the paper. Thus, paper chromatography represents a form of partition chromatography only.
The mobile phase is always a liquid
Paper Chromatography
a technique used for the separation & identification of relatively small chemical substances by a moving solvent on sheets or strips of filter paper
Paper Chromatography
Substances to be identified are 'spotted' near one end of the filter paper
As the solvent moves up the paper, different molecules move at different rates with the smallest molecules moving the fastest.
PaperChromatography
The technique is used for small molecules such as amino acids, small peptides and sugars
Thin LayerChromatography
The stationary phase is a thin layer of material spread across a plastic sheet or glass or metal plate.
Thin Layer Chromatography can be any of the four types: including adsorption, partition, ion exchange, and size exclusion