Partition chromatography

By: Dale Faith O. Dumalagan

Chromatography Chromatography is based on the Greek

word chroma, for colour. This technique is extensively used to separate mixtures into their components, purify compounds and also to test its purity.

Chromatography works on the same principle as extraction, but one phase remains stationary and the other flows past.

Chromatography The stationary phase is the phase that stays in

place inside the column and is usually viscous liquid chemically bonded to the inside of a capillary column or onto the surfacee of solid particles packed into the column.

The mobile phase is the solvent moving through the column and is either liquid or gas.

Fluid entering the column is the eluent, while fluid leaving the column is the eluate.

Elution is the process of passing liquid or gas through the column.

Adsorption Chromatography Adsorption chromatography is a process

of separation of components in a mixture introduced into chromatography system based on the relative differences in adsorption of components to the stationary phase present in the chromatography column.

This adsorption chromatography applies to only solid-liquid or solid-gas chromatography. Because the adsorption phenomenon is inherent property of solids and hence it is used with only solid stationary phase chromatographies.

TypesChromatography

________________________

Adsorption Partition Chromatography Chromatography

Column Thin Layer Gas-Liquid Liquid-Liquid

Chromatography Chromatography Chromatography Chromatography

Adsorption Chromatography1. Column Chromatography

It involves the separation of a mixture over a column of adsorbent (stationary phase) packed in a glass tube.

The mixture adsorbed on adsorbent is placed on the top of the adsorbent column packed in a glass tube.

An appropriate eluant which is a liquid is allowed to flow down the column slowly

The most readily adsorbed substance are retained near the top and the others come down to various distance in the column

1. _2. Thin Layer Chromatography

This is separation of substances of a mixture over a thin layer of an adsorbent coated on glass plate.A thin layer about 0.2mm thick of an adsorbent (silica gel or alumina) is spread over a glass plate of suitable size.The plate is known as thin layer chromatography plate or chromaphate.The solution of the mixture to be separated is applied as a small spot about 2cm above one end of the TLC plate.

Partition Chromatography Definition:

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 the stationary liquid.

Partition ChromatographyPartition chromatography is process of separation whereby the components of the mixture get distributed into two liquid phases due to differences in partition coefficients during the flow of mobile phase in the chromatography column.

This mode of partition chromatography applies to Liquid-liquid, liquid-gas chromatography and not to solid-gas chromatography. Because partition is the phenomenon in between a liquid and liquid or liquid and gas or gas and gas. But not in solid involvement.

Partition Chromatography Priciple:

Separation of components of a sample mixture occurs because of partition. Stationary phase is coated with a liquid which is immiscible in mobile phase.

Partition of component of a sample between sample and liquid/gas stationary phase retard some components of sample more as compared to others. This gives basis for separation.

The stationary phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phases passes over the coated adsorbent and depending upon relative solubility in the coated liquid, separation occurs. The component of sample mixture appear separated because of differences in their partition coefficient.

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.

Gas Liquid Chromatography A form of chromatography in which the mobile phase is a gas and the stationary phase is a liquid, usually on small beads packed in a long column Used to separate and analyze compounds

Sample has to be able to be vaporized without decomposition Based on boiling point/vapor pressure

Typical uses: Testing purity of particular substance Separation of different components of a mixture Prepare pure compounds from a mixture

Mobile phase Inert carrier gas (usually He or N)

Stationary phase Layer of liquid or polymer on inert solid

support Inside a glass or metal tubing (COLUMN)

• Compound is injected with syringe into sample injector

• Compound is carried by carrier gas and vaporized• Vaporized sample interacts with walls of column

– Some samples interact more some less• Due to interaction samples elute at different times

– Retention times– Comparison of retentions times is what is useful

• A detector monitors the outlet stream from the column

Carrier Gas Pressure Regulator and Flow Meter

H2 : It has a distinctly better thermal conductivity and lower density. Demerits are its reactivity with unsaturated compounds and hazardous explosive nature He : It has an excellent thermal conductivity, low density, inertness and it permits greater flow rates. It is highly expensive N2 : It offers reduced sensitivity and is inexpensive Air : It is employed only when the atmospheric O2 is beneficial to the detector separation.

Sample Injection System (a) Liquid Samples : They are usually injected by hypodermic syringes through a self-sealing silicon-rubber septum into a preheated-metal-block flash evaporator. The sample is vapourize as a ‘plug’ and carried right into the column by the respective carrier gas. Sample size ranges between 1–10 μl. (b) Solid Samples : These are either dissolved in volatile liquids (solvents) or temporarily liquefied by exposure to infrared heat. (c) Gas Samples : They are best handled and injected by gas-light syringes or a gas-sampling valve, usually termed as a stream-splitter.

Separation Column Types:

Packed Packed columns contain a finely divided, inert, solid

support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

Capillary a.k.a Open Tubular Capillary columns have an internal diameter of a

few tenths of a millimeter. Wall-coated open tubular (WCOT) Support-coated open tubular (SCOT)

Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed.

SCOT columns are generally less efficient than WCOT columns.

Both types of capillary column are more efficient than packed columns.

Properties of Gas Chromatography Columns

Computer Generated Image of a FSWC column (specialized for measuring BAC levels)

Computer Generated Image of a FSWC column (specialized to withstand extreme heat)

Detector Types of selectivity:

Non-selective detector - responds to all compounds except the carrier gas Selective detector - responds to a range of compounds with a common physical or chemical property Specific detector - responds to a single chemical compound

Liquid-Liquid Chromatography Components within a mixture are separated in

a column based on each component's affinity for the mobile phase. So, if the components are of different polarities and a mobile phase of a distinct polarity is passed through the column, one component will migrate through the column faster than the other. Because molecules of the same compound will generally move in groups, the compounds are separated into distinct bands within the column. If the components being separated are colored, their corresponding bands can be seen.

Other varieties of Liquid Chromatography Partition Chromatography Liquid-Solid Chromatography Ion Exchange or Ion Chromatography Size Exclusion Chromatograhy Affinity Chromatography Chiral Chromatography Plate Theory or Rate Theory