Chromatography

Chromatography
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.

Chromatography
Mobile phase: phase which sample is dissolved in may be gas, liquid, or supercritical fluid Stationary phase: phase which mobile phase is forced through Mobile and stationary phases are chosen so the analyte will distribute itself between the two phases

The general principle.

Use to separate and identify components of mixtures. Several different types - paper, thin layer, gas-liquid. All use the principle of partition affinity between two phases, to separate mixtures of substances. Stationary phase & mobile phase.

Compounds with greatest affinity for mobile phase travel further.

Chromatography
Separates components in mixture: Based on - polarity - boiling point - ionic strength - size

All chromatography needs:

support material stationary phase solvent (or carrier gas) mobile phase.

What happens in practice.

Compounds that have high affinity for mobile phase emerge first, (most volatile). Chromatogram charts recorder response against time. Each component - separate peak. Retention time characteristic of the compound under given conditions.

Thin Layer Chromatography

Here the mobile phase is a liquid Flowing past a thin layer of powder on a solid support. Substances that are less attracted to the solid or are more soluble in the liquid move faster. And so move further up the plate by the time that the process has been stopped by taking the plate out of the liqiud. - larger Rf

Thin Layer Chromatography - t.l.c.

Series of spots forms Compare samples in mixture with known substances. Measure Rf values. Coloured compounds & colourless compounds.

Rf = distance moved by substance distance moved by solvent front

For substances that are very soluble in the liquid Rf will be close to ....

For substances that are rather insoluble in the liquid Rf will be close to ....

Separation and identification.

Gas Liquid Chromatography

Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube. And the stationary phase is an involatile liquid held on particles of a solid support.

Injection port
Oven

Recorder

Detector

Nitrogen cylinder

Colum n

Gas - Liquid Chromatography G.l.c.

Sample introduced by syringe.

Column separates components. (Heated in oven)

Detector monitors compounds emerging from outlet.

Recorder plots signals as a chromatogram.

Uses of G.l.c.
Very sensitive - small quantities of substances detected, explosives, drugs etc. Separation of pure substances for collection. Can be connected to mass spectrometer for direct identification of substances.

Factors affecting retention time:

length of column
packing material type of carrier gas flow rate of carrier gas temperature of column.

Interpreting the trace

Calibration known compounds are added to the column and conditions kept constant. Amount of substance area under peak / peak height. Relative proportions can be determined.

Chromatogram of petrol

Suggest identities of some of the unlabelled peaks.

Typical Gas Chromatogram

Typical Liquid Chromatogram

Partition Chromatography
Used in GC & LC Molecules will partition into the stationary phase based upon affinity for stationary phase & eventually partition into mobile phase again Thin layer is coated onto inside of GC column or on small particles on LC column

Adsorption Chromatography
Very similar to partition chromatography Adsorption just on surface, partition into thin layer Not used as widely as partition used mainly in TLC & very small particles in LC

Ion Exchange Chromatography

Separation of either cations or anions Separtion based on relative strength of ionic bond Anion exchange has cations on surface Used in LC exclusively

Molecular Exclusion Chromatography

Separation based on size Small molecules get trapped in pores & take longer to get out

Gel Electrophoresis
Separation based on size and charge Smaller molecules will migrate further, less tangled

Affinity Chromatography
Very selective Specific binding site is used to concentrate analyte on column Used a lot in biological applications