Notes on chromatography

  • Created by: Tassawar
  • Created on: 16-05-13 20:13



Partition is all about differences in and relative solubilities of components in a sample which causes them to dissolve and partition onto the stationary or mobile phases accordingly. The more soluble/ volatile the component, the faster it moves. 

Stationary phase: non-volatile liquid. Mobile phase: inert solid surface

Examples: paper chromatography and gas liquid chromatography. 

Adsorption chromatography

Adsorption is all about relative bonding and reactivity with the stationary and mobile phases. Some components of the mixture are attracted to the solid surface and the other components are less strongly attracted to the stationary phase travel faster with the mobile phase. 

Stationary phase: solid stationary phase. Mobile phase: liquid or gas phase.

Examples: Thin-layer chromatography.

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Paper chromatography

Stationary phase: chromatographic adsorbent paper. Mobile phase: suitable solvent (water/ ethanol).

A small spot is placed on the chromatographic paper which is dipped in the solvent. The component spots dissolve in the solvent and move up with the solvent, moving different distances depending on their solubilities. The solvent front marks the maximum distance moved by the solvent. The chromatogram is finally treated with a dye/ UV light if the different components aren't visible e.g. ninhydrin to identify amino acids. 

R_f value: this is the (distance moved by the component from the base line) / (distance moved by the solvent front). 

Limitations: Components having similar R_f values are indistinguishable, unknown/ new substances have no R_f values to test against. 

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Thin-layer chromatography - adsorption

Stationary phase: alumina/ silica. Mobile phase: liquid solvent

Small spots are placed with a capillary tube. The different components separate and can be identified. The technique is used in qualitative analysis to determien substance purity. TLC has advantages over paper chromatography:

  • 3 times quicker than paper chromatography.
  • More efficient - works on small samples which can be separated and recovered in a pure form.
  • Resutls are easily reproduced.
  • A range of mixtures can be separated by changing the mobile and stationary phases. 
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Column chromatography

Stationary phase: alumina/ silica. Mobile phase: Suitable organic solvent. 

Chromatography column filled and saturated with solvent and alumina. The sample  to be separated is dissolved in a volume of solvent and added to the column. Solvent added from the top added to replace the one dripping down and to wash sample down the stationary phase. 

The different components are separated as they pass through the column and collected at the bottom in different beakers. 

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Gas-liquid chromatography

Stationary phase: liquid adsorbed on an inert solid support. Mobile phase: inert gas (N/ He)

Used to separate and identify small samples of gases/ volatile liquids. The sample is injected through a self-sealing cap into an oven where it is vaporized. The vapour is carried by a unreactive gas mobile phase over the non-volatile liquid stationary phase.

The components of the sample partition between the inert gas/ non-volatile liquid depending on their relative boiling points and solubilities in relation to the phases. The time taken to travel from column to detector is called the retention time. Measured from the time sample is injected to the time its peak shows the maximum height. 

Improvements: chromatogram is calibrated by passing a substance of known concentration under the same conditions e.g. a standard solution of propan-1-ol used to test for blood alcohol level. Ratio of the areas under the peaks are proportional to their relative concentrations, the concentration of ethanol can be accurately determined .

Used: to identify components that vaporise without decomposition in the oven. Used to test urine samples for illegal substances e.g. steroids and stimulants and blood for alcohol levels.

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High-performance liquid chromatography

Stationary phase: silica/ alumina. Mobile phase: suitable solvent.

This is an improved form of column chromatography. High pressures are used instead of gravity to force the mixture being analysed through a column tightly packed with fine solid particles.

The method is used to separate components which are very similar  to each other. The chromatogram produced is similar to that in GLC but UV light is used to detect the different components. 

Used: analysis of oil pollutants; alcoholic beverages; and antioxidants, sugars and vitamins in food. It is used in the pharmaceutical and polymer industries for quality control of insecticides and herbicides and in biochemical and biotech research 

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