Unit 28: Chromatography — Short Questions

Paper chromatography works on the principle of relative solubility (partition) of the components of a mixture between two phases: a mobile phase (the solvent moving up the paper) and a stationary phase (water held within the cellulose fibres of the paper). Components that are more soluble in the mobile phase move further up the paper, while those more soluble in the stationary phase move less, allowing the mixture to separate.

Ink is itself a mixture of coloured, soluble dyes, so if it were used to mark the baseline it would dissolve in the solvent and travel up the paper along with the sample, contaminating the chromatogram and making the spots impossible to interpret. Pencil (graphite) is insoluble in the solvent, so the baseline mark stays fixed in place and can be used as a reliable reference point for measuring distances and calculating Rf values.

1) TLC is faster than paper chromatography. 2) Separation in TLC occurs by a combination of adsorption (onto the solid silica/alumina layer) and solubility, rather than partition alone as in paper chromatography, which generally gives sharper, better-resolved spots.

Polar substances have a strong attraction for the polar stationary phase (silica or alumina) and are therefore adsorbed more strongly onto it. This strong adsorption holds them back, so they travel more slowly up the plate compared with non-polar substances, which are more soluble in the non-polar solvent and move faster.

In TLC only a small amount of sample can be applied to and analysed on the plate, so it is essentially a qualitative, small-scale analytical technique rather than one that can isolate large quantities of a substance.

The injector port is where the sample is introduced into the gas chromatograph. It is heated by an oven so that the sample is rapidly vaporised before being swept into the column by the carrier gas.

In GC, separation depends on the sample's vapours being carried through the column by the inert carrier gas. Only volatile substances can be readily vaporised at the injector/oven and transported through the column; non-volatile substances cannot be converted to the gas phase, so they cannot be analysed by GC.

1) GC works only for volatile substances — large biomolecules such as proteins and polymers cannot be analysed because they do not vaporise easily. 2) Samples must be thermally stable; compounds that decompose at the high temperatures used cannot be analysed by GC.

Adsorption is a surface phenomenon in which particles of a substance accumulate only on the outer surface of another material (they do not dissolve into its bulk). Example: activated charcoal adsorbs gases and impurities onto its surface, which is why it is used in filters and purification systems.

This is the stem for parts (I) and (ii) below; see those parts for the worked answers.

The Rf values would increase, i.e. the spots would travel further up the plate (closer to the solvent front). Amino acids carry polar groups (–NH2 and –COOH), so in a more polar solvent they dissolve more readily and partition more strongly into the (now more polar) mobile phase. Being carried more effectively by the mobile phase relative to the stationary phase, they move further up the plate, giving higher Rf values.

Mobile phase an inert carrier gas such as helium or nitrogen. Stationary phase: a non-volatile, high-boiling, long-chain non-polar hydrocarbon liquid (e.g. a high-boiling liquid such as a silicone oil) coated onto small particles of a solid support such as silica (SiO2) or alumina (Al2O3).

Retention time is the time taken by a component of the mixture to pass through the GC column, from injection until it reaches the detector — i.e. the time at which its peak appears on the chromatogram. It is characteristic of a substance under a given set of GC conditions (column, stationary phase, temperature, carrier gas flow rate) and is used, by comparison with known retention times, to identify the component.