MCAT Content / Separations And Purifications / Chromatography Basic Principles Involved In Separation Process

Chromatography: Basic principles involved in separation process

Topic: Separations And Purifications

Chromatography is a separation technique that takes advantage of the different products solubilities and relative affinities for the stationary phase used.

Chromatography separates a mixture using solids and liquids to separate its parts. All chromatography works by having a mobile phase (the part that moves), and a stationary phase (the part that stays still) which allow for separation of the different fractions of the original mixture.

Chromatography works because of the different affinities (strength of adhesion) of the various components of the mixture towards the stationary and mobile phases, which are dictated by two properties of the molecule: ‘adsorption’ and ‘solubility’. These different affinities allow for separation of the components.

We can define adsorption as the property of how well a component of the mixture sticks to the stationary phase, while solubility is the property of how well a component of the mixture dissolves in the mobile phase. The higher the adsorption to the stationary phase, the slower the molecule will move through the column. The higher the solubility in the mobile phase, the faster the molecule will move through the column. The retention factor (Rf) of a component is the distance travelled by the component over the distance travelled by the solvent (mobile phase).

Paper chromatography uses paper as a stationary phase. When samples are placed on the bottom of the paper all at the same level, the paper is put into a solvent (mobile phase), and the mobile phase moves up the stationary phase carrying the mixture with it. The mixture begins to separate at different points based on its solubility. More soluble compounds are carried the furthest and end up at the top and less soluble compounds end up at the bottom. The number of components that can be seen depends on the complexity of the mixture.

Thin layer chromatography (TLC) is a type of chromatography, where the stationary phase is a glass plate coated in the absorbent material (often silica gel or alumina) and the mobile phase is an organic solvent. In this case, non polar compounds are more soluble (higher Rf values) and polar compounds are more adsorbent (lower Rf values).

Column chromatography uses an analyte which is loaded over the silica beads packed into a glass column. Here, silica acts as the stationary phase, and the solvent (mobile phase) is then allowed to flow through the silica bed (under gravity or added pressure). The different components of the analyte exhibit varying degrees of adhesion to the silica, and as a result, they travel at different speeds through the stationary phase as the solvent flows through it. The components that adhere more strongly to the stationary phase travel more slowly compared to those with a weaker adhesion. Once fractions pass all the way through the column, they can be collected one at a time, and the compound can be isolated. A more accurate version of column chromatography is HPLC, which stands for high-pressure liquid chromatography. HPLC uses a pump to force the solvent through the column at high pressure. This is particularly useful for mixtures containing very small particles which have a higher resistance to the mobile phase flow.

Gas chromatography involves samples being vaporized and passed through a liquid or solid stationary phase using a gaseous (nitrogen or argon) mobile phase. The molecules with the lowest boiling points come out of the column first. The molecules with the higher boiling points come out of the column last. At the end of the stationary phase, there is a detector that works out how many particles of each component are found at different times.


Practice Questions


MCAT Official Prep (AAMC)

Chemistry Question Pack Question 19

Practice Exam 4 B/B Section Question 28


Key Points

• Chromatography is an analytical technique commonly used for separating a mixture of chemical substances into its components.

• Chromatography has two parts, the mobile phase often a gas or liquid solvent that moves the mixture through the stationary phase. The stationary phase is often a solid or liquid that sticks to components of the mixture with varying degrees of affinity.

• Chromatography works because of the interplay between absorption of a component and the stationary phase and its solubility in the mobile phase.

• Paper, thin layer and column chromatography use solid stationary phases and a liquid solvent. Gas chromatography uses gaseous samples, a liquid or gas mobile phase, and a solid or liquid stationary phase.

Key Terms

mobile phase or carrier: solvent moving through the column

stationary phase: a substance that stays fixed inside the column

fraction: a component of a mixture

column chromatography: chromatography methods using a column instead of paper filled with absorbent material

affinities: how strong a substance is attracted to another substance

retention factor (Rf): measure of how far a molecule travels in chromatography, compared to the mobile phase

thin layer chromatography: a type of chromatography that uses a silica-coated plate with an organic solvent to separate molecules by polarity

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