QAC - Chapter 23 - What is chromatography

Chromatography

• A versatile laboratory technique used to separate, identify, and quantify components in a mixture by passing it through a stationary phase using a mobile phase.

Elution

The process of passing liquid or gas through a chromatography column.

Chromatography

A versatile laboratory technique used to separate, identify, and quantify components in a mixture by passing it through a stationary phase using a mobile phase.

Mobile Phase

(A liquid or a gas flowing through the column)

The stationary phase (stays in place inside the column):

• A viscous liquid chemically bonded to the inside of a silica capillary column

• A viscous liquid coated onto the surface of solid particles packed in the column

• The solid particles themselves

Elution

Packed Column

Filled with particles of the stationary phase.

Open Tubular Column

Narrow, hollow capillary with a stationary phase coated on the inside walls.

Absorption Chromatography

• Solute is adsorbed on the solid particles.

• Stationary Phase: Solid

• Mobile Phase: Liquid or gas

The more strongly a solute is adsorbed, the slower it travels through the column (greater retention time, t_R)

Partition Chromatography (Example: Gas Chromatography)

• Stationary Phase: Liquid bonded to a solid support

• Mobile phase: liquid or gas

Solute partitions between the stationary liquid and the mobile phase, which is a flowing gas in gas chromatography.

Ion-exchange chromatography

• Stationary solid phase: : A resin containing covalently attached anions (-SO3-) or cations (–N(CH3)3+ ).

• Mobile phase: Liquid

Ions of the opposite charge are attracted to the stationary phase by electrostatic force.

Molecular exclusion chromatography or gel filtration/permeation chromatography

• Stationary phase: porous gel

• Mobile phase: liquid or gas

This technique separates molecules by size, with larger solutes passing through most quickly.

Affinity Chromotography

Very selective/specific interactions

Partition Coefficient, K_D

Chromatogram:

A graph of detector’s response as a function of elution time

Retention time, tR

For each component, it is the time between injection of the mixture onto the column and when that component reaches the detector.

Adjusted Retention time (t'_R)

For a retained solute, it is the additional time required to travel the length of the column, beyond that required by the solvent

t_m

The time that unretained mobile phase travels through the column (minimum possible time).

The greater α

The greater the separation between the two components.

α is independent of flow rate and can be used to help identify peaks when the flow rate changes.

Diffusion Coefficient, D

The rate at which a substance moves randomly from a region of high concentration to a region of lower concentration.

Plate height (H)

The constant of proportionality between the variance, σ2, of the band and the distance it has traveled, x.