Separations

Separations Introduction A sample that requires analysis is often a mixture of many components in a complex matrix. For samples containing unknown compounds, the components must be separated from each other so that each individual component can be identified by other analytical methods. The separation properties of the components in a mixture are constant under constant conditions, and therefore once determined they can be used to identify and quantify each of the components. Such procedures are typical in chromatographic and electrophoretic analytical separations. A mixture can be separated using the the differences in physical or chemical properties of the individual components. As an example, dumping spaghetti and water in a colander separates the two components because the liquid water can run through the colander but the solid spaghetti cannot (assuming that it is not grossly overcooked as prepared in some university dining halls). Some water will stick to the spaghetti and some spaghetti may go down the drain because the colander is not 100% efficient. An analagous example is the filtering of a solid precipitate to separate it from a solution. These separations are based on the states of matter of the two components, other physical properties that are useful for separations are density and size. Some useful chemical properties by which compounds can be separated are solubility, boiling point, and vapor pressure.

Simple separation procedures • • • • •

Centrifugation Crystallization Distillation Extraction Filtering

Instrumental separation procedures • •

Chromatography Electrophoresis

Chromatography Introduction

Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the the components in a mixture. A column (or other support for TLC, see below) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column they can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on-line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC-MS and LC-MS), Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array UV-VIS absorption spectroscopy (HPLC-UV-VIS).

Specific chromatographic methods: Gas chromatography (GC) Applied to volatile organic compounds. The mobile phase is a gas and the stationary phase is usually a liquid on a solid support or sometimes a solid adsorbent. High-performance liquid chromatography (HPLC) A variation of liquid chromatography that utilizes high-pressure pumps to increase the efficiency of the separation. Liquid chromatography (LC) Used to separate analytes in solution including metal ions and organic compounds. The mobile phase is a solvent and the stationary phase is a liquid on a solid support, a solid, or an ion-exchange resin. Size-exclusion chromatography (SEC) Also called gel-permeation chromatography (GPC), the mobile phase is a solvent and the stationary phase is a packing of porous particles. Thin-layer chromatography (TLC) A simple and rapid method to monitor the extent of a reaction or to check the purity of organic compounds. The mobile phase is a solvent and the stationary phase is a solid adsorbent on a flat support.

Chromatography Theory Introduction

Chromatography theory consists of empirical relationships to describe chromatographic columns and the separation of peaks in chromatograms. The underlying principle that controls chromatographic separations is a dynamic behavior that depends upon partitioning and mass transport.

Description of Chromatograms • •

Resolution of peaks in a chromatogram Relationship of resolution to k', Rs, a (Alpha)

Description of chromatographic columns • •

N+H Golay Equation