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