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CHROMATOGRAPHY
Introduction
Chromatography is the name given to a family of analytical chemistry techniques employed in the
separation of mixtures. Though there are many different types of chromatography, they all employ
the basic strategy of passing a "mobile phase" mixture to be separated (called the analyte)
through a "stationary phase" that differentially retards movement of mobile phase components to
allow their physical separation.
The mobile phase chemical mixture is carried in either a liquid or gas medium, while the
stationary phase can be any of several absorbing media such as paper, gelatin, alumina, or silica.
Each of the mobile phase components in a mixture exhibits a characteristic rate of movement, or
"retention time," and chromatographic separation of the components occurs because of differences
in the retention time of each. Retention time is determined by the physical and chemical
attributes of the mixture components and those of the stationary phase through which they travel.
Attributes of significance influencing component mobility include charge, relative solubility, and
adsorption.
Compounds are identified in chromatography by comparing the relative speed of migration of unknown
compounds to speeds of known standards. This is typically done through comparing terms called Rf
values which are calculated as the distance traveled by the unknown compound divided by the
distance traveled by the leading edge of the solvent (the "solvent front"). Because compound
mobility varies between laboratories and even between experimental runs in the same lab,
chromatographic analyses are usually set up to include one or more standard compounds as controls
with which to compare the test compound mobilities.
There is a bewildering array of different chromatographic techniques in laboratory use today. A
couple of the most common techniques are briefly described below.
Paper Chromatography
In this simple chromatographic method, a sample containing a mixture of compounds (often pigments)
is placed in solution, and this is applied to one end of a strip of chromatography paper. The
paper is dipped into the appropriate solvent (e.g., water, ethanol). As the solvent is pulled
upward through the paper (through capillary action), it pulls the various compounds in the mixture
up with it. The compounds migrate up the paper at different rates, however, because they vary in
their solubility and in the degree to which they are attracted to the chromatography paper.
Once chromatographic separation is completed, discreet spots containing each of the compounds from
the sample mixture can be visually ascertained, sometimes with the aid of UV light or through the
addition of chemicals that render the spots visible.
Thin Layer Chromatography (TLC)
Thin layer chromatography is similar to paper chromatography, but it allows separations to be run
faster and also offers a choice of stationary phase adsorbents. Cellulose, silica gel, and
alumina (aluminum oxide) are commonly used adsorbents. The absorbent is bound in a thin layer
(hence the name of the process) to a flat substrate such as a glass or plastic plate, dried, and
then activated via heating. The sample mixture in solution is applied to one end of the
adsorbent-plate and chromatographic separation of components occurs. Fluorescent dye and
application of UV light may be used to visualize spots after separation, or various chemical color
reagents can be applied to turn spots visible.
Column Chromatography
An appropriate solvent is placed in a glass column containing silica gel or other solid supporting
medium. A sample mixture is placed on top of the support and gravity causes the sample to migrate
downward through the column. Differential solubilities of individual compounds within the
selected solvent and affinities to the silica gel produce chromatographic separation as component
solutes exit the bottom of the column at different times.
In a modern variation of column chromatography, positive pressure is applied to the top of the
column and the separation is sped up. This variation is referred to as flash column
chromatography.
High performance liquid chromatography (below) is another column chromatography variant.
High Performance Liquid Chromatography (HPLC)
As in flash column chromatography, HPLC employs positive pressure to force the sample through the
column at a more rapid than gravity alone would produce. Shortening the residence time allows
separated mixture components to remain on the stationary phase for only a brief period before the
separation is completed. This minimizes diffusion of separated compounds within the column,
producing narrower separation peaks and enhanced resolution between compounds.
Two different types of HPLC are normal phase (NP-HPLC) and reversed phase (RP-HPLC)
chromatography. They differ from one another in the polarities of the mobile and liquid phases
employed in the separation. NP-HPLC, the older of the two strategies) uses a nonpolar mobile phase
and a polar stationary phase. It is favored when separation of a polar analyte is the goal.
RP-HPLC employs a polar mobile phase and a nonpolar stationary phase and is preferred when the
sample mixture contains large nonpolar compounds.
Gas-Liquid Chromatography
As in flash column chromatography, HPLC employs positive pressure to force the sample through the
column at a more rapid than gravity alone would produce. Shortening the residence time allows
separated mixture components to remain on the stationary phase for only a brief period before the
separation is completed. This minimizes diffusion of separated compounds within the column,
producing narrower separation peaks and enhanced resolution between compounds.
Two different types of HPLC are normal phase (NP-HPLC) and reversed phase (RP-HPLC)
chromatography. They differ from one another in the polarities of the mobile and liquid phases
employed in the separation. NP-HPLC, the older of the two strategies) uses a nonpolar mobile phase
and a polar stationary phase. It is favored when separation of a polar analyte is the goal.
RP-HPLC employs a polar mobile phase and a nonpolar stationary phase and is preferred when the
sample mixture contains large nonpolar compounds.
Related Weblinks
Sheffield Hallam University UK Department of Chemistry Introduction to Chromatography
http://www.protocol-online.org/prot/Molecular_Biology/Electrophoresis/index.html
University of Akron Hardy Research Group Chromatography Tutorial
http://ull.chemistry.uakron.edu/analytical/Chromatography
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