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| Chromatography
techniques can be characterized in a variety of ways.
Many of the possible categories are used only
infrequently, so we will deal only with the most commonly
used varieties.
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The first
broad distinction among chromatographic techniques is
based on the mobile phase:
- Gas Chromatography (GC)
uses a moving gas.
- Liquid Chromatography
(LC) uses a moving liquid.
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Liquid
Chromatography, in turn, is most commonly categorized on
the basis of the physical arrangement of the stationary
phase:
- A sheet of paper,
through which the mobile phase moves by
capillarity (Paper Chromatography). Paper
Chromatography today is more often used as a
teaching tool than as an analytical tool.
- A thin layer of
powdered material bound to a glass or plastic
support, through which the mobile phase moves by
capillarity (Thin Layer Chromatography, or TLC).
TLC is used occasionally as a rapid technique for
qualitative analysis.
- A bed of powdered
material packed in a glass or plastic column,
through which the mobile phase is pulled by
gravity ("Classical" or "Open
Column" Chromatography). This is used
primarily as a preparative technique.
- A bed of powdered
material packed in a small, disposable, plastic
cartridge, through which mobile phase is pulled
by vacuum or pushed with a small syringe (Solid
Phase Extraction, or SPE). This is used primarily
as a sample cleanup technique prior to GC or HPLC
- A bed of powdered
material packed in a steel or high-strength
plastic column through which the mobile phase is
pushed by a constant-flow pump. ("High
Performance Liquid Chromatography" or HPLC).
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HPLC is
subdivided on the basis of separation chemistry. All of
these techniques can be done using the same
instrumentation.
- Reversed-phase
chromatography uses a non-polar (hydrophobic)
stationary phase and a polar (usually including
some water) mobile phase. This is the most common
type of HPLC separation in use today.
- Normal-phase
chromatography uses a polar (hydrophilic)
stationary phase and a non-polar (usually with no
water) mobile phase. This was the type of
separation to which the term "chromatography"
was first applied (remember, the stationary phase
was powdered chalk -- calcium carbonate, a very
polar material -- and the mobile phase was
petroleum ether -- mainly hexane, a very non-polar
hydrocarbon solvent). This technique is still
used, but is less common than reversed-phase.
- Ion-exchange
chromatography uses a stationary phase support
which has been derivatized so as to permanently
bind charged groups to the surface. The mobile
phase is typically an aqueous buffer. This
technique is used primarily for the analysis of
ions such as strong acids or bases or for
separation of large molecules such as nucleic
acids, proteins, or large peptides.
- Ion-pair
chromatography is a "hybrid" technique
in which charged groups are temporarily bound to
the surface of a "reversed-phase" type
of column packing. This technique is often used
for the analysis of small, weak-acid or weak-base
compounds.
- Size-exclusion
chromatography uses a stationary phase of
controlled pore-size distribution. Separation is
based on the extent to which analyte molecules
can penetrate the network of pores.
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| Another way to
subdivide HPLC is based on whether the mobile phase
composition is held constant during the course of the
separation ("isocratic" HPLC) or is changed
during the course of the separation ("gradient"
HPLC).
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