Chromatography
Principle
Chromatography is a family of chemistry techniques, analytically and
preparatively, for the separation of mixtures. It involves passing the mixture
containing the analyte, in the "mobile phase", often in a stream of
solvent, through the "stationary phase." The stationary phase retards
the passage of the sample components. When components pass through the system
at different rates they become more and more separated in time. Each component
has a characteristic time of passage through the system, called the
"retention time." Chromatographic separation is achieved when the
retention time of the analyte differs from that of other components in the
sample.
The mixture is carried by liquid or gas and is separated into its component
parts as a result of differential distributions of the solutes as they flow
over a stationary liquid or solid phase. Various techniques rely on the
differential affinities of substances for a gas or liquid mobile medium and for
a stationary absorbing medium through which they pass, such as paper, gelatin,
alumina or silica.
The (chemical) physics underlying all types of chromatography concern
various cohesive and adhesive forces (see Cohesion
and adhesion), capillary forces,
surface tension and diffusion with the analyte, mobile and stationary
phase playing the key roles.
Application
In many clinical disciplines but especially in internal medicine. Also in
many biomedical, biochemical and chemical research and industrial.
More Info
Retention
The retention is a measure of the speed at which a substance moves in a
chromatographic system. In continuous development systems where the compounds
are eluted with the eluent, the retention is usually measured as the retention time Rt or tR,
the time between injection and detection. In interrupted development systems
like thin layer chromatography, the retention is measured as the retention factor Rf, defined by
Rf = distance moved by compound / distance moved by eluent.
Since it is hard to standardize retention, a comparison is made with a
standard compounds under absolutely identical conditions.
A chromatographic system can be described as the mobile
and stationary phases being in equilibrium. The partition coefficient K is based on this equilibrium, defined as
K = [solute in stationary phase] / [solute in mobile phase]. K is assumed to be independent of the
concentration of the analyte, and can change if experimental conditions are
changed, for example temperature. As K
increases, it takes longer for solutes to separate. For a column of fixed
length and flow, the retention time (tR) and retention volume (Vr) can be measured
and used to calculate K.
Chromatographic techniques
Fig. 1 Principle
of a paper chromatograph
In paper chromatography a small spot of solution containing the sample is
applied to a strip of chromatography paper (Fig. 1). This sample is adsorbed
onto the paper. The paper is then dipped into a suitable solvent (such as
ethanol or water) and placed in a sealed container. As the solvent rises
through the paper by capillary forces it meets the sample mixture. This starts
to travel up the paper with the solvent. Cohesive and adhesive interactions
with the paper make different compounds travel at different rates. The process
takes some hours. The final chromatogram can be compared with other known
mixture chromatograms for identification. This technique demonstrates very well
the principle, but at the moment it has only educational relevance.
Thin layer chromatography (TLC)
Fig. 2
Separation of black ink on a TLC plate
In TLC the stationary phase is a thin adsorbent layer like silica gel,
alumina, etc. on a flat carrier like a glass plate, a thick aluminum foil, etc.
(Fig. 2). The process is similar to paper chromatography but runs faster and
separate better. It is used for monitoring chemical reactions and analysis of
reaction products. Colorless spots of the compounds are made visible by a
fluorescent dye (see Fluorescence)
in the adsorbent under UV light. Rf
values
should be the same regardless of the extent of travel of the solvent, and
in theory are independent of a single experimental run. They do depend on the
solvent used, and the type of TLC plate. Nowadays relevance is mainly
educational.
Column chromatography
Column chromatography utilizes a vertical glass column filled with some
form of solid support with the sample to be separated placed on top of this
support. The rest of the column is filled with a solvent which, under the
influence of gravity, moves the sample through the column. Similarly to other
forms of chromatography, differences in rates of movement through the solid
medium are translated to different exit times from the bottom of the column for
the various elements of the original sample.
Fig. 3 Principle
of a flash column chromatography setup
The flash column chromatography (Fig. 3) is very similar to the traditional
column chromatography, except for that the solvent is driven through the column
by applying positive pressure. It is faster and gives better separation.
Miniaturized (disposable columns), this technique is widely applied.
Gas(-liquid) chromatography G(L)C
Gas-liquid chromatography is based on a partition equilibrium of analyte
between a liquid stationary phase and a mobile gas. The mobile
phase is a carrier gas, usually an inert gas such as He or N2, and
the stationary phase is a microscopic layer of liquid on an inert solid support
inside of a very long very thin tube known as a column. It is useful for a wide range of
non-polar analytes, but poor for thermally labile molecules. It is often
combined with mass
spectrography.
Ion exchange chromatography is a column chromatography that uses a charged
stationary phase. It is used to separate charged compounds including amino
acids, peptides and proteins. The stationary phase is usually an ion exchange
resin that carries charged functional groups which interact with oppositely
charged groups of the compound to be retained. Bound compounds can be eluted
from the column by gradient elution (solvent composition with a time-gradient,
e.g. in salt concentration or pH) or isocratic elution
(solvent composition constant). Ion exchange chromatography is commonly used to
purify proteins using Fast
Protein Liquid Chromatography (FPLC).
Immobilized metal ion affinity chromatography, IMAC
IMAC is a popular and powerful way to purify proteins. It is based on the
specific covalent binding between histidine or other unique amino acids (either
naturally or grafted with recombinant DNA techniques) and various immobilized
metal ions, such as copper, nickel, zinc, or iron.
High performance liquid chromatography, HPLC
HPLC is a form of column chromatography used frequently in biochemistry and
analytical chemistry. The analyte is forced through a column (stationary phase)
by a liquid (mobile phase) at high pressure, which decreases the time the
analytes have to diffuse within the column. Diffusion within the column leads
to broad peaks and loss of resolution. Less time on the column then translates
to narrower peaks in the resulting chromatogram and thence to better resolution
and sensitivity (discrimination from ‘’background noise’). Another way to
decrease time the analyte stays on the column is to change the composition of
the mobile phase over a period of time (a solvent time-gradient). HPLC is often
combined within one apparatus with a mass spectrograph or gas chromatograph.
Reversed phase
(RP) liquid chromatography
RP-HPLC was developed for large polar biomolecules. Like the name implies
the nature of the stationary phase is reversed. RP-HPLC consists of a nonpolar
stationary phase and a polar mobile phase. One common stationary phase is special
treated silica. The retention time is longer when the mobile phase is more
polar. This is the reverse of the situation which exists when normal silica is
used as the stationary phase.
Gel permeation
chromatography GPC
GPC also known as size exclusion chromatography or Sephadex gel
chromatography, separates molecules on basis of size. Smaller molecules enter a
porous media and take longer to exit the column, hence larger particles leave
the column first. GPC is good for determining polymer molecular weight
distribution, but has a low resolution.
Affinity chromatography is based on selective non-covalent interaction
between an analyte and specific molecules. It is often used in the purification
of proteins (or better protein constructs).
There are many other versions of chromatography, see Wikipedia or textbooks
on analytic chemistry.
After Wikipedia and other web-sources