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Chromatography: Jump To Navigationjump To Search
Thin layer chromatography is used to separate components of a plant extract, illustrating the experiment
with plant pigments that gave chromatography its name
Contents
History[edit]
Main article: History of chromatography
Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in
1900.[4] He continued to work with chromatography in the first decade of the 20th century,
primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls.
Since these components have different colors (green, orange, and yellow, respectively) they
gave the technique its name. New types of chromatography developed during the 1930s and
1940s made the technique useful for many separation processes.[5]
Chromatography technique developed substantially as a result of the work of Archer John Porter
Martin and Richard Laurence Millington Synge during the 1940s and 1950s, for which they won
the 1952 Nobel Prize in Chemistry.[6] They established the principles and basic techniques of
partition chromatography, and their work encouraged the rapid development of several
chromatographic methods: paper chromatography, gas chromatography, and what would
become known as high-performance liquid chromatography. Since then, the technology has
advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could
be applied in many different ways, resulting in the different varieties of chromatography
described below. Advances are continually improving the technical performance of
chromatography, allowing the separation of increasingly similar molecules. Chromatography has
also been employed as a method to test the potency of cannabis.[7]
Chromatography terms[edit]
The analyte is the substance to be separated during chromatography. It is also normally
what is needed from the mixture.
Analytical chromatography is used to determine the existence and possibly also the
concentration of analyte(s) in a sample.
A bonded phase is a stationary phase that is covalently bonded to the support particles or to
the inside wall of the column tubing.
A chromatogram is the visual output of the chromatograph. In the case of an optimal
separation, different peaks or patterns on the chromatogram correspond to different
components of the separated mixture.
Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example
obtained by a spectrophotometer, mass spectrometer or a variety of other detectors)
corresponding to the response created by the analytes exiting the system. In the case of
an optimal system the signal is proportional to the concentration of the specific analyte
separated.
Displacement chromatography[edit]
The basic principle of displacement chromatography is: A molecule with a high affinity for
the chromatography matrix (the displacer) competes effectively for binding sites, and
thus displaces all molecules with lesser affinities.[14] There are distinct differences
between displacement and elution chromatography. In elution mode, substances
typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks,
preferably to baseline, is desired for maximum purification. The speed at which any
component of a mixture travels down the column in elution mode depends on many
factors. But for two substances to travel at different speeds, and thereby be resolved,
there must be substantial differences in some interaction between the biomolecules and
the chromatography matrix. Operating parameters are adjusted to maximize the effect of
this difference. In many cases, baseline separation of the peaks can be achieved only
with gradient elution and low column loadings. Thus, two drawbacks to elution mode
chromatography, especially at the preparative scale, are operational complexity, due to
gradient solvent pumping, and low throughput, due to low column loadings.
Displacement chromatography has advantages over elution chromatography in that
components are resolved into consecutive zones of pure substances rather than
“peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a
larger column feed can be separated on a given column with the purified components
recovered at significantly higher concentrations.
Affinity chromatography[edit]
Further information: Affinity chromatography
Affinity chromatography[15] is based on selective non-covalent interaction between an
analyte and specific molecules. It is very specific, but not very robust. It is often used in
biochemistry in the purification of proteins bound to tags. These fusion proteins are
labeled with compounds such as His-tags, biotin or antigens, which bind to the stationary
phase specifically. After purification, some of these tags are usually removed and the
pure protein is obtained.
Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe,
etc.). Columns are often manually prepared. Traditional affinity columns are used as a
preparative step to flush out unwanted biomolecules.
However, HPLC techniques exist that do utilize affinity chromatography properties.
Immobilized Metal Affinity Chromatography (IMAC)[16][17] is useful to separate
aforementioned molecules based on the relative affinity for the metal (i.e. Dionex IMAC).
Often these columns can be loaded with different metals to create a column with a
targeted affinity.
Supercritical fluid chromatography[edit]
Further information: Supercritical fluid chromatography
Supercritical fluid chromatography is a separation technique in which the mobile phase is
a fluid above and relatively close to its critical temperature and pressure.
Special techniques[edit]
Reversed-phase chromatography[edit]
Main article: Reversed-phase chromatography
Reversed-phase chromatography (RPC) is any liquid chromatography procedure in
which the mobile phase is significantly more polar than the stationary phase. It is so
named because in normal-phase liquid chromatography, the mobile phase is significantly
less polar than the stationary phase. Hydrophobic molecules in the mobile phase tend to
adsorb to the relatively hydrophobic stationary phase. Hydrophilic molecules in the
mobile phase will tend to elute first. Separating columns typically comprise a C8 or C18
carbon-chain bonded to a silica particle substrate.
Hydrophobic interaction chromatography[edit]
Hydrophobic interactions between proteins and the chromatographic matrix can be
exploited to purify proteins. In hydrophobic interaction chromatography the matrix
material is lightly substituted with hydrophobic groups. These groups can range from
methyl, ethyl, propyl, octyl, or phenyl groups.[19] At high salt concentrations, non-polar
sidechains on the surface on proteins "interact" with the hydrophobic groups; that is,
both types of groups are excluded by the polar solvent (hydrophobic effects are
augmented by increased ionic strength). Thus, the sample is applied to the column in a
buffer which is highly polar. The eluant is typically an aqueous buffer with decreasing salt
concentrations, increasing concentrations of detergent (which disrupts hydrophobic
interactions), or changes in pH.
In general, Hydrophobic Interaction Chromatography (HIC) is advantageous if the
sample is sensitive to pH change or harsh solvents typically used in other types of
chromatography but not high salt concentrations. Commonly, it is the amount of salt in
the buffer which is varied. In 2012, Müller and Franzreb described the effects of
temperature on HIC using Bovine Serum Albumin (BSA) with four different types of
hydrophobic resin. The study altered temperature as to effect the binding affinity of BSA
onto the matrix. It was concluded that cycling temperature from 50 degrees to 10
degrees would not be adequate to effectively wash all BSA from the matrix but could be
very effective if the column would only be used a few times.[20] Using temperature to
effect change allows labs to cut costs on buying salt and saves money.
If high salt concentrations along with temperature fluctuations want to be avoided you
can use a more hydrophobic to compete with your sample to elute it. [source] This so-
called salt independent method of HIC showed a direct isolation of Human
Immunoglobulin G (IgG) from serum with satisfactory yield and used Beta-cyclodextrin
as a competitor to displace IgG from the matrix.[21] This largely opens up the possibility of
using HIC with samples which are salt sensitive as we know high salt concentrations
precipitate proteins.
Two-dimensional chromatography[edit]
In some cases, the chemistry within a given column can be insufficient to separate some
analytes. It is possible to direct a series of unresolved peaks onto a second column with
different physico-chemical (chemical classification) properties.[citation needed] Since the
mechanism of retention on this new solid support is different from the first dimensional
separation, it can be possible to separate compounds by two-dimensional
chromatography that are indistinguishable by one-dimensional chromatography.
The sample is spotted at one corner of a square plate, developed, air-dried, then rotated
by 90° and usually redeveloped in a second solvent system.
Simulated moving-bed chromatography[edit]
Further information: Simulated moving bed
The simulated moving bed (SMB) technique is a variant of high performance liquid
chromatography; it is used to separate particles and/or chemical compounds that would
be difficult or impossible to resolve otherwise. This increased separation is brought about
by a valve-and-column arrangement that is used to lengthen the stationary phase
indefinitely. In the moving bed technique of preparative chromatography the feed entry
and the analyte recovery are simultaneous and continuous, but because of practical
difficulties with a continuously moving bed, simulated moving bed technique was
proposed. In the simulated moving bed technique instead of moving the bed, the sample
inlet and the analyte exit positions are moved continuously, giving the impression of a
moving bed. True moving bed chromatography (TMBC) is only a theoretical concept. Its
simulation, SMBC is achieved by the use of a multiplicity of columns in series and a
complex valve arrangement, which provides for sample and solvent feed, and also
analyte and waste takeoff at appropriate locations of any column, whereby it allows
switching at regular intervals the sample entry in one direction, the solvent entry in the
opposite direction, whilst changing the analyte and waste takeoff positions appropriately
as well.
Pyrolysis gas chromatography[edit]
Pyrolysis–gas chromatography–mass spectrometry is a method of chemical analysis in
which the sample is heated to decomposition to produce smaller molecules that are
separated by gas chromatography and detected using mass spectrometry.
Pyrolysis is the thermal decomposition of materials in an inert atmosphere or a vacuum.
The sample is put into direct contact with a platinum wire, or placed in a quartz sample
tube, and rapidly heated to 600–1000 °C. Depending on the application even higher
temperatures are used. Three different heating techniques are used in actual pyrolyzers:
Isothermal furnace, inductive heating (Curie Point filament), and resistive heating using
platinum filaments. Large molecules cleave at their weakest points and produce smaller,
more volatile fragments. These fragments can be separated by gas chromatography.
Pyrolysis GC chromatograms are typically complex because a wide range of different
decomposition products is formed. The data can either be used as fingerprint to prove
material identity or the GC/MS data is used to identify individual fragments to obtain
structural information. To increase the volatility of polar fragments, various methylating
reagents can be added to a sample before pyrolysis.
Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquid samples can
be performed directly inside Programmable Temperature Vaporizer (PTV) injectors that
provide quick heating (up to 30 °C/s) and high maximum temperatures of 600–650 °C.
This is sufficient for some pyrolysis applications. The main advantage is that no
dedicated instrument has to be purchased and pyrolysis can be performed as part of
routine GC analysis. In this case quartz GC inlet liners have to be used. Quantitative
data can be acquired, and good results of derivatization inside the PTV injector are
published as well.
Fast protein liquid chromatography[edit]
Further information: Fast protein liquid chromatography
Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is
often used to analyze or purify mixtures of proteins. As in other forms of
chromatography, separation is possible because the different components of a mixture
have different affinities for two materials, a moving fluid (the "mobile phase") and a
porous solid (the stationary phase). In FPLC the mobile phase is an aqueous solution, or
"buffer". The buffer flow rate is controlled by a positive-displacement pump and is
normally kept constant, while the composition of the buffer can be varied by drawing
fluids in different proportions from two or more external reservoirs. The stationary phase
is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical
glass or plastic column. FPLC resins are available in a wide range of bead sizes and
surface ligands depending on the application.
Countercurrent chromatography[edit]
Further information: Countercurrent chromatography