INTRODUCTION
Chromatography
is a powerful analytical tool because it can be used to separate complex
mixtures. Why is this important? Because even sophisticated detection
techniques are most definitive when used to identify single, pure components.
With mixtures, identification becomes problematic. To better understand how chromatography
techniques work, this presentation is centered on the basic concepts in chromatography.
is a powerful analytical tool because it can be used to separate complex
mixtures. Why is this important? Because even sophisticated detection
techniques are most definitive when used to identify single, pure components.
With mixtures, identification becomes problematic. To better understand how chromatography
techniques work, this presentation is centered on the basic concepts in chromatography.
Chromatography can be defined as the
separation of components of a mixture by difference in partitioning or
distribution between two phases [1]. For example, liquid/liquid chromatography
could be used to separate components of a mixture based on their polarity. If
you wanted to identify a non-polar organic contaminate mixed with a polar
material, a simple way to separate them would be to add the mixture to a
container of oil and water and shake it. On the principle that like dissolves
like, the non-polar material would partition into the oil; the polar material
would move into the water. Once the oil and water phases separated, the water
phase could be removed, fresh water added, and the process of washing the oil
phase repeated until essentially all of the polar material was removed. The
purified non-polar contaminant in the oil phase could then be analyzed and
identified. The technique could be reversed to identify a polar component. The
concept of chromatography, as it is generally applied today, involves the
partitioning of mixtures between a stationary phase (the sorbent) and the mobile
phase (a liquid or gas).
separation of components of a mixture by difference in partitioning or
distribution between two phases [1]. For example, liquid/liquid chromatography
could be used to separate components of a mixture based on their polarity. If
you wanted to identify a non-polar organic contaminate mixed with a polar
material, a simple way to separate them would be to add the mixture to a
container of oil and water and shake it. On the principle that like dissolves
like, the non-polar material would partition into the oil; the polar material
would move into the water. Once the oil and water phases separated, the water
phase could be removed, fresh water added, and the process of washing the oil
phase repeated until essentially all of the polar material was removed. The
purified non-polar contaminant in the oil phase could then be analyzed and
identified. The technique could be reversed to identify a polar component. The
concept of chromatography, as it is generally applied today, involves the
partitioning of mixtures between a stationary phase (the sorbent) and the mobile
phase (a liquid or gas).
Chromatography involves a sample (or sample extract)
being dissolved in a mobile phase (which may be a gas, a
liquid or a supercritical fluid). The mobile phase is then forced through an
immobile, immiscible stationary phase. The phases are chosen such
that components of the sample have differing solubilities in each phase. A
component which is quite soluble in the stationary phase will take longer to
travel through it than a component which is not very soluble in the stationary
phase but very soluble in the mobile phase. As a result of these differences in
mobilities, sample components will become separated from each other as they
travel through the stationary phase.
being dissolved in a mobile phase (which may be a gas, a
liquid or a supercritical fluid). The mobile phase is then forced through an
immobile, immiscible stationary phase. The phases are chosen such
that components of the sample have differing solubilities in each phase. A
component which is quite soluble in the stationary phase will take longer to
travel through it than a component which is not very soluble in the stationary
phase but very soluble in the mobile phase. As a result of these differences in
mobilities, sample components will become separated from each other as they
travel through the stationary phase.
Techniques such as H.P.L.C. (High Performance Liquid
Chromatography) and G.C. (Gas Chromatography) use columns –
narrow tubes packed with stationary phase, through which the mobile phase is
forced. The sample is transported through the column by continuous addition of
mobile phase. This process is called elution. The average rate at
which an analyte moves through the column is determined by the time it spends
in the mobile phase.
Chromatography) and G.C. (Gas Chromatography) use columns –
narrow tubes packed with stationary phase, through which the mobile phase is
forced. The sample is transported through the column by continuous addition of
mobile phase. This process is called elution. The average rate at
which an analyte moves through the column is determined by the time it spends
in the mobile phase.
DISTRIBUTION OF ANALYTES
BETWEEN PHASES
BETWEEN PHASES
The distribution of analytes between phases can often be
described quite simply. An analyte is in equilibrium between the two phases;
described quite simply. An analyte is in equilibrium between the two phases;
Amobile
Astationary
Astationary The equilibrium constant, K, is termed
the partition coefficient; defined as the molar concentration of
analyte in the stationary phase divided by the molar concentration of the
analyte in the mobile phase. The time between sample injection and an analyte
peak reaching a detector at the end of the column is termed the retention
time (tR ). Each analyte in a sample will have a different
retention time. The time taken for the mobile phase to pass through the column
is called tM.
the partition coefficient; defined as the molar concentration of
analyte in the stationary phase divided by the molar concentration of the
analyte in the mobile phase. The time between sample injection and an analyte
peak reaching a detector at the end of the column is termed the retention
time (tR ). Each analyte in a sample will have a different
retention time. The time taken for the mobile phase to pass through the column
is called tM.
A term called the retention factor, k’, is
often used to describe the migration rate of an analyte on a column. You may also
find it called the capacity factor. The retention factor for
analyte A is defined as;
often used to describe the migration rate of an analyte on a column. You may also
find it called the capacity factor. The retention factor for
analyte A is defined as;
k’A = t R – tM / tM
t R and tM are
easily obtained from a chromatogram. When an analytes retention factor is less
than one, elution is so fast that accurate determination of the retention time
is very difficult. High retention factors (greater than 20) mean that elution
takes a very long time. Ideally, the retention factor for an analyte is between
one and five.
easily obtained from a chromatogram. When an analytes retention factor is less
than one, elution is so fast that accurate determination of the retention time
is very difficult. High retention factors (greater than 20) mean that elution
takes a very long time. Ideally, the retention factor for an analyte is between
one and five.
We define a quantity called the selectivity
factor, , which describes the separation of two species (A and B), on
the column;
factor, , which describes the separation of two species (A and B), on
the column;
= k
‘B / k ‘A
‘B / k ‘A
When calculating the selectivity factor, species A elutes faster than species B. The
selectivity factor is always greater than one.
selectivity factor is always greater than one.
FOUNDATION OF CHROMATOGRAPHY
Chromatography was first employed in Russia by the
Italian-born scientist Mikhail
Tsvet in 1900[2]. 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.
Italian-born scientist Mikhail
Tsvet in 1900[2]. 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.
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. They established the principles and basic techniques of
partition chromatography, and their work encouraged the rapid development of
several chromatographic methods:
substantially as a result of the work of Archer
John Porter Martin and Richard
Laurence Millington Synge during
the 1940s and 1950s. They established the principles and basic techniques of
partition chromatography, and their work encouraged the rapid development of
several chromatographic methods:
Paper chromatography, Thin layer 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.
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.
COMMON TERMS USED IN CHROMATOGRAPHY
ANALYTE: The analyte is the substance to be
separated during chromatography. It is also normally what is needed from the
mixture.
separated during chromatography. It is also normally what is needed from the
mixture.
ANALYTICAL CHROMATOGRAPHY: Analytical chromatography is
used to determine the existence and possibly also the concentration of
analyte(s) in a sample.
used to determine the existence and possibly also the concentration of
analyte(s) in a sample.
BONDED
PHASE: A bonded
phase is a stationary phase that is covalently bonded to the
support particles or to the inside wall of the column tubing.
PHASE: A bonded
phase is a stationary phase that is covalently bonded to the
support particles or to the inside wall of the column tubing.
CHROMATOGRAM: 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.
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.
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.
CHROMATOGRAPH: A chromatograph is equipment that
enables a sophisticated separation, e.g. gas chromatographic or liquid
chromatographic separation.
enables a sophisticated separation, e.g. gas chromatographic or liquid
chromatographic separation.
Chromatography is
a physical method of separation that distributes components to separate between
two phases, one stationary (stationary phase), the other (the mobile phase)
moving in a definite direction.
a physical method of separation that distributes components to separate between
two phases, one stationary (stationary phase), the other (the mobile phase)
moving in a definite direction.
ELUATE: The eluate is the mobile phase
leaving the column.
leaving the column.
ELUENT: The eluent is the solvent that carries
the analyte.
the analyte.
ELUOTROPIC
SERIES: An eluotropic series is a list of solvents ranked
according to their eluting power.
SERIES: An eluotropic series is a list of solvents ranked
according to their eluting power.
IMMOBILIZED
PHASE: An immobilized phase is a stationary
phase that is immobilized on the support particles, or on the inner wall of the
column tubing.
PHASE: An immobilized phase is a stationary
phase that is immobilized on the support particles, or on the inner wall of the
column tubing.
THE
MOBILE PHASE: The mobile phase is the phase that moves in a definite direction.
It may be a liquid (LC and Capillary Electrochromatography (CEC)), a gas (GC),
or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile
phase consists of the sample being separated/analyzed and the solvent that
moves the sample through the column. In the case of HPLC the
mobile phase consists of a non-polar solvent(s) such as hexane in normal phase
or polar solvents in reverse phase chromatography and the sample being separated.
The mobile phase moves through the chromatography column (the stationary phase)
where the sample interacts with the stationary phase and is separated.
MOBILE PHASE: The mobile phase is the phase that moves in a definite direction.
It may be a liquid (LC and Capillary Electrochromatography (CEC)), a gas (GC),
or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile
phase consists of the sample being separated/analyzed and the solvent that
moves the sample through the column. In the case of HPLC the
mobile phase consists of a non-polar solvent(s) such as hexane in normal phase
or polar solvents in reverse phase chromatography and the sample being separated.
The mobile phase moves through the chromatography column (the stationary phase)
where the sample interacts with the stationary phase and is separated.
PREPARATIVE CHROMATOGRAPHY: Preparative chromatography is
used to purify sufficient quantities of a substance for further use, rather
than analysis.
used to purify sufficient quantities of a substance for further use, rather
than analysis.
RETENTION
TIME: The retention time is the
characteristic time it takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions.
TIME: The retention time is the
characteristic time it takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions.
THE
SAMPLE: The sample is
the matter analyzed in chromatography. It may consist of a single component or
it may be a mixture of components. When the sample is treated in the course of
an analysis, the phase or the phases containing the analytes of interest is/are
referred to as the sample whereas everything out of interest separated from the
sample before or in the course of the analysis is referred to as waste.
SAMPLE: The sample is
the matter analyzed in chromatography. It may consist of a single component or
it may be a mixture of components. When the sample is treated in the course of
an analysis, the phase or the phases containing the analytes of interest is/are
referred to as the sample whereas everything out of interest separated from the
sample before or in the course of the analysis is referred to as waste.
THE
SOLUTE: The solute refers
to the sample components in partition chromatography.
SOLUTE: The solute refers
to the sample components in partition chromatography.
THE
SOLVENT: The solvent refers
to any substance capable of solubilizing another substance, and especially the
liquid mobile phase in liquid chromatography.
SOLVENT: The solvent refers
to any substance capable of solubilizing another substance, and especially the
liquid mobile phase in liquid chromatography.
THE
STATIONARY PHASE: The stationary phase is the substance fixed in place for the
chromatography procedure. Examples include the silica layer in thin layer chromatography
STATIONARY PHASE: The stationary phase is the substance fixed in place for the
chromatography procedure. Examples include the silica layer in thin layer chromatography
THE
DETECTORS: The detector refers
to the instrument used for qualitative and quantitative detection of analytes
after separation.
DETECTORS: The detector refers
to the instrument used for qualitative and quantitative detection of analytes
after separation.
Chromatography is based on
the concept of partition coefficient. Any solute partitions between two
immiscible solvents. When we make one solvent immobile (by adsorption on a
solid support matrix) and another mobile it results in most common applications
of chromatography. If matrix support is polar (e.g. paper, silica etc.) it is
forward phase chromatography, and if it is non-polar (C-18) it is reverse
phase.
the concept of partition coefficient. Any solute partitions between two
immiscible solvents. When we make one solvent immobile (by adsorption on a
solid support matrix) and another mobile it results in most common applications
of chromatography. If matrix support is polar (e.g. paper, silica etc.) it is
forward phase chromatography, and if it is non-polar (C-18) it is reverse
phase.
REFERENCES
1. Roberts, J.D., and Caserio, M.C. (1965). “Basic
Principles of Organic Chemistry,” W.A, Benjamin, Inc. New York.
Principles of Organic Chemistry,” W.A, Benjamin, Inc. New York.
2. Mikhail Tswett (1906). “Physikalisch-Chemische Studien über das Chlorophyll. Die
Adsorption.” (Physical-chemical
studies of chlorophyll. Adsorption.) Berichte
der Deutschen botanischen Gesellschaft, vol. 24, pp. 316–326.
Adsorption.” (Physical-chemical
studies of chlorophyll. Adsorption.) Berichte
der Deutschen botanischen Gesellschaft, vol. 24, pp. 316–326.
3. Ettre, L. S. (1993).
“Nomenclature for chromatography (IUPAC Recommendations 1993)”. Pure and Applied Chemistry 65 (4).doi:10.1351/pac199365040819.
“Nomenclature for chromatography (IUPAC Recommendations 1993)”. Pure and Applied Chemistry 65 (4).doi:10.1351/pac199365040819.
