Chromatography terms
The analyte is the substance to be separated during chromatography.
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.
A chromatograph is equipment that 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.
The eluate is the mobile phase leaving the column.
The eluent is the solvent that carries the analyte.
An eluotropic series is a list of solvents ranked according to their eluting power.
An immobilized phase is a stationary phase that is immobilized on the support particles, or on the
inner wall of the column tubing.
Techniques by chromatographic bed shape
Column chromatography
For more details on this topic, see Column chromatography.
Column chromatography is a separation technique in which the stationary bed is within a tube. The particles
of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside
volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open,
unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in
rates of movement through the medium are calculated to different retention times of the sample.
In 1978, W. [Link] introduced a modified version of column chromatography called flash column
chromatography (flash). The technique is very similar to the traditional column chromatography, except for
that the solvent is driven through the column by applying positive pressure. This allowed most separations to
be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash
chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the
cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The
introduction of gradient pumps resulted in quicker separations and less solvent usage.
In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This
allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of
broken cells.
Phosphocellulose chromatography utilizes the binding affinity of many DNA-binding proteins for
phosphocellulose. The stronger a protein's interaction with DNA, the higher the salt concentration needed to
elute that protein.[6]
Planar chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or on a plane.
The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper
chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer
chromatography). Different compounds in the sample mixture travel different distances according to how
strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention
factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
Paper chromatography
For more details on this topic, see Paper chromatography.
Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip
of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As
the solvent rises through the paper, it meets the sample mixture, which starts to travel up the paper with the
solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel
farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and
therefore do not travel as far.
Thin layer chromatography
For more details on this topic, see Thin layer chromatography.
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper
chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a
thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has
the advantage of faster runs, better separations, and the choice between different adsorbents. For even better
resolution and to allow for quantification, high-performance TLC can be used.
APPLICATIONS OF CHROMATOGRAPHY
Chromatography is a powerful and versatile tool for separating closely related chemical species. In addition,
it can be employed for the qualitative identification and quantitative determination of separated species.
Qualitative Analysis
Chromatography is widely used for recognizing the presence or absence of components in mixtures that
contain a limited number of species whose identities are known. For example, 30 or more amino acids in a
protein hydrolysate can be detected with a reasonable degree of certainty by means of a chromatogram. On
the other hand, because a chromatogram provides but a single piece of information about each species in a
mixture (the retention time), the application of the technique to the qualitative analysis of complex samples
of unknown composition is limited. This limitation has been largely overcome by linking chromatographic
columns efuently with ultraviolet, infrared, and mass spectrometers.
The resulting hyphenated instruments are powerful tools for identifying the components of complex
mixtures. It is important to note that while a chromatogram may not lead to positive identification of the
species in a sample; it often provides sure evidence of the absence of species. Thus, failure of a sample to
produce a peak at the same retention time as a standard obtained under identical conditions is strong evidence
that the compound in question is absent (or present at a concentration below the detection limit of the
procedure).
Quantitative Analysis
Quantitative chromatography is based on a comparison of either the height or the area of an analyte peak with
that of one or more standards. The conditions are controlled properly; both of these parameters vary linearly
with concentration. "
Analyses Based on Peak Height
The height of a chromatographic peak is obtained by connecting the base lines on the two sides of the peak
by a straight line and measuring the perpendicular distance from this line to the peak. This measurement can
ordinarily be made with reasonably high precision and yields accurate results, provided variations in column
conditions do not alter peak width during the period required to obtain chromatograms for sample and
standards. The variables that must be controlled closely are column temperature, eluent flow rate, and rate of
sample injection.
In addition, care must be taken to avoid overloading the column. The effect of sample-injection rate is
particularly critical for the early peaks of a chromatogram. Relative errors of 5% to 10% due to this cause are
not unusual with syringe injection.
Analyses Based on Peak Area
Peak area is independent of broadening effects caused by the variables mentioned in the previous paragraph.
From this standpoint, therefore, area is a more satisfactory analytical parameter than peak height. On the
other hand, peak heights are more easily measured and, for narrow peaks, more accurately determined. Most
modem chromatographic instruments are equipped with electronic integrators that provide precise
measurements of relative peak areas. If such equipment is not available, a manual estimate must be made. A
simple method that works well for symmetric peaks of reasonable widths is to multiply peak height by the
width at one-half peak height.
Calibration with Standards
The most straightforward method for quantitative chromatographic analyses involves the preparation of a
series of standard solutions that approximate the composition of the unknown. Chromatograms for the
standards are then obtained, and peak heights or areas are plotted as a function of concentration. A plot of the
data should yield a straight line passing through the origin; analyses are based on this plot. Frequent
standardization is necessary for highest accuracy.
The Internal-Standard Method
The highest precision for quantitative chromatography is obtained by using internal standards because the
uncertainties introduced by sample injection, flow rate, and variations in column conditions are minimized.
In this procedure, a carefully measured quantity of an internal-standard is introduced into each standard and
sample, and the ratio of analyte peak area (or height) to internal-standard peak area (or height) is the
analytical parameter. For this method to be successful, it
is necessary that the internal-standard peak be well separated from the peaks of all other components in the
sample, but it must appear close to the analyte peak. With a suitable internal standard, precisions of 0.5% to
1 % relative are reported.
Activity
1. What is the description of chromatography
2. Classify the different chgromatographic techniques, and give examples of principle types of
application.
3. What is the van Deemter equation? Define terms.
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Summary
Few methods of chemical analysis are truly specific to a particular analyte. It is often found that the analyte
of interest must be separated from the myriad of individual compounds that may be present in a sample. As
well as providing the analytical scientist with methods of separation, chromatographic techniques can also
provide methods of analysis.
We discovered that a technique known as 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. We have also look at the
varoius forms of chromatograph which included GC, HPLC, TLC and Planar Chromatograph. We have also
looked at parameter that cause peak broadening using Van Deemter equation. Chromatrography can be used
for both qualitative analysis basis on the retention time of the analyte and quantitative analysis which is basis
on the peak area or height.
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 chromotagraphy 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 is used to purify sufficient quantities of a substance for
further use, rather than analysis.
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 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 refers to the sample components in partition chromatography.
The solvent refers to any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography.
The stationary phase is the substance fixed in place for the chromatography procedure.
Examples include the silica layer in thin layer chromatography
The detector refers to the instrument used for qualitative and quantitative detection of
analytes after separation.
Liquid Chromatograph (LC), 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.
Distribution of analytes between phases
The distribution of analytes between phases can often be described quite simply. An analyte is in
equilibrium between the two phases;
The equilibrium constant, K, is termed the partition coefficient; defined as the molar
concentration of analyte in the stationary phase (Cs) divided by the molar concentration of the
analyte in the mobile phase(Cm).
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;
tR 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.
Example
Calculate the retention factor for the chromatographic peak with tR = 52.3 min and tM =8.0 min
Solution
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We define a quantity called the selectivity factor, α , which describes the separation of two
species (A and B) on the column;
α = k 'B / k 'A
When calculating the selectivity factor, species A elutes faster than species B. The selectivity
factor is always greater than one.
Band broadening and column efficiency
To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obtained. This
means that band broadening must be limited. It is also beneficial to measure the efficiency of the
column.
The Theoretical Plate Model of Chromatography
The plate model supposes that the chromatographic column is contains a large number of
separate layers, called theoretical plates. Separate equilibrations of the sample between the
stationary and mobile phase occur in these "plates". The analyte moves down the column by
transfer of equilibrated mobile phase from one plate to the next.
It is important to remember that the plates do not really exist; they are a figment of the
imagination that helps us understand the processes at work in the column. They also serve as a
way of measuring column efficiency, either by stating the number of theoretical plates in a
column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a
Theoretical Plate (the smaller the better).
If the length of the column is L, then the HETP is
The number of theoretical plates that a real column possesses can be found by examining a
chromatographic peak after elution;
An alternative way is given by
Where w1/2 is the peak width at half-height.
As can be seen from this equation, columns behave as if they have different numbers of plates
for different solutes in a mixture.
Example
Calculate the number of plates in the column resulting in the chromatographic peak t R = 52.3 min
and wb = 9.0 min.
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The Rate Theory of Chromatography
A more realistic description of the processes at work inside a column takes account of the time
taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate
model, which assumes that equilibration is infinitely fast). The resulting band shape of a
chromatographic peak is therefore affected by the rate of elution. It is also affected by the
different paths available to solute molecules as they travel between particles of stationary phase.
If we consider the various mechanisms which contribute to band broadening, we arrive at the
Van Deemter equation for plate height;
HETP = A + B / u + C u
whereu is the average velocity of the mobile phase. A, B, and C are factors which contribute to
band broadening.
A Eddy diffusion
The mobile phase moves through the column which is packed with stationary phase. Solute
molecules will take different paths through the stationary phase at random. This will cause
broadening of the solute band, because different paths are of different lengths.
B - Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses
out from the center to the edges. This causes band broadening. If the velocity of the mobile phase
is high then the analyte spends less time on the column, which decreases the effects of
longitudinal diffusion.
C - Resistance to mass transfer
The analyte takes a certain amount of time to equilibrate between the stationary and mobile
phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the
stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the
stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the
worse the broadening becomes.
Van Deemter plots
A plot of plate height vs. average linear velocity of mobile phase.
Such plots are of considerable use in determining the optimum mobile phase flow rate.
Resolution
Although the selectivity factor, , describes the separation of band centres, it does not take into
account peak widths. Another measure of how well species have been separated is provided by
measurement of the resolution. The resolution of two species, A and B, is defined as
Baseline resolution is achieved when R = 1.5
It is useful to relate the resolution to the number of plates in the column, the selectivity factor
and the retention factors of the two solutes;
To obtain high resolution, the three terms must be maximised. An increase in N, the number of
theoretical plates, by lengthening the column leads to an increase in retention time and increased
band broadening - which may not be desirable. Instead, to increase the number of plates, the
height equivalent to a theoretical plate can be reduced by reducing the size of the stationary
phase particles.
It is often found that by controlling the capacity factor, k', separations can be greatly improved.
This can be achieved by changing the temperature (in Gas Chromatography) or the composition
of the mobile phase (in Liquid Chromatography).
The selectivity factor, α, can also be manipulated to improve separations. When α is close to
unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable
time. In these cases, k' is optimised first, and then α is increased by one of the following
procedures:
1. Changing mobile phase composition
2. Changing column temperature
3. Changing composition of stationary phase
4. Using special chemical effects (such as incorporating a species which complexes with
one of the solutes into the stationary phase)
This equation can be rearranged to give the number of plates needed torealize a given resolution
Review your learning
You should now be familiar with the terms used in chromatography, how species become
separated from one another, and how various conditions can be manipulated to obtain well-
resolved chromatograms with a minimum elution time.
Example
Ethanol and Methanol are separated in a capillary GC column with retention times of 370 s and
385 s, respectively, and base widths (w b) of 16,0 and 17.0 s. An unretained air peak occurs at
10.0 s .Calculate the separation factor and the resolution.
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Solution
Use the longest eluting peak to calculate N
plates
Separation factor
Capacity factor
Average capacity factor will be
Resolution
Or alternatively resolution can be determined
Techniques by chromatographic bed shape
Column chromatography
For more details on this topic, see Column chromatography.
Column chromatography is a separation technique in which the stationary bed is within a tube.
The particles of the solid stationary phase or the support coated with a liquid stationary phase
may fill the whole inside volume of the tube (packed column) or be concentrated on or along the
inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the
tube (open tubular column). Differences in rates of movement through the medium are calculated
to different retention times of the sample.
In 1978, W. C. Still introduced a modified version of column chromatography called flash
column chromatography (flash). The technique is very similar to the traditional column
chromatography, except for that the solvent is driven through the column by applying positive
pressure. This allowed most separations to be performed in less than 20 minutes, with improved
separations compared to the old method. Modern flash chromatography systems are sold as pre-
packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be
linked with detectors and fraction collectors providing automation. The introduction of gradient
pumps resulted in quicker separations and less solvent usage.
In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed
bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture
broths or slurries of broken cells.
Phosphocellulose chromatography utilizes the binding affinity of many DNA-binding proteins
for phosphocellulose. The stronger a protein's interaction with DNA, the higher the salt
concentration needed to elute that protein.[6]
Planar chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or
on a plane. The plane can be a paper, serving as such or impregnated by a substance as the
stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a
glass plate (thin layer chromatography). Different compounds in the sample mixture travel
different distances according to how strongly they interact with the stationary phase as compared
to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the
identification of an unknown substance.
Paper chromatography
For more details on this topic, see Paper chromatography.
Paper chromatography is a technique that involves placing a small dot or line of sample solution
onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of
solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which
starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance,
and the compounds within the mixture travel farther if they are non-polar. More polar substances
bond with the cellulose paper more quickly, and therefore do not travel as far.
Thin layer chromatography
For more details on this topic, see Thin layer chromatography.
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to
paper chromatography. However, instead of using a stationary phase of paper, it involves a
stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert
substrate. Compared to paper, it has the advantage of faster runs, better separations, and the
choice between different adsorbents. For even better resolution and to allow for quantification,
high-performance TLC can be used.
Applications of Chromatography
Chromatography is a powerful and versatile tool for separating closely related chemical species.
In addition, it can be employed for the qualitative identification and quantitative determination of
separated species.
Qualitative Analysis
Chromatography is widely used for recognizing the presence or absence of components in
mixtures that contain a limited number of species whose identities are known. For example, 30
or more amino acids in a protein hydrolysate can be detected with a reasonable degree of
certainty by means of a chromatogram. On the other hand, because a chromatogram provides but
a single piece of information about each species in a mixture (the retention time), the application
of the technique to the qualitative analysis of complex samples of unknown composition is
limited. This limitation has been largely overcome by linking chromatographic columns efuently
with ultraviolet, infrared, and mass spectrometers.
The resulting hyphenated instruments are powerful tools for identifying the components of
complex mixtures. It is important to note that while a chromatogram may not lead to positive
identification of the species in a sample; it often provides sure evidence of the absence of
species. Thus, failure of a sample to produce a peak at the same retention time as a standard
obtained under identical conditions is strong evidence that the compound in question is absent
(or present at a concentration below the detection limit of the procedure).
Quantitative Analysis
Quantitative chromatography is based on a comparison of either the height or the area of an
analyte peak with that of one or more standards. The conditions are controlled properly; both of
these parameters vary linearly with concentration. "
Analyses Based on Peak Height
The height of a chromatographic peak is obtained by connecting the base lines on the two sides
of the peak by a straight line and measuring the perpendicular distance from this line to the peak.
This measurement can ordinarily be made with reasonably high precision and yields accurate
results, provided variations in column conditions do not alter peak width during the period
required to obtain chromatograms for sample and standards. The variables that must be
controlled closely are column temperature, eluent flow rate, and rate of sample injection.
In addition, care must be taken to avoid overloading the column. The effect of sample-injection
rate is particularly critical for the early peaks of a chromatogram. Relative errors of 5% to 10%
due to this cause are not unusual with syringe injection.
Analyses Based on Peak Area
Peak area is independent of broadening effects caused by the variables mentioned in the previous
paragraph. From this standpoint, therefore, area is a more satisfactory analytical parameter than
peak height. On the other hand, peak heights are more easily measured and, for narrow peaks,
more accurately determined. Most modem chromatographic instruments are equipped with
electronic integrators that provide precise measurements of relative peak areas. If such equipment
is not available, a manual estimate must be made. A simple method that works well for
symmetric peaks of reasonable widths is to multiply peak height by the width at one-half peak
height.
Calibration with Standards
The most straightforward method for quantitative chromatographic analyses involves the
preparation of a series of standard solutions that approximate the composition of the unknown.
Chromatograms for the standards are then obtained, and peak heights or areas are plotted as a
function of concentration. A plot of the data should yield a straight line passing through the
origin; analyses are based on this plot. Frequent standardization is necessary for highest
accuracy.
The Internal-Standard Method
The highest precision for quantitative chromatography is obtained by using internal standards
because the uncertainties introduced by sample injection, flow rate, and variations in column
conditions are minimized. In this procedure, a carefully measured quantity of an internal-
standard is introduced into each standard and sample, and the ratio of analyte peak area (or
height) to internal-standard peak area (or height) is the analytical parameter. For this method to
be successful, it
is necessary that the internal-standard peak be well separated from the peaks of all other
components in the sample, but it must appear close to the analyte peak. With a suitable internal
standard, precisions of 0.5% to 1 % relative are reported.
Activity
1. What is the description of chromatography
2. Classify the different chromatographic techniques, and give examples of principle types of
application.
3. What is the van Deemter equation? Define terms.
