Chromatography, Principle, Methods, Different types, classification

1. Chromatography
Course: B.Sc Biotechnology
Subject: Bio Analytical Technique
Unit: IV

2. Analytic technique to discover chemical components:
a method of finding out which components a gaseous
or liquid mixture contains that involves passing it
through or over something that absorbs the different
components at different rates
Chromatography

3. Chromatography
• Chromatography is the collective term for a set of laboratory techniques
for the separation of mixtures.
• The mixture is dissolved in a fluid called the mobile phase, which carries it
through a structure holding another material called the stationary phase.
• The various constituents of the mixture travel at different speeds, causing
them to separate.
• The separation is based on differential partitioning between the mobile
and stationary phases.
• Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus changing the
separation.
• A partition-coefficient is the ratio of concentrations of a compound in a
mixture of two immiscible phases at equilibrium. These coefficients are a
measure of the difference in solubility of the compound in these two
phases.

4. 1.

5. • Chromatography may be preparative or analytical.
– The purpose of preparative chromatography is to separate the
components of a mixture for more advanced use.
– Analytical chromatography is done normally with smaller amounts of
material and is for measuring the relative proportions of analytes in a
mixture. The two are not mutually exclusive.
• Chromatography was first employed by the Russian scientist Mikhail Tsvet
in 1900.
• 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 they gave the technique its name.
• New types of chromatography developed during the 1930s and 1940s
made the technique useful for many separation processes.

6. Chromatography terms
• 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 corresponding to
the response created by the analytes exiting
the system.
2.

7. • 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 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 is the phase that moves in a definite direction.
It may be a liquid (LC and Capillary Electro chromatography), or a gas (GC).
The mobile phase consists of the sample being separated and the solvent
that moves the sample through the column. In the case of HPLC the
mobile phase consists of a non-polar solvent 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 is used to purify sufficient quantities of a
substance for further use, rather than analysis.

8. • The retention time is the characteristic time it takes for a particular
analyte to pass through the system 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.
• 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.
• 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.

9. CHROMATOGRAPHIC PERFORMANCE
PARAMETERS
• The successful chromatographic separation of analytes in a mixture
depends upon the selection of the most appropriate process of
chromatography followed by the optimisation of the experimental
conditions associated with the separation. Optimisation requires an
understanding of the processes that are occurring during the
development and elution, and of the calculation of a number of
experimental parameters characterising the behaviour of each
analyte in the mixture.
1. Retention time
A chromatogram is a pictorial record of the detector response as a
function of elution volume or retention time. It consists of a series
of peaks or bands, ideally symmetrical in shape, representing the
elution of individual analytes.

10. 2. Retention factor
The retention factor, Rf, is a quantitative indication of how far a
particular compound travels in a particular solvent. The Rf value is a
good indicator of whether an unknown compound and a known
compound are similar, if not identical. If the Rf value for the unknown
compound is close or the same as the Rf value for the known compound
then the two compounds are most likely similar or identical.
3. Plate height
Chromatography columns are considered to consist of a number of
adjacent zones in each of which there is sufficient space for an analyte
to completely equilibrate between the two phases. Each zone is called a
theoretical plate (of which there are N in total in the column). The
length of column containing one theoretical plate is referred to as the
plate height, H, which has units of length normally in micrometres.
The smaller the value of H and the greater the value of N, the more
efficient is the column in separating a mixture of analytes. The numerical
value of both N and H for a particular column is expressed by reference
to a particular analyte.
Plate height is simply related to the width of the analyte peak, expressed
in terms of its standard deviation s

11. 4. Resolution
The purpose of chromatography is to separate or resolve compounds.
The separation or distance between two peaks is known as their
resolution and is a function of the 3 factors discussed previously:
retention (the time it takes for the analytes to elute, related to k),
selectivity (how different the analytes are from each other and related to
α), and efficiency (how good the column is, related to N).

12. Qualitative and quantitative analysis
• Chromatographic analysis can be carried out on either a qualitative or
quantitative basis.
Qualitative analysis
• The objective of this approach is to confirm the presence of a specific
analyte in a test sample. This is achieved on the evidence of:
• A comparison of the retention time of the peaks in the chromatograph
with that of an authentic reference sample of the test analyte obtained
under identical chromatographic conditions. Confirmation of the presence
of the analyte in the sample can be obtained by spiking a second sample
of the test sample with a known amount of the authentic compound. This
should result in a single peak with the predicted increase in area.
• The use of either a mass spectrometer or nuclear magnetic resonance
(NMR) spectrometer as a detector so that structural evidence for the
identity of the analyte responsible for the peak can be obtained.

13. Quantitative analysis
• The objective of this approach is to confirm the presence of a specific
analyte in a test sample and to quantify its amount.
• Quantification is achieved on the basis of peak area coupled with an
appropriate calibration graph. The area of each peak in a chromatogram
can be shown to be proportional to the amount of the analyte producing
the peak. The area of the peak may be determined by measuring the
height of the peak (hP) and its width at half the height (wh).
• This procedure is time consuming when complex and/or a large number
of analyses are involved and dedicated integrators or microcomputers
best perform the calculations.
• These can be programmed to compute retention time and peak area and
to relate them to those of a reference standard enabling relative retention
ratios and relative peak area ratios to be calculated.
• These may be used to identify a particular analyte and to quantify it using
previously obtained and stored calibration data.

14. Purpose of Chromatography
• AnalyticalAnalytical - determine chemical composition
of a sample
• PreparativePreparative - purify and collect one or more
components of a sample

15. Classification of Methods
• There are two classification schemes:
–mobile phase
–attractive forces

16. Mobile Phase
• gas (GC)
• water (LC)
• organic solvent (LC)
• supercritical fluid (SCFC)

17. Classification based on Mobile Phase
Gas ChromatographyGas Chromatography
Gas - solidGas - solid Gas - liquidGas - liquid
Stationary Phase
Sample MUST be volatile at temperatures BELOW 3500
C
Pyrolysis GC -
heat solid materials
to 500 - 10000
C
so they decompose
into gaseous products

18. Classification based on Attractive
Forces
• Adsorption - for polar non-ionic compounds
• Ion Exchange - for ionic compounds
– Anion - analyte is anion; bonded phase has positive charge
– Cation – analyte is cation; bonded phase has negative charge
• Partition - based on the relative solubility of analyte in
mobile and stationary phases
– Normal – analyte is nonpolar organic; stationary phase MORE polar
than the mobile phase
– Reverse – analyte is polar organic; stationary phase LESS polar than
the mobile phase
• Size Exclusion - stationary phase is a porous matrix; sieving

19. Detectors
• UV-vis
• Refractive Index (RI)
• Mass spectrometry (MS)
• Electrochemical (EC)
– amperometric
• NMR - novel

20. Problem:
• What would be a good method for
determining the following:
– identity of accelerant at a suspected arson scene
– amount of caffeine in Coca Cola
– identifying active ingredient in an illicit drug
preparation (LSD is heat sensitive)
– purification and characterization of novel thermophilic
plant enzyme from South America
– identifying explosive materials used in Oklahoma
bombing

21. ColumnChromatography
• Column chromatography in chemistry is a method used to purify individual chemical
compounds from mixtures of compounds.
• It is often used for preparative applications on scales from micrograms up to kilograms.
• The main advantage of column chromatography is the relatively low cost and
disposability of the stationary phase used in the process. The latter prevents cross-
contamination and stationary phase degradation due to recycling.
• The classical preparative chromatography column is a glass tube with a diameter from
5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter at the
bottom.
• Two methods are generally used to prepare a column: the dry method and the wet
method.

22. • For the dry method, the column is first filled with dry stationary phase powder,
followed by the addition of mobile phase, which is flushed through the column
until it is completely wet, and from this point is never allowed to run dry.
• For the wet method, a slurry is prepared of the eluent with the stationary phase
powder and then carefully poured into the column. Care must be taken to avoid
air bubbles. A solution of the organic material is pipetted on top of the stationary
phase. This layer is usually topped with a small layer of sand or with cotton or glass
wool to protect the shape of the organic layer from the velocity of newly added
eluent. Eluent is slowly passed through the column to advance the organic
material.
• The individual components are retained by the stationary phase differently and
separate from each other while they are running at different speeds through the
column with the eluent. At the end of the column they elute one at a time. During
the entire chromatography process the eluent is collected in a series of fractions.
Fractions can be collected automatically by means of fraction collectors.
• The composition of the eluent flow can be monitored and each fraction is analyzed
for dissolved compounds, e.g. by analytical chromatography, UV absorption, or
fluorescence. Colored compounds can be seen through the glass wall as moving
bands.

23. 3.

24. Stationary phase
• The stationary phase or adsorbent in column chromatography is a solid. The most
common stationary phase for column chromatography is silica gel, followed by
alumina. Cellulose powder has often been used in the past. Also possible are ion
exchange chromatography, reversed-phase chromatography (RP), affinity
chromatography or expanded bed adsorption (EBA).
Mobile phase
The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is
chosen so that the retention factor value of the compound of interest is roughly around 0.2 -
0.3 in order to minimize the time and the amount of eluent to run the chromatography.
The eluent has also been chosen so that the different compounds can be separated
effectively. The eluent is optimized in small scale pretests, often using thin layer
chromatography (TLC) with the same stationary phase.
There is an optimum flow rate for each particular separation. A faster flow rate of the eluent
minimizes the time required to run a column and thereby minimizes diffusion, resulting in a
better separation.
However, the maximum flow rate is limited because a finite time is required for the analyte
to equilibrate between the stationary phase and mobile phase

25. • A simple laboratory column runs by gravity flow. The flow rate of such a
column can be increased by extending the fresh eluent filled column
above the top of the stationary phase or decreased by the tap controls.
Faster flow rates can be achieved by using a pump or by using
compressed gas (e.g. air, nitrogen, or argon) to push the solvent through
the column.
• The particle size of the stationary phase is generally finer in flash column
chromatography than in gravity column chromatography. For example,
one of the most widely used silica gel grades in the former technique is
mesh (40 – 63 µm), while the latter technique typically requires mesh (63
– 200 µm) silica gel.
• Column chromatography is an extremely time consuming stage in any lab
and can quickly become the bottleneck for any process lab. Therefore,
several manufacturers have developed automated flash chromatography
systems that minimize human involvement in the purification process.
Automated systems will include components normally found on more
expensive high performance liquid chromatography (HPLC) systems such
as a gradient pump, sample injection ports, a UV detector and a fraction
collector to collect the eluent.

26. • Typically these automated systems can separate samples from a few
milligrams up to an industrial many kilogram scale and offer a much
cheaper and quicker solution to doing multiple injections on prep-HPLC
systems.
• The resolution (or the ability to separate a mixture) on an LPLC system will
always be lower compared to HPLC, as the packing material in an HPLC
column can be much smaller, typically only 5 micrometre thus increasing
stationary phase surface area, increasing surface interactions and giving
better separation.
• The software controlling an automated system will coordinate the
components, allow a user to only collect the fractions that contain their
target compound and help the user to find the resulting purified material
within the fraction collector. The software will also save the resulting
chromatograph from the process for archival and/or later recall purposes.

27. • Typically, column chromatography is set up with peristaltic pumps, flowing buffers
and the solution sample through the top of the column. The solutions and buffers
pass through the column where a fraction collector at the end of the column setup
collects the eluted samples. Prior to the fraction collection, the samples that are
eluted from the column pass through a detector such as a spectrophotometer or
mass spectrometer so that the concentration of the separated samples in the
sample solution mixture can be determined.
• The ultimate goal of chromatography is to separate different components from a
solution mixture. The resolution expresses the extent of separation between the
components from the mixture. The higher the resolution of the chromatogram,
the better the extent of separation of the samples the column gives. This data is a
good way of determining the column’s separation properties of that particular
sample. The resolution can be calculated from the chromatogram.

28. Paper chromatography
• Paper chromatography is an analytical method technique for separating and
identifying mixtures that are or can be coloured, especially pigments.
• This can also be used in secondary or primary colours in ink experiments. This
method has been largely replaced by thin layer chromatography, but is still a
powerful teaching tool.
• Double-way paper chromatography, also called two-dimensional
chromatography, involves using two solvents and rotating the paper 90° in
between.
• This is useful for separating complex mixtures of compounds having similar
polarity, for example, amino acids. If a filter paper is used, it should be of a high
quality paper.
• The mobile phase is developing solutions that can travel up to the stationary
phase carrying the sample along with it.

29. • Paper chromatography is a useful technique because it is relatively quick and
requires small quantities of material.
• Separations in paper chromatography involve the same principles as those in thin
layer chromatography. In paper chromatography, like thin layer chromatography,
substances are distributed between a stationary phase and a mobile phase. The
stationary phase is usually a piece of high quality filter paper.
• The mobile phase is a developing solution that travels up the stationary phase,
carrying the samples with it. Components of the sample will separate readily
according to how strongly they adsorb onto the stationary phase versus how
readily they dissolve in the mobile phase.

30. Types of Paper Chromatography
1.Descending Paper Chromatography-In this type, development of the chromatogram
is done by allowing the solvent to travel down the paper is called Descending
Chromatography. Here, the mobile phase is present in the upper portion.
2. Ascending Paper Chromatography-Here the solvent travel upward direction of the
Chromatographic paper. Both the Descending and Ascending Paper
Chromatography are used for separation of Organic and Inorganic substances.
3. Ascending-Descending Paper Chromatography-It is the hybrid of both the above
techniques. The upper part of the Ascending chromatography can be folded over a
rod and allowing the paper to become descending after crossing the rod.
4. Horizontal Paper Chromatography-It is also called as Circular chromatography.
Here a circular filter paper is taken and the sample is given at the center of the
paper. After drying the spot the filter paper is tied horizontally on a Petridish
containing solvent. So that Wick of the paper is dipped inside the solvent. The
solvent rises through the wick and the component get separated in form of
concentrate circular zone.
5. Two-Dimensional Paper Chromatography-In this technique a square or rectangular
paper is used. Here the sample is applied to one of the corners and development
is performed at right angle to the direction of first run.

31. Thin-layer chromatography
• Thin-layer chromatography (TLC) is a chromatography technique used to separate
non-volatile mixtures.
• Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium
foil, which is coated with a thin layer of adsorbent material, usually silica gel,
aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary
phase.
• After the sample has been applied on the plate, a solvent or solvent mixture
(known as the mobile phase) is drawn up the plate via capillary action. Because
different analytes ascend the TLC plate at different rates, separation is achieved.
• Thin-layer chromatography can be used to monitor the progress of a reaction,
identify compounds present in a given mixture, and determine the purity of a
substance.
• Specific examples of these applications include: analyzing ceramides and fatty
acids, detection of pesticides or insecticides in food and water, analyzing the dye
composition of fibers in forensics, assaying the radiochemical purity of
radiopharmaceuticals, or identification of medicinal plants and their constituents
• A number of enhancements can be made to the original method to automate the
different steps, to increase the resolution achieved with TLC and to allow more
accurate quantitative analysis. This method is referred to as HPTLC,

32. 4.

33. • Different compounds in the sample mixture travel at different rates due to the
differences in their attraction to the stationary phase, and because of differences
in solubility in the solvent.By changing the solvent, or perhaps using a mixture, the
separation of components (measured by the Rf value) can be adjusted. Also, the
separation achieved with a TLC plate can be used to estimate the separation of a
flash chromatography column.
• The process is similar to paper chromatography with the advantage of faster runs,
better separations, and the choice between different stationary phases. Because
of its simplicity and speed TLC is often used for monitoring chemical reactions and
for the qualitative analysis of reaction products.

34. Analysis
• As the chemicals being separated may be colorless, several methods exist to
visualize the spots:
• fluorescent analytes like quinine may be detected under blacklight (366 nm)
• Often a small amount of a fluorescent compound, usually manganese-activated
zinc silicate, is added to the adsorbent that allows the visualization of spots under
UV-C light (254 nm). The adsorbent layer will thus fluoresce light-green by itself,
but spots of analyte quench this fluorescence.
• Iodine vapors are a general unspecific color reagent

35. The Beginning: Paper Chromatography
The unifying aspect of chromatographic methods is that the
separation is not with an applied electric field
The very first type of chromatography was the ‘adsorption’
variety, which includes paper and TLC
Mikhail Tsvet
1872-1919
Separated chlorophylls
and karatenoids using
calcium carbonate as the
‘stationary phase’
About 20 years later, Richard L.M.
Synge and Archer Martin develop
paper chromatography and win the
nobel prize 5.

36. Adsorption Chromatography Mechanism
Adsorption chromatography uses a stationary phase and a
mobile phase
The stationary phase is usually a solid ‘sheet’ either cellulose
(paper), silica gel (standard TLC) or aluminum oxide
The stationary phase must have ‘moderately charged’
functional groups:
NN C
Cellulose Silica Gel Al2O3 6.

37. Adsorption Chromatography Mechanism
To move the analytes over the stationary phase, we use a
non-polar solvent which is drawn up via capillary action
Some popular mobile phase solvents are:
Non-polar: Alkanes (up to octane), Diethyl ether
Moderate polarity: Alcohols, Ketones (not acetone)
Polar: H2O, Weak Salt Buffers
7.

38. TLC and Paper Visualization
Once you’ve run the molecule, how do you see it?
If you’re very lucky, then your analytes are chromophoric
If you’re somewhat lucky, then your analytes absorb UV light
If you’re unlucky then you have to use a stain (like iodine)
8.

39. “Paper Chromatography” 2000-2007: 175 results
TLC and Paper Chromatography Nowadays
In Biochemistry, TLC and paper chromatography had their
heyday in the 50s (Remember Sanger).
Nonetheless they are still routinely used as basic analytical
tools, particularly in organic chemistry (TLC)
We don’t separate proteins this way – electrophoretic
separations work much better
1960-1967: 720 results
“TLC” 2000-2007: 420 results
1980-1987: 394 results

40. Gas chromatography
• Gas chromatography (GC), is a common type of chromatography used in analytical
chemistry for separating and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include testing the purity of a particular
substance, or separating the different components of a mixture.
• In some situations, GC may help in identifying a compound.
• In preparative chromatography, GC can be used to prepare pure compounds from
a mixture.
• In gas chromatography, the mobile phase is a carrier gas, usually an inert gas such
as helium or an unreactive gas such as nitrogen.
• The stationary phase is a microscopic layer of liquid or polymer on an inert solid
support, inside a piece of glass or metal tubing called a column. The instrument
used to perform gas chromatography is called a gas chromatograph.
• The gaseous compounds being analyzed interact with the walls of the column,
which is coated with a stationary phase.
• This causes each compound to elute at a different time, known as the retention
time of the compound. The comparison of retention times is what gives GC its
analytical usefulness.

41. • Gas chromatography is in principle similar to column chromatography, but has
several notable differences.
• First, the process of separating the compounds in a mixture is carried out between
a liquid stationary phase and a gas mobile phase, whereas in column
chromatography the stationary phase is a solid and the mobile phase is a liquid.
• Second, the column through which the gas phase passes is located in an oven
where the temperature of the gas can be controlled, whereas column
chromatography has no such temperature control. Finally, the concentration of a
compound in the gas phase is solely a function of the vapor pressure of the gas.
• Gas chromatography is also similar to fractional distillation, since both processes
separate the components of a mixture primarily based on boiling point
differences. However, fractional distillation is typically used to separate
components of a mixture on a large scale, whereas GC can be used on a much
smaller scale.
• Gas chromatography is also sometimes known as vapor-phase chromatography
(VPC), or gas–liquid partition chromatography (GLPC). These alternative names,
as well as their respective abbreviations, are frequently used in scientific
literature.

42. 9.

43. Data reduction and analysis
Qualitative analysis
• Generally chromatographic data is presented as a graph of detector response (y-
axis) against retention time (x-axis), which is called a chromatogram.
• This provides a spectrum of peaks for a sample representing the analytes present
in a sample eluting from the column at different times.
• Retention time can be used to identify analytes if the method conditions are
constant.
• Also, the pattern of peaks will be constant for a sample under constant conditions
and can identify complex mixtures of analytes.
• However, in most modern applications, the GC is connected to a mass
spectrometer or similar detector that is capable of identifying the analytes
represented by the peaks.

44. Quantitative analysis
• The area under a peak is proportional to the amount of analyte present in the
chromatogram.
• By calculating the area of the peak using the mathematical function of integration,
the concentration of an analyte in the original sample can be determined.
• Concentration can be calculated using a calibration curve created by finding the
response for a series of concentrations of analyte, or by determining the relative
response factor of an analyte.
• The relative response factor is the expected ratio of an analyte to an internal
standard and is calculated by finding the response of a known amount of analyte
and a constant amount of internal standard.
• In most modern GC-MS systems, computer software is used to draw and integrate
peaks, and match MS spectra to library spectra.

45. AFFINITY CHROMATOGRAPHY
Principle
• Separation and purification of analytes by affinity chromatography is unlike most
other forms of chromatography and such techniques as electrophoresis and
centrifugation in that it does not rely on differences in the physical properties of
the analytes.
• Instead, it exploits the unique property of extremely specific biological interactions
to achieve separation and purification.
• As a consequence, affinity chromatography is theoretically capable of giving
absolute purification, even from complex mixtures, in a single process.
• The technique was originally developed for the purification of enzymes, but it has
since been extended to nucleotides, nucleic acids, immunoglobulins, membrane
receptors and even to whole cells and cell fragments.
• The technique requires that the material to be isolated is capable of binding
reversibly to a specific ligand that is attached to an insoluble matrix

46. Under the correct experimental conditions, when a complex mixture containing
the specific compound to be purified is added to the immobilised ligand, generally
contained in a conventional chromatography column, only that compound will bind
to the ligand.
All other compounds can therefore be washed away and the compound
subsequently recovered by displacement from the ligand.
The method requires a detailed preliminary knowledge of the structure and
biological specificity of the compound to be purified so that the separation
conditions that are most likely to be successful may be carefully planned.

47. • In the case of an enzyme, the ligand may be the substrate, a competitive
reversible inhibitor or an allosteric modifier.
• The conditions chosen would normally be those that are optimal for enzyme–
ligand binding.
• Since the success of the method relies on the reversible formation of the complex
and on the numerical values of the first-order rate constants kþ1 and k–1, as the
enzyme is added progressively to the insolubilised ligand in a column, the enzyme
molecules will be stimulated to bind and a dynamic situation develops in which the
concentration of the complex and the strength of the binding increase.

48. 10.

49. Materials and applications
Matrix
• An ideal matrix for affinity chromatography must have the following
characteristics:
• possess suitable and sufficient chemical groups to which the ligand may be
covalently coupled, and be stable under the conditions of the attachment;
• be stable during binding of the macromolecule and its subsequent elution;
• interact only weakly with other macromolecules to minimise non-specific
adsorption;
• exhibit good flow properties.
In practice, particles that are uniform, spherical and rigid are used. The most
common ones are the cross-linked dextrans and agarose, polyacrylamide,
polymethacrylate, polystyrene, cellulose and porous glass and silica.

50. Ligand
• The chemical nature of a ligand is dictated by the biological specificity of the
compound to be purified. In practice it is sometimes possible to select a ligand
that displays absolute specificity in that it will bind exclusively to one particular
compound.
• More commonly, it is possible to select a ligand that displays group selectivity in
that it will bind to a closely related group of compounds that possess a similar in-
built chemical specificity.
• An example of the latter type of ligand is 50AMP, which can bind reversibly to
many NADþ-dependent dehydrogenases because it is structurally similar to part of
the NADþ molecule.
• It is essential that the ligand possesses a suitable chemical group that will not be
involved in the reversible binding of the ligand to the macromolecule, but which
can be used to attach the ligand to the matrix.
• The most common of such groups are NH2, COOH, SH and OH.

51. Practical procedure
• The procedure for affinity chromatography is similar to that used in other forms of
liquid chromatography.
• The buffer used must contain any cofactors, such as metal ions, necessary for
ligand–macromolecule interaction.
• Once the sample has been applied and the macromolecule bound, the column is
eluted with more buffer to remove nonspecifically bound contaminants.
• The purified compound is recovered from the ligand by either specific or non-
specific elution.
• Non-specific elution may be achieved by a change in either pH or ionic strength.
pH shift elution using dilute acetic acid or ammonium hydroxide results from a
change in the state of ionisation of groups in the ligand and/or the macromolecule
that are critical to ligand–macromolecule binding.
• A change in ionic strength, not necessarily with a concomitant change in pH, also
causes elution due to a disruption of the ligand–macromolecule interaction; 1M
NaCl is frequently used for this purpose.

52. 11.

53. Applications
• Many enzymes and other proteins, including receptor proteins and
immunoglobulins, have been purified by affinity chromatography.
• The application of the technique is limited only by the availability of immobilised
ligands.
• The principles have been extended to nucleic acids and have made a considerable
contribution to developments in molecular biology.
• Messenger RNA, for example, is routinely isolated by selective hybridisation on
poly(U)-Sepharose 4B by exploiting its poly(A) tail.
• Immobilised single-stranded DNA can be used to isolate complementary RNA and
DNA.
• Whilst this separation can be achieved on columns, it is usually performed using
singlestranded DNA immobilised on nitrocellulose filters.
• Immobilised nucleotides are useful for the isolation of proteins involved in nucleic
acid metabolism.

54. ION-EXCHANGE
CHROMATOGRAPHY
Principle
• This form of chromatography relies on the attraction between oppositely charged
stationary phase, known as an ion exchanger, and analyte.
• It is frequently chosen for the separation and purification of proteins, peptides,
nucleic acids, polynucleotides and other charged molecules, mainly because of its
high resolving power and high capacity.
• There are two types of ion exchanger, namely cation and anion exchangers.
• Cation exchangers possess negatively charged groups and these will attract
positively charged cations.
• These exchangers are also called acidic ion exchangers because their negative
charges result from the ionisation of acidic groups.
• Anion exchangers have positively charged groups that will attract negatively
charged anions.
• The term basic ion exchangers is also used to describe these exchangers, as
positive charges generally result from the association of protons with basic groups.

55. Materials and applications
• Matrices used include polystyrene, cellulose and agarose. Functional ionic groups
include sulphonate (–SO3
–
) and quaternary ammonium (–N+
R3), both of which are
strong exchangers because they are totally ionised at all normal working pH
values, and carboxylate (–COO-
) and diethylammonium (–HN+
(CH2CH3)2 , both of
which are termed weak exchangers because they are ionised over only a narrow
range of pH values.
• Examples are given in Table 11.3. Bonded phase ion exchangers suitable for
• HPLC, containing a wide range of ionic groups, are commercially available.
• Porous varieties are based on polystyrene, porous silica or hydrophilic polyethers,
and are particularly valuable for the separation of proteins.
• They have a particle diameter of 525 mm.
• Most HPLC ion exchangers are stable up to 60 C and separations are often carried
out at this temperature, owing to the fact that the raised temperature decreases
the viscosity of the mobile phase and thereby increases the efficiency of the
separation.

56. Examples of commonly used ion exchangers

57. Choice of exchanger
• The choice of the ion exchanger depends upon the stability of the test analytes,
their relative molecular mass and the specific requirements of the separation.
• Many biological analytes, especially proteins, are stable within only a fairly narrow
pH range so the exchanger selected must operate within this range.
• Generally, if an analyte is most stable below its isoionic point (giving it a net
positive charge) a cation exchanger should be used, whereas if it is most stable
above its isoionic point (giving it a net negative charge) an anion exchanger should
be used.
• Either type of exchanger may be used to separate analytes that are stable over a
wide range of pH values.
• The choice between a strong and weak exchanger also depends on analyte
stability and the effect of pH on analyte charge.

58. • Weak electrolytes requiring a very low or high pH for ionisation can be separated
only on strong exchangers, as they only operate over a wide pH range.
• In contrast, for strong electrolytes, weak exchangers are advantageous for a
number of reasons, including a reduced tendency to cause protein denaturation,
their inability to bind weakly charged impurities and their enhanced elution
characteristics.
• Although the degree of cross-linking of an exchanger does not influence the ion-
exchange mechanism, it does influence its capacity.
• The relative molecular mass and hence size of the proteins in the sample therefore
determines which exchanger should be used.

59. Eluent pH
• The pH of the buffer selected as eluent should be at least one pH unit above or
below the isoionic point of the analytes.
• In general, cationic buffers such as Tris, pyridine and alkylamines are used in
conjunction with anion exchangers, and anionic buffers such as acetate,
barbiturate and phosphate are used with cation exchangers.
• The precise initial buffer pH and ionic strength should be such as just to allow the
binding of the analytes to the exchanger.
• Equally, a buffer of the lowest ionic strength that effects elution should initially be
used for the subsequent elution of the analytes.
• This ensures that initially the minimum numbers of contaminants bind to the
exchanger and that subsequently the maximum number of these impurities
remains on the column.
• If, however, gradient elution is to be used, the initial conditions chosen are such
that the exchanger binds all the test analytes at the top of the column.

60. Elution
• Gradient elution is far more common than isocratic elution. Continuous or
stepwise pH and ionic strength gradients may be employed but continuous
gradients tend to give better resolution with less peak tailing.
• Generally with an anion exchanger, the pH gradient decreases and the ionic
strength increases, whereas for cation exchangers both the pH and ionic gradients
increase during the elution.

61. Book and Web References
• Book Name : Wilson and Wlaker
• Book Name : B.D.Singh
• en.wikipedia.org/wiki/Chromatography
• http://www.slideshare.net/suniu/principles-and-application-of-
chromatography
• http://www.google.co.in/PPT
• http://www.chem.iitkgp.ernet.in/faculty/SDG/Protein%20isolation
%20chromatography.pdf
• http://davisson.nat.uni-magdeburg.de/Downloads/Chromatographie.pdf

62. Image References
• 1.http://www.micromountain.com/sci_diagrams/sci_app/sci_
app_assets/ctography_lab_none.jpg
• 2.http://upload.wikimedia.org/wikipedia/commons/thumb/3
/39/Rt_5_9.png/300px-Rt_5_9.png
• 3.http://upload.wikimedia.org/wikipedia/commons/thumb/f/
f1/Cromatografia_su_colonna.jpg/800px-
Cromatografia_su_colonna.jpg
• 4.,7.http://www.biorad.com/webroot/web/images/lsr/soluti
ons/technologies/lab_scale_chromatography/chromatograph
y/technology_detail/thin-layer-chromatography-liquid-
chromatography.jpg
• 5.http://upload.wikimedia.org/wikipedia/commons/d/d2/Chr
omatography_tank.png

63. • 6.http://sci.waikato.ac.nz/farm/images/cellulose
%20structure%20RF.jpg
• 8.http://biologycorner.com/resources/chromatography.gif
• 9.,10.Principles and Techniques of Biochemistry and
Molecular Biology, by Wilson n Walker
• 11.http://www.biorad.com/webroot/web/images/lsr/solutio
ns/technologies/lab_scale_chromatography/chromatography
/technology_detail/chromt11_img1.jpg