Chromatography

Course: B.Sc Biotechnology

Subject: Bio Analytical Technique

Unit: IV

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

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.

1.

• 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.

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.

• 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.

• 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.

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 timeA 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.

2. Retention factorThe 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 heightChromatography 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

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).

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.

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.

Purpose of Chromatography

• AnalyticalAnalytical - determine chemical composition of a sample

• PreparativePreparative - purify and collect one or more components of a sample

Classification of Methods

• There are two classification schemes:–mobile phase– attractive forces

Mobile Phase

• gas (GC)• water (LC)• organic solvent (LC)• supercritical fluid (SCFC)

Classification based on Mobile Phase

Gas ChromatographyGas Chromatography

Gas - solidGas - solid Gas - liquidGas - liquid

Stationary Phase

Sample MUST be volatile at temperatures BELOW 3500C

Pyrolysis GC -heat solid materialsto 500 - 10000Cso they decomposeinto gaseous products

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

Detectors

• UV-vis• Refractive Index (RI)• Mass spectrometry (MS)• Electrochemical (EC)– amperometric

• NMR - novel

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

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.

• 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.

3.

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 phaseThe 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

• 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.

• 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.

• 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.

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.

• 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.

Types of Paper Chromatography1.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.

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,

4.

• 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 R f 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.

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

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 Tsvet1872-1919

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.

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.

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.

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.

“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 results1980-1987: 394 results

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.

• 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.

9.

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.

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.

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

Under the correct experimental conditions, when a complex mixture containingthe 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.

• 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.

10.

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.

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.

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.

11.

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.

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.

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.

Examples of commonly used ion exchangers

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.

• 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.

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.

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.

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

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/solutions/technologies/lab_scale_chromatography/chromatography/technology_detail/thin-layer-chromatography-liquid-chromatography.jpg

• 5.http://upload.wikimedia.org/wikipedia/commons/d/d2/Chromatography_tank.png

• 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