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Chromatography is the science which is studies the separation of molecules based on differences in their structure and/or composition. In general, chromatography involves moving a preparation of the materials to be separated - the "test preparation" - over a stationary support. The molecules in the test preparation will have different interactions with the stationary support leading to separation of similar molecules. Test molecules which display tighter interactions with the support will tend to move more slowly through the support than those molecules with weaker interactions. In this way, different types of molecules can be separated from each other as they move over the support material. Chromatographic separations can be carried out using a variety of supports, including immobilized silica on glass plates (thin layer chromatography), volatile gases (gas chromatography), paper (paper chromatography), and liquids which may incorporate hydrophilic, insoluble molecules (liquid chromatography).
Chromatography is a separation method that exploits the differences in partitioning behavior between a mobile phase and a stationary phase to separate the components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption. There are two theories of chromatography, the plate and rate theories.
How it works
In all chromatography there is a mobile phase and a stationary phase. The stationary phase is the phase that doesn't move and the mobile phase is the phase that does move. The mobile phase moves through the stationary phase picking up the compounds to be tested. As the mobile phase continues to travel through the stationary phase it takes the compounds with it. At different points in the stationary phase the different components of the compound are going to be absorbed and are going to stop moving with the mobile phase. This is how the results of any chromatography are gotten, from the point at which the different components of the compound stop moving and separate from the other components. In paper and thin-layer chromatography the mobile phase is the solvent. The stationary phase in paper chromatography is the strip or piece of paper that is placed in the solvent. In thin-layer chromatography the stationary phase is the thin-layer cell. Both these kinds of chromatography use capillary action to move the solvent through the stationary phase.
The retention is a measure of the speed at which a substance moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time Rt or tR, the time between injection and detection. In interrupted development systems like TLC the retention is measured as the retention factor Rf, the run length of the compound divided by the run length of the eluent front:
The retention of a compound often differs considerably between experiments and laboratories due to variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important to compare the retention of the test compound to that of one or more standard compounds under absolutely identical conditions.
What is the Retention Factor, Rf ?
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 R f 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. The retention factor, Rf, is defined as: Rf = distance the solute (D1) moves divided by the distance traveled by the solvent front (D2)
R f = D1 / D 2
where D1 = distance that color traveled, measured from center of the band of color to the point where the food color was applied D2 = total distance that solvent traveled
Simple Theory of Partition Chromatography
In this chromatography there are two physically distinguishable compounds, a mobile phase and a stationary phase. Molecular species separate because they differ in their distribution between these two phases. The relative movement of each molecule is result of a balance between a driving force (the movement of the mobile phase) and the retarding forces. The stationary phase is the sorbent. If the sorbent is a liquid held stationary by a solid, the solid is called the support or matrix. The mobile phases is the solvent or developer and the components in the mixture to be separated constitute then solute.
The theory of partition chromatography is that, in general, if two phases are in contact with one another and if one or both phases contain a solute, the solute will distribute itself between the two phases. This is called partitioning and is described by the partition coefficient (K), the ratio of the concentrations of the solute in the two phases.
Techniques in Partition Chromatography
Partition chromatography will be described in terms of the operation of a column – that is, a tube filled with a sorbent and a solvent. A solution containing the solute is layered on the top of the sorbent and allowed to enter the sorbent. The solvent is hen allowed to pass continually through the column. Although he sorbent and solvent within the column are certainly continuous from he top of the column to the bottom, the column can e thought of as consisting of a large number of individual layers (theoretical plates), each containing he two phases. Consider 256 identical molecules that distribute themselves equally between the two phases, one stationary and one mobile and a column with 18 plates. In the uppermost plate (the origin) the 256 molecules are distributed so that 128 are in each phase. When the mobile 128 molecules from plate 1 enter the second plate, they redistribute 64 and 64 in that plate, the 128 remaining in plate 1 also redistribute 64 and 64, as shown in figure. After 20 successive transfers, the situation shown in the graph is achieved. The distributions of these two kinds of molecules are different and a substantial fraction of the molecules have separated. As the number of the theoretical plates increased (i.e. if the column length is increased), greater separation will result.
Partition chromatography can be carried out in columns by using a matrix that does not adsorb the solutes. Common supporting materials are diatomaceous earth (e.g. Celite), silica gel, cellulose powder and certain cross – linked dextrans (e.g. Sephadex LH20). The stationary phase is created by suspending the support or washing the column with the appropriate sorbent. Typical stationary-phase materials are hydrophobic solvents such as benzene for the separation of nonpolar materials or hydrophilic solvents such as an alcohol, for polar materials. Typical mobile phases are alcohols or amides for ye nonpolar material or water for polar substances. The stationary phase materials are liquid.
Types of partition chromatography
The most common types of partition chromatography are 1) 2) 3) 4) Paper Chromatography Thin – layer chromatography Gas-liquid chromatography Gel chromatography
Special types of partition chromatography are
1) 2) 3) 4)
Liquid chromatography (LC) Hi Performance Liquid chromatography (HPLC) Size exclusion chromatography Column chromatography
This is an older technique which involves placing a small spot of sample solution onto a strip of chromatography paper. The paper is placed into 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. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.
Techniques in Paper Chromatography
In paper chromatography there is no effluent and substances are distinguished by their relative positions in the paper after the solvent has moved a given distance. A tiny volume of a solution of the mixture to be separated is placed at a marked spot on a strip or sheet of paper (Fig) and allowed to dry. This spot defines the origin. The paper is then placed in closed chamber and one end is immersed in a suitable solvent (the mobile phase). Capillarity draws the solvent through the paper, dissolves the sample as it passes the origin and moves the components in direction of flow. After the solvent front has reached a point near the other end of the paper, the sheet is removed and dried. The spots which may or may not be visible are then detected and their positions marked. The
relative distance traveled by a spot and by the solvent front is called the Rf. Values of depend on the substance, he paper and the solvent.
RF = sample spot distance
solvent front distance
VARIATIONS IN COLOUR of spots when sprayed (developed) with reagent (e.g. ninhydrin for amino acids.)
Paper chromatograms can be developed by either ascending or descending solvent flow. Descending chromatography has two advantages: (1) it is faster because gravity aids in the flow and (2) for quantitative separations of materials with very small Rf values, which therefore require long runs, the solvent can run off the paper. Its only disadvantages is the care with which the apparatus must be assembled because dirt or poor contact where the paper passes over the support bar can result in the inhomogeneous flow and consequent streaking.
A particularly useful variant is two-dimensional paper chromatography. In this method, after chromatography has carried out in a single direction, the paper is dried and then rechromatographed at right angles to the original direction of flow, using a different solvent system. (FIG) In this way substances that fail to separate in he first solvent can often be separated in the second.
Detection and Identification of spots
Spots in paper chromatograms can be detected by their color, by their fluorescence, by the chemical reactions that take place after the paper has been sprayed with various reagents, or by the radioactivity. Identification is usually based on comparison with standards of known Rf or by elution. Elution is accomplished by cutting out the spot and soaking the paper in the appropriate solvent.
Fingerprinting: A special application of paper chromatography to study of Proteins.
An important problem in molecular biology is to identify an isolated protein or to identify the site of an amino acid change in a mutant protein. The fingerprint technique developed by Vernon Ingram allows this to be done relatively simply.
The fingerprint of a mutant protein with a single amino acid change differs from that of a wild type protein. If the amino acid change does not affect the site of cleavage by the protease, a single spot in the fingerprint will disappear and one new spot will appear. If it does not affect the site of cleavage, several spots will be altered. By eluting the original and mutant spots and determining the amino acid composition of each, the amino acid change can be determined.
The fingerprinting technique has had several important applications in molecular biology, among which are:
Proof that a Protein (A) is a product of cleavage of a larger Protein (B)
If all spots in a fingerprint of protein A are found in protein B, it is likely that protein A is a part of the amino acid sequence of protein B. Hence, either protein B is made from protein A by the addition of amino acids or protein A is derived from protein B by hydrolysis.
Fig: A paper chromatogram in which four amino acids have been separated.
Identification of the amino acids inserted by various suppressor transfer RNAs
Certain mutations cause the premature termination of amino acid sequence in proteins. This termination is reversed by the presence of suppressor tRNA molecules, which insert an amino acid at the site of premature termination and thereby allow continued synthesis to the natural terminus. A protein results that has a single amino acid
replacement. Fingerprints of such proteins allow the identification of the amino acid inserted by each of he known suppressor tRNAs.
Identification of base changes produced by particular mutagens
It is possible to identify base changes that produce particular mutations because, the genetic code is well known. Suppose that, a mutagen is found to produce frequent changes from phenylalanine to isoleucine. The DNA triplets corresponding to phenylalanine are AAA and AAG and to isoleucine are TAA, TAG and TAT. Because most mutations change only a single base, the particular mutagen would have to lead to frequent replacement of A with T.
THIN –LAYER CHROMATOGRAPHY Simple theory of TLC:
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. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance. In thin-layer chromatography, the stationary phase is a layer (0.25-05) of sorbent spread uniformly over the surface of a glass or plastic plate. The TLC plate is placed in a chamber containing the solvent and developed by ascending chromatography.
After the solvent front has almost reached the top, the plate is removed from the chamber and dried. Spots are usually located as in paper chromatography by natural color, fluorescence or by spraying various reagents that react with the substances in the spot to produce color. Commonly used sprays are ninhydrin for amino acids, rhodamine B for lipids, antimony chloride for steroids and terpinoids, H2SO4 plus heating for almost any organic substance; K2MnO4 in H2SO4 for hydrocarbons, anisaldehyde in H2SO4 for carbohydrates, bromine vapor for olefins and so forth. Materials can be eluted from he chromatogram by scraping off the sorbent and eluting the powder with suitable solvent. A typical thinlayer chromatogram is shown in figure:
Fig: A typical thin-layer chromatogram
Advantages of TLC:
Thin – layer chromatography is widely used because, compared with paper or column chromatography, it offers the following advantages: 1) 2) 3) 4) 5) Greater resolving power because spots are smaller Greater speed of separation and has higher resolution. A wider choice of materials as sorbent Easy detection of spots and Easy isolation of substances from chromatogram.
TLC will be used to examine the composition of various common over-the-counter medications. These medications are classified into one or more basic categories: analgesics (pain relieving), antipyretic (fever reduction), anti-inflammatory (reduces swelling), or uricosuric (relieves symptoms of arthritis and gout). The best known of these is aspirin, but several other chemically similar compounds are (or were) also used. Among these are phenacetin, salicylamide and acetaminophen.
Caffeine is sometimes added to these formulations to overcome drowsiness. A few other compounds such as N-cinnamylephedrine (cinnamedrine) and diphenylpyrilene are included for other therapeutic effects, such as antispasmodic or slight sedative action. In addition to the active ingredients, the tablets of these drugs contain starch, lactose, and other substances that act as binders and permit rapid solution, and sometimes also inorganic bases. The objective of this experiment is to identify an unknown drug tablet by a TLC comparison with standard compounds.
The main uses of TLC
1. Monitoring reactions. Simplest and quickest way to monitor a reaction Reaction should be chromatographed against starting-materials (and a co-spot). Allows you to follow the progress of the reaction, and to assess the best time for work-up. 2. Indicate identity of a compound. The unknown is compared with a known materials. Each substance is spotted separately and also together (co-spot). 3. Indication of purity. Diasteroisomers can usually be distinguished (But not always). 4. For flash chromatography. a. TLC is used to determine the solvent system and quantity of silica required b. TLC is used to monitor the column fractions
GAS – LIQUED CHROMATOGRAPHY
Gas chromatography (GC) is based on a partition equilibrium of analyses between a solid stationary phase and a mobile gas. The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers, frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.
Simple Theory of Gas Liquid Chromatography
A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and identified electronically. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, and the temperature. In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance" (head) of the column, usually using a micro syringe (or, solid phase micro extraction fibers, or a gas source switching system). Although the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time).
A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.
The method is the collection of conditions in which the GC operates for a given analysis. Method development is the process of determining what
conditions are adequate and/or ideal for the analysis required. Conditions which can be varied to accommodate a required analysis include inlet temperature, detector temperature, column temperature and temperature program, carrier gas and carrier gas flow rates, the column's stationary phase, diameter and length, inlet type and flow rates, sample size and injection technique. Depending on the detector(s) (see below) installed on the GC, there may be a number of detector conditions that can also be varied. Some GCs also include valves which can change the route of sample and carrier flow, and the timing of the turning of these valves can be important to method development.
Identification of compounds from the Detector output
Each of the detectors indicates the amount of material emerging from the column as a function of time. However, there is no direct indication of the identify of the material producing a particular peak nor of he amount in the peak. There are various means of identifying peaks. For example, if the substances present in a mixture are known in advance, peaks may be identified by preparing a duplicate sample containing a small amount of an added known substance to the mixture and rechromatographing the mixture.
Advantages of Gas-liquid chromatography
The separation in gas-liquid chromatography is excellent. Sensitivity and speed are extraordinary, with 10-12 gram being detectable for many substances. Because the rapidity of development of chromatograms depends on the rate of diffusion between the mobile and stationary phases and because the diffusion rate of gases is much greater than that of liquids, the gas chromatogram can be run approximately one thousand times as fast as that produce by liquid column chromatography. Hence separation is frequently achieved in less then a minute. Furthermore, by using a nondestructive detector and condensing the samples at the collection end, it is possible to use gas-liquid chromatography preoperatively. Large preparative instruments can purify gram quantities of material.
In general, substances that vaporize below ca. 300 °C (and therefore are stable up to that temperature) can be measured quantitatively. The samples are also required to be salt-free; they should not contain ions. Very minute amounts of a substance can be measured, but it is often required that the sample must be measured in comparison to a sample containing the pure, suspected substance. Various temperature programs can be used to make the readings more meaningful; for example to differentiate between substances that behave similarly during the GC process. Professionals working with GC analyze the content of a chemical product, for example in assuring the quality of products in the chemical industry; or measuring toxic substances in soil, air or water. GC is very accurate if used properly and can measure Pico moles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.
In practical courses at colleges, students sometimes get acquainted to the GC by studying the contents of Lavender oil or measuring the ethylene that is secreted by Nicotiana benthamiana plants after artificially injuring their leaves. These GC analyses are done rather quickly (1 to 15 minutes per sample) and therefore suited for such courses. One example of the use of gas chromatography is in the study of the selectivity of Fischer-Tropsch synthesis catalysts. The outlet from this process contains a number of light gases including H2, CO, CO2 and CH4, as well as heavier parafinic and olefinic hydrocarbons (C2-C40+). In a typical experiment, a packed column is used to separate the light gases, which are then detected with a TCD. The hydrocarbons are separated using a capillary column and detected with an FID. A complication with light gas analyses that include H2 is that He, which is the most common and most sensitive inert carrier (sensitivity is proportional to molecular mass) has an almost identical thermal conductivity to hydrogen (it is the difference in thermal conductivity between two separate filaments in a Wheatstone Bridge type arrangement that shows when a component has been eluted). For this reason, dual TCD instruments are used with a separate channel for hydrogen that uses nitrogen as a carrier are common. Argon in often used when analysing gas phase chemistry reactions such as F-T synthesis so that a single carrier gas can be used rather than 2 separate ones. The sensitivity is less but this is a tradeoff for simplicity in the gas supply.
Gel chromatography (or molecular sieve chromatography) is a special type of partition chromatography in which separation is based on molecular size.
Simple Theory of Gel Chromatography
The basis of gel chromatography is quite simple. A column is prepared of tiny particles of an inert substance that contains small pores. If a solution containing molecules of various dimensions is passed through the column, molecules larger than the pores move only in the space between the particles and hence are not retarded by the column material. However molecules smaller than the pores diffuse in and out of particles with a probability that increases with decreasing molecular size; in this way, they are slowed down in their movement down the column. As long as the material of which the particles are made (i.e. the gel) does not absorb the molecules, the probability of penetration is the principal factor determining the rate of movement through the column. Hence, molecules are eluted from the column in order to decreasing size or, if the shape is relatively constant (e.g. globular or rod like), decreasing molecular weight. In detailed analysis of the mechanism of gel chromatography, it is clear that this steric effect, although the principal factor, does not alone explain the chromatographic behavior of all molecules. Another important factor is the charge of the molecule, although this is only manifested at very low ionic strength when highly charged small molecules seem to be excluded from the pores even though the size is sufficient. This is probably due to electrostatic repulsion between the molecules, this limiting the number of molecules in a pore at any given time. At very low ionic strength, there are also apparently adsorptive effects with some types of gel.
Materials of Gel Chromatography
A gel is a three dimensional network whose structure is usually random. The gels used as molecular sieves consist of cross – linked polymers that are generally inert, do not react or bid with the material being analyzed, and are uncharged. The space within the gel is file with liquid and this liquid occupies most of the gel volume. The gel currently in use is of three types: a) Dextran b) Agarose c) Polyacrylamide They are use for aqueous solution.
Dextran is a polysaccharide composed of glucose residues and produced by the fermentation of sucrose by the microorganism Leuconostoc mesenteroides. It is prepared with various degrees of cross – linking to control pore size and is supplied in the form of dry beads of various degrees of fineness that swell when water is added. Swelling is the process by which the pores become filled with the liquid to be used as eluant. It is commercially available under the trade name Sephadex.
Obtain from certain seaweeds, is a linear polymer of D – galactose and 3, 6 – anhydro – 1 – galactose and forms a gel that is held together without cross links by hydrogen bonds. It is dissolved in boiling water and forms a gel when cooled. The concentration of the material in the gel determines the size of the pores – which are much larger than those of Sephadex. This makes it useful for the analysis or separation of large globular proteins or long, linear molecule such as DNA. Agarose is useless as a solid gel
because the flow rate is too low; so it is supplied as wet beads called Sepharose and Bio – gel.
Polyacrylamide gels are prepared by cross linking acrylamide with N, N’-methylene-bis-acerylamide. Again, the pore size is determined by the degree of cross linking. These gels differ from Dextran and Agarose gels in that they contain a polar, carboxylamide group on alternate carbon atoms, but their separation properties are much the same as those of the dextrans. Polyacrylamide gels, which are marketed as Bio-gel P, seem to be as useful as dextrans, although they have used less frequently. They do have advantages over the dextrans in that they are commercially available in a wider range of pore sizes.
Fig: Mechanism of gel chromatography
Red particles – sample molecules smaller than the pore size Light green particles – sample molecules bigger than red particles but equal to the pore size Green particles – bigger than all particles and also from the pore size.
Advantages of Gel Chromatography
For the separation of molecules whose molecular weights differ, gel chromatography is unsurpassed for the following reasons: 1. Because the chromatographic behavior of almost all substances in gels is independent of temperature, pH, ionic strength, and buffer composition, separations can be carried out under virtually all conditions. For very labile materials (e.g., enzymes), this means that the conditions for maximum stability can be maintained. 2. Because there is virtually no adsorption, very labile substances are not affected by the chromatography. For example, some enzymes are inactivated or altered by binding to adsorbent surfaces or ionic – exchange resins. 21
3. There is less zone spreading than with other chromatographic techniques. 4. The elution volume is related in a simple manner to molecular weight.
Application of Gel Chromatography
1. In the chemical synthesis of various reagents, it is usually necessary to separate the product from the reactants. For example, in preparing fluorescent antibodies by reacting antibody with fluorescein isothiocyanate, the conjugated protein must be separated from unreacted dye. This can be done with Sephadex, using gels that pass large proteins in the void volume. The unreacted protein is not separated from the conjugation protein but, for the fluorescent antibody technique, this is usually unnecessary. 2. In the assay of enzymes or the determination of cofactor requirements, the enzyme preparation sometimes contains inhibitors of small molecular size or the cofactors themselves. Also, I physical studies of some molecules (e.g. in fluorescence spectroscopy), interfering substances may be present. Such small molecules are easily removed with the Dextran or Polyacrylamide gels. 3. Similarly there are frequently contaminants of large molecular size in mixtures being assayed for small molecules. He small – pore dextrans are useful in such cases. Also protein must often be freed to nucleic acids; this can sometimes be done by using an Agarose gel, which impedes all proteins and passes nucleic acids in the void volume. 4. The most common use of gel chromatography is in the purification of proteins. To purify a protein from a cell extract, it is usually necessary to use a sequence of separation procedures based on such parameters as solubility in certain solutions, chare, molecular weight, and so forth. The step in which size separation takes place almost invariably uses gel chromatography. Gel Chromatography is also a valuable analytical tool. The determination of molecular weight mentioned previously is an important example of this. Other examples are: 1. In studying RNA metabolism, various fractions of RNA are usually distinguished by zone centrifugation or even better by Polyacrylamide gel electrophoresis. Gel chromatography with Agarose is also of great use. 2. Plasma protein fractions must often be determined quantitatively in the diagnosis of certain human diseases. This can be done directly with Dextran gels and as been developed as a reliable test for macro and hyperglobulinemia. 3. The tritium exchange method for examining protein or DNA structure requires that the macromolecule be rapidly separated from 3H2O. This can be done in about ten seconds using charged gels because the 3H2O is strongly retarded in all gels. IF the
smallest pore size is used, the macromolecule comes through rapidly in the void volume or shortly thereafter. 4. Gel chromatography can be used to study biding between proteins and small molecules either by separating the product and he reactants or by passing protein through a column equilibrated with the small molecule. A simple calculation allows the determination of binding constants. The great value of gel chromatography in studies of chemical equilibrium is that a gel column can be operated over a wide range of concentrations, pH, ionic strength, and temperature e because the pore size of the el is unaffected by the factors.
Liquid chromatography (LC) is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column.
Simple theory of Liquid Chromatography
Simple liquid chromatography consists of a column with a fritted bottom that holds a stationary phase in equilibrium with a solvent. Typical stationary phases are: solids (adsorption), ionic groups on a resin (ion-exchange), liquids on an inert solid support (partitioning), and porous inert particles (size-exclusion). The mixture to be separated is loaded onto the top of the column followed by more solvent. The different components in the sample mixture pass through the column at different rates due to differences in their partioning behavior between the mobile liquid phase and the stationary phase. The compounds are separated by collecting aliquots of the column effluent as a function of time.
Conventional LC is most commonly used in preparative scale work to purify and isolate some components of a mixture. It is also used in ultra trace separations where small disposable columns are used once and then discarded. Analytical separations of solutions for detection or quantification typically use more sophisticated highperformance liquid chromatography instruments.
HI – PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
High performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. The analyte is forced through a column (stationary phase) by a liquid (mobile phase) at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to diffuse within the column. Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase.
General characteristics of reversed phase chromatography
• • • •
Broad scope which allows sample types with a wide range of polarities and molecular weights to be separated. General rapidity of mobile phase column equilibration during methods development and gradient regeneration. General ease of use. Applicability to separation of ionic or ionizable compounds by manipulating secondary chemical equilibrium such as ionization control and ion pairing in the aqueous mobile phase. o Buffering the mobile phase in the pH range from 2 to 5 with one of the common buffers, the ionization of the weak acids can be suppressed or controlled allowing them to be retained in their neutral form. Similarly weak bases can be retained in their neutral form at pH 7-7.5. o For strong acids and bases ionization control cannot be employed because the stability of alkyl bonded phases is diminished below pH 2 and above pH 7.5. Highly hydrophilic weak acids and bases often remain difficult to retain with ionization control. In such cases ion pair reversed phase chromatography can be used. In this method, counterions (species of opposite charge to the solutes) thereby regulate the retention. Typically alkyl amines or tetra alkyl amines are added to ion pair with acids whereas alkyl sulfates, sulfonates, or phosphates are used to ion pair with bases. The possibility of special selectivity such as structural or steric are achievable by specific mobile phase additives: o Metal ions are capable of binding to organic compounds in a very selective method which is used for ligand exchange chromatography. The selectivity generated in these metal ion phase systems is based in part on differences of the solute (ligand) binding strength to the metal ion. An alternate approach is the addition of various chelating agents (4dodecyldiethylene-triamine - C12 dien) in combination with a metal ion. The type and strength of the metal chelate complex-solute binding can be greatly varied depending upon the chemical environment surrounding the metal ion as determined by the chelating agent added.
Solvents must be degassed to eliminate formation of bubbles. The pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation. The liquid sample is introduced into a sample loop of an injector with a syringe. When the loop is filled, the injector can be injecting the sample into the stream by placing the sample loop in line with the mobile phase tubing. The different types of HPLC columns are described below. The presence of analytes in the column effluent is recorded by detecting a change in refractive index, UVVIS absorption at a set wavelength, fluorescence after excitation with a suitable wavelength, or electrochemical response.
Columns -- Conventional liquid chromatography uses plastic or glass columns
that can range from a few centimeters to several meters. The most common lengths are 10-100 cm, with the longer columns finding use for preparative-scale separations.
High-performance liquid chromatography (HPLC) columns are stainless steel tubes, typically of 10-30 cm in length and 3-5 mm inner diameter. Short, fast analytical columns, and guard columns, which are placed before an analytical column to trap junk and extend the lifetime of the analytical column, are 3-10 cm long. 26
Fig: Picture of an HPLC column The applicability of chemiluminescence reactions as a means of detecting compounds in liquid chromatography (LC) is based to a large degree on post column reactions. A primer on liquid chromatography (and high performance LC) can be found here; however, a brief description follows. This describes, in the main, HPLC chromatographic systems.
Components of High Performance Liquid Chromatography
Liquid phase samples (mixtures) are injected onto an LC column usually using a syringe and specially devised injection valve. The sample is swept onto the chromatographic column by the flowing mobile phase and chromatographic separation occurs as the mixture travels down the column. Normal HPLC detectors detect the elution of a compound from the end of the column based on some physical characteristic such as ultraviolet light absorption, ability to fluoresce, or the difference in index of refraction between the analyte and the mobile phase itself. The majority of HPLC systems work this way. An example schematic of an HPLC system is shown below:
Need for HPLC Chemiluminescence’s Detection
The use of chemiluminescence detection for HPLC comes from the need to detect compounds either very sensitively (at very low concentrations) or very selectively, that is, a target compound that must be determined in the presence of co-eluting compounds that just can not be successfully separated from the analyte.
Since chemiluminescence derives from the generation of light cause by a chemical reaction, there is no source lamp light that must be filtered out (as in the case of fluorescence detection) in order to detect the analyte emission. This means that the photons coming from the de-exciting analyte molecule are detected against a black background, and this detection can be accomplished by a photomultiplier which can detect a large percentage of the emitted photons.
Methods of HPLC Post Column Chemiluminescence Detection
IF a target analyte can be determined via HPLC chemiluminescence then it probably has one of three characteristics: 1) it either chemiluminesces when mixed with a specific reagent; 2) it catalyzes chemiluminescence between other reagents; or 3) is suppresses chemiluminescence between other reagents. Examples of all three will be given below using the well explored luminol reaction.
Luminol based chemiluminescence’s detection
Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants like hydrogen peroxide (H2O2) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA) which gives off light at approximately 425 nm. If luminol is the target analyte (seldom) then a schematic of a post column detector based on its solution phase reaction would look like this:
In this case one reagent pump would send a solution containing a dissolved metal ion like copper(II) or iron(III) to the mixer at the end of the LC column, while the other reagent pump would send a solution containing the oxidant such as H 2O2 or hypochlorite (another oxidant) and a base. Depending on the catalyst used (which basically controls the time necessary for maximum light emission to develop and the decay profile of that emission) the distance from the mixer to the detection cell is carefully determined to allow for the most sensitive detection-in this case the detection of luminol arriving from the LC column where it could have been separated from interfering compounds. More realistically, some important chemical species can be derivatized using luminol itself or luminol like reagents that can be detected in the same or similar ways. 29
Detection based on luminol suppression
What follows is a method of chemiluminescence detection in which the suppression of a background chemiluminescence signal could be used to determine a compound that elutes from the LC column. For instance, many organic molecules will complex metal cations and thereby make them less available as catalysts in the luminol reaction. This is a nifty way to determine the concentration of the organic molecule: Mix a constant concentration of a metal cation, luminol, base, and an oxidant. This will create a baseline light signal that is relatively constant.
With the LC column output fed into the mixer, the amount of light detected will decrease when an organic analyte (which can complex with the metal ion) elutes from the column.
The amount of light decrease depends directly on the amount of the analyte. This is true as long as the amount of metal cation is not completely complexed. At this point the light decrease will no longer be linearly related to the amount of organic analyte. Basically the same schematic seen above is seen here with the metal catalyst coming from the first reagent pump and feeding into a second mixer placed upstream of the first mixer. This is to allow the eluting organic molecules (e.g., analytes like amino acids) to have time to tie up the metal catalyst before they are mixed with the other reagents. The second reagent pump adds luminol, base and oxidant. When that metal/organic complex gets to the second mixer and ultimately to the detection cell, the baseline light intensity will drop off. Voila! An "antisignal"-proportional to the amount of the (analyte) organic molecules eluting from the column.
SIZE – EXCLUTION CHROMATOGRAPHY
Size exclusion chromatography (SEC) is also known as gel permeation chromatography or gel filtration chromatography and separates particles on the basis of size. Smaller molecules enter a porous media and take longer to exit the column, whereas larger particles leave the column earlier. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.
giving chromatogram as below
Mild, non-denaturing conditions - very suitable for separating proteins of different molecular masses.
Also used for:
De-salting or buffer exchange of, protein solutions Determination of Molecular mass of biological macromolecules - calibrate column with similar molecules of known molecular mass
Quaternary structure usually remains intact. Types of matrix for forming stationary phase:
• • •
Cross-linked Dextran polymer (Sephadex G-10 to G-200) Cross-linked Polyacrylamide (Bio-gel P-2 to P-300) Agarose - the largest pore size
Column chromatography encompasses a number of techniques based around utilizing a vertical glass column filled with some form of solid support, with the sample to be separated placed on top of this support. The rest of the column is filled with a solvent which, under the influence of gravity, moves the sample through the column. Similarly to other forms of chromatography, differences in rates of movement through the solid medium are translated to different exit times from the bottom of the column for the various elements of the original 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
The protein separation actually occurs on a chromatography column that contains a charged stationary phase. Oppositely charged proteins bind to the stationary phase and like charged or uncharged proteins wash through the column with the void volume. The mobile phase travels through the column, carrying unbound proteins with it. The bound proteins are then eluted from the column by increasing the salt concentration in the mobile phase.
The chromatography column is the heart of the separation process. For protein separations the column usually contains porous beads of a hydrophilic polymer, such as cellulose or some other type of carbohydrate polymer. The surface of the polymer beads is chemically modified to give it properties that would make it suitable for various types of chromatography: ion exchange, molecular exclusion, hydrophobic interaction or affinity. The appropriate stationary phase is suspended in the desired mobile phase and poured into the chromatography column. Once the stationary phase has been fully equilibrated with the mobile phase, the protein sample can be introduced onto the column. The separation then occurs based on the attraction between the protein, the stationary phase and the mobile phase. For example, a positively charged protein would bind to a negatively charged stationary phase when the mobile phase has a low ionic strength (see Salts). An increase in the salt concentration may displace the protein from the stationary phase when positive ions in the mobile phase compete with the protein for binding sites on the stationary phase. In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.
The mixture to be analyzed by column chromatography is applied to the top of the column. The liquid solvent (the eluant) is passed through the column by gravity or by the
application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or eluant, are collected as the solvent drips from the bottom of the column. Column chromatography is separated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, it is called flash chromatography, a "state of the art" method currently used in organic chemistry research laboratories The term "flash chromatography" was coined by Professor W. Clark Still because it can be done in a “flash."
Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the organic chemist for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles. Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography; larger particles (lower mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400 mesh for flash columns. Alumina is used more frequently in column chromatography than it is in TLC. Alumina is quite sensitive to the amount of water which is bound to it: the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity I and deactivated with water before use according to specific procedures. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed. Silica gel and alumina are the only column chromatography adsorbents used in the CU organic chemistry teaching labs; please refer to the references for information on other column chromatography adsorbents.
The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the successful application of column chromatography as a separation technique. TLC is generally used to determine the system for a column chromatography separation. The choice of a solvent for the elution of compounds by column chromatography is covered in the Chromatography Overview section. Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent is first used to elute a less-polar compound. Once the less-polar compound is off the column, a more-polar solvent is added to the column to elute the more-polar compound.
Analysis of Column Eluant
If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. More commonly, the compounds to be isolated from column chromatography are colorless. In this case, small fractions of the eluant are collected sequentially in labeled tubes and the composition of 39
each fraction is analyzed by thin layer chromatography. (Other methods of analysis are available; this is the most common method and the one used in the organic chemistry teaching labs.)
Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.
Simple theory of Adsorption Chromatography
Consider a solid surface containing a wide varity of binding sites – for example, regions that are electron – rich (negatively charged), electron – poor (positively charged), nonpolar and so forth, and a liquid containing solute in contact with the surface. If binding is reversible, the number of molecules bound to the surface will depend on the solute concentration. This dependency is shown in figure. Curves of this sort are called adsorption isotherms. The most common is the convex curve – that is, binding sites with high affinity are filled first so that additional amounts of solute are bound less tightly. The binding isotherm is a characteristic of a particular molecule and sorbent.
If a given concentration of a molecule is applied to the surface and solvent is allowed to flow across the surface, a fixed amount will bind ad the remainder will move along. The advancing material will bind with two differences: (1) the retarding force is binding or adsorption and (2) the fraction bound is not a constant fraction but decreases with decreasing concentration. The rate at which the substance moves is related to the strength of binding – that is, the tighter the binding, the slower the movement. Clearly then, molecules can be separated if they have different adsorption isotherms.
Solute in liquid (or gas) phase interacts with adsorption sites on solid surface (finely divided particles for maximum surface area). Material Alumina Silica Gel Activated carbon Calcium phosphate gel Hydroxyapatite Substance separated Small organic molecules, Proteins Sterols, amino acids Peptides, amino acids, carbohydrates Proteins, Polynucleotide Nucleic acids
Polar groups on solid form dipolar interactions (e.g. hydrogen bonds) with sample dissolved (usually) in organic solvent. Elute by increasing polarity of the solvent (e.g. if using acetonitrile CH3CN, add methanol (CH3OH)) --> competing bonds with adsorption sites. Gradient elution useful (also for ion-exchange and partition chromatography).
Types of Adsorption Chromatography
Adsorption chromatography uses a mobile liquid phase and a solid stationary phase. Separation is either in columns or on thin layers. An important variation of adsorption chromatography is ion – exchange chromatography. This differs mainly in that the composition of the mobile phase such that, as the material is being applied to the adsorbent, the solute becomes immobilized. Migration does not begin until a new mobile phase is added.
Fig : A simple fraction collector
Operation of columns
The chromatogram is developed by flowing a solvent (the mobile phase) through the column. The process is called eluting the column. As different substances move through the column, they separate and appear in the effluent when particulars volume of material, both solid and liquid in the column is called the bed volume. The volume of the mobile phase is the void, retention or hold up volume.
Fig: Operation of a column showing the loading of the column and various stages of elution.
The liquid leaving the column (the eluent) is usually collected as discrete fractions, using an automatic collector. The separated components are then found and identified by testing aliquots of each fraction – for example, spectral measurements, chemical tests, radioactivity and so forth. In cases in which analysis is by the absorption of light, an automatic, continuously recording spectrophotometer is used.
Ion exchange chromatography (IEC) is applicable to the separation of almost any type of charged molecule, from large proteins to small nucleotides and amino acids. It is very frequently used for proteins and peptides, under widely varying conditions. However, for amino acids standardized conditions are used. In protein structural work the consecutive use of gel permeation chromatography (GPC) and IEC is quite common.
Basic Principles of Ion Exchange Chromatography
In ion exchange chromatography, the stationary phase of the column has a charge (either + or -). A mixture of proteins is added to the column and everything which has the same charge passes through the column due to electrostatic repulsion. If the charge of the matrix is positive, it will bind negatively charged molecules. This technique is called anion exchange. If the matrix is negatively charged, it will bind positively charged molecules (cation exchange). Thus, a scientist picks the resin to use based on the properties of the protein of interest. During the chromatography, the protein binds to the oppositely charged beads. Contaminating proteins which have the same net charge as the matrix can be separated from the bound proteins by washing the column with buffer. Proteins which remain bound to the matrix can be differentially eluted by increasing the salt concentration or by altering the pH of the mobile phase. Ion exchange containing diethyl aminoethyl (DEAE) or carboxymethyl (CM) groups is frequently used in biochemistry.
The ionic properties of both DEAE and CM are dependent on pH, but both are sufficiently charged to work well as ion exchangers within the pH range 4 to 8 where most protein separations take place.
Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups) which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. 45
When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge. A basic protein, which is positively charged, will bind to a support which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge.
Families of molecules (acidic, basics and neutrals) can be easily separated by this technique. This is perhaps the most frequently used chromatographic technique used for protein purification.
Retention by attraction between groups on stationary phase with opposite charge to sample molecules. Stationary phase = insoluble, but solvent permeable polymer matrix (e.g. cellulose) chemically modified to introduce ionizable groups (e.g. -COOH).
• • •
Change of pH to neutralize charged group on either solute or stationary phase. Increase [salts] (especially polyvalent) in eluant buffer --> Displace by competing ions. pH or salt gradient to enhance separation.
Ion-exchange media are classified according to whether the attached ionizable group is strongly or weakly acidic or basic --> determines the usable pH range
Medium (X = matrix) Anion exchangers X-CH2N+(CH3)3 X-CH2NH+(CH3)2 (CH3CH2)2 X-CH2CH2NH+ diethylamino-ethyl (DEAE) Cation exchangers X-SO3X-COOX-CH2COOcarboxymethyl (CM)
pH range 2 - 11 2-7 3-6
strong intermediate weak
nucleotides organic acids proteins
strong intermediate weak
2 - 11 6 - 10 7 - 10
amino acids peptides proteins
Note DEAE-cellulose and CM-cellulose popular for chromatographic separation of proteins - mild, non-denaturing procedure.
pH and Selectivity in Ion Exchange Chromatography
The property of a protein which governs its adsorption to an ion exchanger is the net surface charge. Since surface charge is the result of weak acidic and basic groups of protein; separation is highly pH dependent. Going from low to high pH values the surface charge of proteins shifts from a positive to a negative charge surface charge. The pH vs. net surface curve is an individual property of a protein, and constitutes the basis for selectivity in IEC. At a pH value below its isoelectric point a protein (+ surface charge) will adsorb to a cation exchanger (-) such as one containing CM-groups. Above the isoelectric point protein (- surface charge) will adsorb to a anion exchanger (+), e.g. one containing DEAE-groups.
Ionic Strengths and Selectivity in Ion Exchange Chromatography
As in all forms of liquid chromatography, conditions are employed that permit the sample components to move through the column with different speeds. At low ionic strengths, all components with affinity for the ion exchanger will be tightly adsorbed at the top of the ion exchanger and nothing will remain in the mobile phase. When the ionic strength of the mobile phase is increased by adding a neutral salt (e.g., NaCl), the salt 48
ions will compete with the protein and more of the sample components will be partially desorbed and start moving down the column. Increasing the ionic strength even more causes a larger number of the sample components to be desorbed, and the speed of the movement down the column will increase. The higher the net charge of the protein, the higher the ionic strength needed to bring about desorption. At a certain high level of ionic strength, all the sample components are fully desorbed and move down the column with the same speed as the mobile phase. Somewhere in between total adsorption and total desorption one will find the optimal selectivity for a given pH value of the mobile phase. Thus, to optimize selectivity in ion exchange chromatography, a pH value is chosen that creates sufficiently large net charge differences among the sample components. Then, an ionic strength is selected that fully utilizes these charge differences by partially desorbing the components. The respective speed of each component down the column will be proportional to that fraction of the component which is found in the mobile phase.
Very often the sample components vary so much in their adsorption to the ion exchanger that a single value of the ionic strength cannot make the slow ones pass through the column in a reasonable time. In such cases, a salt gradient is applied. This will bring about a continuous increase of ionic strength in the mobile phase. Such a gradient will gradually desorbs the sample components in the order of increasing net charge, until all the components are fully desorbed. At this point, though, we have already separated the components of the sample. Thus a salt gradient compresses a chromatogram so as to elute components with widely different adsorptive properties within a reasonable time. In fact, most IEC experiments utilize a salt gradient. If it is necessary to selectively increase resolution somewhere within the gradient, but still to elute the slow components within a reasonable time, a section of lower gradient slope is built into the gradient so that it covers that part of the chromatogram where increased resolution is desired. This is called the adapted gradient technique and requires an advanced programmable gradient device.
Application of Ion-exchange Chromatography
In principle, any substance that is charged can be chromatographed on an ion exchanger. Resin exchangers are most useful for small organic molecules and can even be used to separate metallic ions (e.g. Ca2+ from Mg2+). Proteins and polysaccharides are best used with the cellulose, Dextran and Polyacrylamide exchangers. The Dextran and Polyacrylamide exchangers have also been widely used for the separation of nucleotides, amino acids and other biologically important small molecules.
Theory of Affinity Chromatography
Affinity chromatography is a type of chromatography that makes use of a specific affinity between a substance to be isolated and a molecule that it can specifically bind (a ligand). The column material is synthesized by covalently coupling a binding molecule (which may be a macromolecule or a small molecule) to an insoluble matrix. The column material s the specifically able to absorb from the solution the substance to be isolated. Elution is accomplished by changing the conditions to those in which binding does not occur. This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solutes containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.
Several requirements must be met for success in affinity chromatography 1) The matrix should be a substance that does not itself adsorb molecules to any significant extent. 2) The ligand must be coupled without altering its binding properties. 3) A ligand should be chosen whose binding is relatively tight because, although weak binding will enhance retardation, it may not be adequate for separation to result. 4) It should be possible to elute without destroying the sample.
The most useful matrix material is Agarose because it exhibits minimal adsorption, maintains good flow properties after coupling, and tolerates the extremes of pH and ionic strength as well as 7.0 M guanidium chloride and urea, which are often needed for successful elution. It is possible to purchase Agarose to which are covalently coupled either reagents for coupling proteins, membranes and steroids or concanavalin A, a ready-to-use adsorbent for polysaccharides and glycoprotein containing α-D-mannosyl and α-D-glucosyl residues (e.g. cell membranes and whole cells), is an additional benefit.
Use of Affinity Chromatography
The major use of affinity chromatography to date has been the purification of proteins, membranes and polysaccharides. Examples of its use are as follows:
Purification of Proteins
This is usually done with Sepharose to which the substance that is being transported is coupled (e.g. thyroxin binding globulins, estradiol-binding proteins and hormone and drug receptors).
Affinity Chromatography Method
Affinity chromatography is designed to purify a particular protein from a mixed sample.
Figure 1. Loading affinity column.
Figure 2. Proteins sieve through matrix of affinity beads.
Figure 3. Proteins interact with affinity ligand with some binding loosely and others tightly.
Figure 4. Wash off proteins that do not bind.
Figure 5. Wash off proteins that bind loosely.
Figure 6. Elute proteins that bind tightly to ligand and collect purified protein of interest.
Purification of enzymes:
The substrate, a tight binding inhibitor, or a cofactor can be coupled to the matrix. If a mixture of proteins or even a crude cell extract is passed through the column, only materials that bid remain on the column. In some cases, enzymes can be substantially purified directly from very complex mixtures.
Purification of Antibodies
This has been accomplished mainly with cyanogens bromide Sepharose to which has been coupled various antigens such as proteins, viruses or bovine serum albumin coupled with haptens; it is the method of choice for antibody purification.
Purification of Membranes and Particles containing known substances
Membranes to which a hormone binds can be purified using Sepharose coupled with that hormone; influenza virus, which contains neuraminidase on its surface, has also been purified using Sepharose to which inhibitors of neuraminidase are coupled.
Purification of Glycoprotein
This is efficiently done with concanavalin A-Sepharose.
Separation of Specific Animal Cells
This has been done using coupled agglutinins, such as concanavalin A, wheat germ agglutinin or phytohemagglutinin. For example, some virus induced tumor cells can be separated fro normal cells because the tumor cells bind more tightly to concanavalin A Sepharose.
Chromatography and Biotechnology
This discussion of chromatography will focus on the separation of proteins into relatively homogeneous groups because proteins are often the target molecules which must be purified for use as "biopharmaceuticals" or medicines. It is important to remember, however, that chromatography can also be applied to the separation of other important molecules including nucleic acids, carbohydrates, fats, vitamins, and more. One of the important goals of biotechnology is the production of the therapeutic molecules known as "biopharmaceuticals," or medicines. There are a number of steps that researchers go through to reach this goal:
• • • • • • • • •
identification of a "target protein" which may have therapeutic value identification of the "target gene" -- the gene responsible for encoding the target protein isolation of the target gene insertion of the target gene into a host cell (such as E. coli) which will both grow well, and continue to produce the protein product encoded for by the target gene separation of the target protein from the many other host cell proteins large scale production of the target protein under controlled manufacturing conditions large scale testing for efficacy as a medicine marketing of a new medicine Many different disciplines, including microbiology, molecular biology, chemistry, and others, are required to complete the steps listed above to bring a protein from
the "scientifically interesting" state to that of a full-fledged drug to be used in treating a specific disease. This discussion will focus on the work and tools of the chromatographer. Chromatographers use many different types of chromatographic techniques in biotechnology as they bring a molecule from the initial identification stage to the stage of a becoming a marketed product. The most commonly used of these techniques is liquid chromatography, which is used to separate the target molecule from undesired contaminants (usually host-related), as well as to analyze the final product for the requisite purity established with governmental regulatory groups (such as the FDA).