Paper Chromatography (6)

BIOPHYSICS

LESSON 37 CHROMATOGRAPHY What is Chromatography welcome to the wonderful world of chromatography! What is chromatography? Well, quite simply, it is a broad range of physical methods used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed. The Russian botanist Mikhail Tswett is credited with the original development of a separation technique that we now recognize as a form of chromatography. In 1903 he first time reported the separation of a mixture of plant pigment using a column of calcium carbonate. How Does It Work A mixture of various components enters a chromatography process, and the different components run through the system at different rates. These differential rates of migration as the mixture moves over adsorptive materials provide separation. Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column. The basis of all forms of chromatography is the partition or distribution coefficient(Kd), which describes the way in which a compound distribute itself between two immiscible phases. For two such phases A and B the value for this coefficient is a constant at a given temperature and is given by the expression. So, Why Is It So Special? In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods in which industries can accomplish these goals. Chromatography is a very special separation process for a multitude of reasons! First of all, it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. In fact, chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. Second, chromatography can be used to separate delicate products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins.

mobile phase is typically a solvent moving through the column which carries the mixture to be separated. This can either be a liquid or a gas, depending on the type of process. The stationary phase is usually a viscous liquid coated on the surface of solid particles which are packed into the column as discussed above, although the solid particles can also be taken as the stationary phase. In any case, the partitioning of solutes between the stationary and mobile phases lead to the desired separations.

1. Feed Injection The feed is injected into the mobile phase. The mobile phase flows through the system by the action of a pump (older analytical chromatorgraphy used capillary action or gravity to move the mobile phase). 2. Separation in the Column As the sample flows through the column, its different components will adsorb to the stationary phase to varying degrees. Those with strong attraction to the support move more slowly than those with weak attraction. This is how the components are separated. 3. Elution from the Column After the sample is flushed or displaced from the stationary phase, the different components will elute from the column at different times. The components with the least affinity for the stationary phase (the most weakly adsorbed) will elute first, while those with the greatest affinity for the stationary phase (the most strongly adsorbed) will elute last. 4. Detection The different components are collected as they emerge from the column. A detector analyzes the emerging stream by measuring a property which is related to concentration and characteristic of chemical composition. For example, the refractive index or ultraviolet absorbence is measured.

Chromatography - Basic Operation The process of a chromatographic separation takes place within a chromatography column. This column, made of glass or metal, is either a packed bed or open tubular column. A packed bed column contains particles which make up the stationary phase. Open tubular columns are lined with a thin film stationary phase. The center of the column is hollow. The

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Types of Chromatography Thin layer Chromatography (TLC) Gas Chromatography Liquid Chromatography

Very polar additives:

Ion Exchange Chromatography

Methanol > Ethanol > Isopropanol

Affinity Chromatography

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Thin Layer Chromatography (TLC) is an extremely useful technique for monitoring reactions. It is also used to determine the proper solvent system for performing separations using column chromatography. TLC uses a stationary phase, usually alumina or silica, that is highly polar (standard) or non-polar (reverse phase). The mobile phase is a solvent whose polarity you will choose. In most lab applications, you will use standard phase silica plates. You will apply your reaction mixture in solution to the plate and then “run” the plate by allowing a solvent (or combination of solvents) to move up the plate by capillary action. Depending on the polarity of the components of the mixture, different compounds will travel different distances up the plate. More polar compounds will “stick” to the polar silica gel and travel short distances on the plate. Non-polar substances will spend more time in the mobile solvent phase and travel larger distances on the plate. The measure of the distance a compound travels is called the Rf value. This number, between zero and one, is defined as the distance the compound moved from the baseline (where it was originally spotted) divided by the distance the solvent front moved from the baseline. The actual run on the slica plate is given in the figure below. Here, the details of the TLC running is described below.

Steps for TLC:

Acetonitrile > Ethyl Acetate > Chloroform > Dichloromethane > Diethyl Ether > Toluene Non-polar additives: Cyclohexane, Petroleum Ether, Hexane, Pentane Common solvent combinations: Ethyl Acetate/Hexane : 0–30% most popular combination, sometimes tough to remove solvents completely on rotary evaporator Ether/Pentane: 0–40% very popular, easy to remove on the rotary evaporator Ethanol/Hexane or Pentane: 5–30% useful for very polar compounds Dichloromethane/Hexane or Pentane : 5–30% sometimes useful when other mixtures fail 3. Fill TLC chamber with 1–2 mL of the desired solvent system. Place a large piece of cut filter paper in the chamber as well. 4. Spot the compound on the baseline of the TLC plate. We will use commercial spotters, but spotters can be pulled from hot Pasteur pipets (you may see this in your UROP). If you are monitoring a reaction, make sure to spot the starting material, the reaction mixture, and a co-spot of both. 5. Run the TLC. Let the solvent go about 90% of the way up the plate.

1. Cut TLC plates. Usually silica plates are bought as square glass pieces that must be cut using a diamond tipped glass cutter and following a template. Before scoring the glass, use a ruler and a pencil to lightly mark baselines on the silica side of the plate (be careful not to remove any silica from the plate). Using a sharp glass cutter and a ruler as a guide, you should have no problem scoring the glass. Once the entire plate is scored, you can thenbreak the glass into individual pieces. (In the beginning this may be frustrating, but after some practice, you should become comfortable with this technique.) 2. Determine an appropriate solvent system. Your compounds will travel different distances up the plate depending on the solvent you choose. In non-polar solvents like pentane and hexane, most polar compounds will not move, while non-polar compounds will travel some distance up the plate. In contrast, polar solvents will usually move non-polar compounds to the solvent front and push the

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Moderately polar additives:

6. Remove the plate from the chamber and mark the solvent front immediately with a pencil. You will use this to calculate the Rf. 7. Let the solvent dry off of the plate. 8. Visualize the TLC using non-destructive technique(s). The best non-destructive method is the UV lamp. Place your plate under the UV lamp and circle any UV active spots with your pencil. Although we won’t do this in 5.301, another popular non-destructive method is staining with iodine. (You might see this in your UROP.) 9. Visualize the TLC using a destructive method. This will be critical for compounds that are not UV-active. There are several varieties of stains that are very useful and will be available to you in 5.301. To use the stain, pick up the dried TLC plate with a pair of tweezers and dip it into the stain, making sure to cover the area from the baseline to the solvent front. Completely dry the back of the plate with a paper towel. Place on a hot plate and watch the development

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Figure: explains the very basic operation of column chromatography.

polar compounds off of the baseline. A good solvent system is one that moves all components of your mixture off the baseline, but does not put anything on the solvent front - Rf values between 0.15 and 0.85. This is not always possible, but should be your goal when running a TLC. (For column chromatography the correct solvent system should give an Rf between 0.2 and 0.3.) Now, which solvents to pick? Here is a list of some standard solvents and their relative polarity (from LLP):

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of the spots. Remove the TLC plate from the heat once the spots are visible and before the background color obscures the spots. 10. Revise your choice of solvent system based on the results of your initial TLC. Make the solvent system more polar if you want a larger Rf or make it less polar if you want to decrease the Rf. Also, if there is “streaking” of your compound on the plate -basically you see large streaks instead of sharp circles - your sample is probably too concentrated. Try diluting your sample and running the TLC again. If this doesn’t work, you will have to move to a different solvent system.

contain only a limited capacity. However, this method also yields rapid separation of mixtures. The mobile phase in gas chromatography is generally an inert gas. The stationary phase is generally an adsorbent or liquid distributed over the surface of a porous, inert support.

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11. Label your TLC, calculate the Rf for each spot and draw a picture of it in your notebook. 49

Liquid Chromatography:There are a variety of types of liquid chromatography. There is liquid adsorption chromatography in which an adsorbent is used. This method is used in largescale applications since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is analogous to gas-liquid chromatography. The three types that will be considered here fall under the category of modern liquid chromatography. They are reverse phase, high performance and size exclusion liquid chromatography, along with supercritical fluid chromatography. Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low polarity stationary phase which is chemically bonded to an inert solid such as silica. The separation is essentially an extraction operation and is useful for separating non-volatile components. High performance liquid chromatography (HPLC) is similar to reverse phase, only in this method, the process is conducted at a high velocity and pressure drop. The column is shorter and has a small diameter, but it is equivalent to possessing a large number of equilibrium stages. Size exclusion chromatography, also known as gel permeation or filtration chromatography does not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of separating large molecules from smaller ones. The larger molecules elute first since they cannot penetrate the pores. This method is common in protein separation and purification.

Figure: Left side of the picture shows the actual run of the samples on the TLC plate. The calculated migration values are drawn in the graph on the left. The sample is loaded on the lower line, which migrated according the polarity of the substance. For the spot ‘A’ the migration value is calculated by the densitometer trace formula.

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Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids, supercritical fluids have solubilities and densities have as large, and they have diffusivities and viscosities quite a bit larger. This type of chromatography has not yet been implemented on a large scale.

Gas Chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to carry the solute through the column. The most common detectors used in this type of chromatography are thermal conductivity and flame ionization detectors. There are three types of gas chromatography that will be discussed here: gas adsorption, gas-liquid and capillary gas chromatography. Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method is commonly used to separate mixtures of gases. Gas-liquid chromatography is a more common type of analytical gas chromatography. In this type of column, an inert porous solid is coated with a viscous liquid which acts as the stationary phase. Diatomaceous earth is the most common solid used. Solutes in the feed stream dissolve into the liquid phase and eventually vaporize. The separation is thus based on relative volatilities.

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Ion exchange chromatography: is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective. Since the resins are fairly inexpensive and high capacities can be used, this method of separation is applied early in the overall process.

Capillary gas chromatography is the most common analytical method. Glass or fused silica comprise the capillary walls which are coated with an absorbent or other solvent. Because of the small amount of stationary phase, the column can

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Here are few examples of affinity column chromatography: ENZYME + INHIBITOR <=> ENZYME-INHIBITOR COMPLEX ANTIBODY + ANTIGEN —> ANTIBODY-ANTIGEN PRECIPITATE LECTIN + CELL WALL ——> LECTIN-CELL-WALL COMPLEX

Figure: Shows the attraction of mobile anion phase towards the stationaly cation phase. Affinity Chromatography: An intriguing chromatographic technique based on the natural specificity of some biopolymers is affinity chromatography. There are a number of proteins and other biological macromolecules that complex with some other biological entity with a high degree of specificity. This fact is made use of in product recovery operations via the use of affinity chromatography. A very good example of purifying the enzymes is given in the diagrammatic form in Figure4. Suppose a certain biomolecule (a) is attached to a solid used to pack a chromatographic column. Now consider a molecule (b) in solution, which has a specific affinity for (a). It is but natural that (b) will want to get out of solution and bind to (a), right? It’s this attraction of (b) for (a) which is defined as the partition coefficient ‘K’. Now since ‘K’ for (b) is going to be much higher than that of any other proteins in solution, it will bind to the column while the rest of the complex solution will merely pass through the column with insignificant amounts of non-specific binding occuring.

Fig: Molecular diagram about the functioning of the affinity chromatography With the advent of Monoclonal Antibody production, which allows the synthesis of a single type of antibody with a very high specific binding constant to its corresponding antigen (a particular protein or other molecule), the preparation of affinity columns has become not only routine, but commercial. With such ‘immunosorbent’ column seperation, some important practical and theoretical differences arise compared to more conventional forms of chromatographic resolution. They are as follows: 1. The dominant cost in the process is the antibody needed to make the immunosorbent column. Generally speaking, this is much more costly than the antigen-containing broth itself. As a result, 2. A small column of repeated, high capacity use is required. 3. Elution of the adsorbed product requires breaking the antigen-antibody complex. Now this means that denaturing conditions must be employed. Since the antibodies themselves are proteins too, loss of some antibody binding affinity typically occurs, resulting in gradual loss of column capacity. 4. A first cycle on a new column gives poorer recovery than successive operations, apparently due to some irreversible binding. 5. A major economic goal in designing any affinity chromatography setup is determination of optimal elution buffer wash volumes and concentrations. Now let us look at a specific example where affinity chromatography is used, namely, in the purification of human leukocyte interferon made in E. coli.

Figure 4: Diagram of purification of enzyme of by affinity column chromatography

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Proteins synthesized in genetically engineered organisms and intended for injection into animals must be stringently purified. Pyrogens from E. coli, including the outer envelope

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lipopolysaccharide (LPS) must be removed or inactivated. Hence product recovery operations such as affinity chromatography are an important step in the manufacturing process. Given below is a schematic representation of the typical steps involved in processing human leukocyte interferon produced by recombinant DNA techniques. This will give you an idea of where exactly affinity chromatography is usually involved in the realm of bioprocessing. HUMAN LEUKOCYTE INTERFERON | E. coli EXTRACTION BY MECHANICAL BREAKAGE | POLYETHELYNEIMINE PRECIPITATION | AMMONIUM SULFATE PRECIPITATION OF SUPERNATENT | DIALYSIS OF PELLET | *IMMUNOADSORBENTCOLUMN (MONOCLONAL ANTIBODIES) | CATION EXCHANGE CELLULOSE CHROMATOGRAPHY Affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds. The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or “affinity tails”, that are inserted into the matrix can be genetically engineered to possess a specific affinity. In a process similar to ion exchange chromatography, the desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Fouling of the matrix can occur when a large number of impurities are present, therefore, this type of chromatography is usually implemented late in the process.

Presentation of Results Till now you have studied the basics of chromatography its types and the functioning and now you will be able to see the results in the form of graphs which is called as the chromatogram. Here a example of the chromatogram is. The figure below will help you to follow along in our discussion.

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Since the sample is separated in the column, different peaks on the chromatogram correspond to different components in the sample mixture. The chromatograms above show the results of separations of protein mixtures by ion exchange chromatography. The lettered peaks correspond to different proteins (A = ovalbumin, B = conalbumin, C = cytochrome c, D = lysozyme). The separation corresponding to the chromatogram on the left was performed at pH 5.85, while the one on the right was performed at pH 6.5. It is evident that operation conditions such as pH and temperature have a significant effect on the output. Analysis of the result:

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The level of complexity of the sample is indicated by the number of peaks which appear.

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Qualitative information about the sample composition is obtained by comparing peak positions with those of standards.

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Quantitative assessment of the relative concentrations of components is obtained from peak area comparisons.

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Column performance is indicated by comparison with standards.

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: 1. identification of a “target protein” which may have therapeutic value

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2. identification of the “target gene” — the gene responsible for encoding the target protein 3. isolation of the target gene 4. insertion of the target gene into a host cell (such as E. coli) which will both grow well, and continue to 5. produce the protein product encoded for by the target gene 6. separation of the target protein from the many other host cell proteins 7. large scale production of the target protein under controlled manufacturing conditions 8. large scale testing for efficacy as a medicine 9. 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).

Notes

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