CHROMATOGRAPHY

Name the carrier gases used in GLC. How is the choice made? (2) The choice of carrier gas depends on the type of detector that is used and the components that are to be determined. Carrier gases for chromatographs must be of high purity and chemically inert towards the sample e.g., helium (He), argon (Ar),nitrogen (N2), carbon dioxide (CO2) and hydrogen (H2). The carrier gas system can contain a molecular sieve to remove water or other impurities.

Give the principle of TCD detector used in GLC. (2) {2012} Thermal conductivity detectors (TCD) were one the earliest detectors developed for use with gas chromatography. The TCD works by measuring the change in carrier gas thermal conductivity caused by the presence of the sample, which has a different thermal conductivity from that of the carrier gas. Their design is relatively simple, and consists of an electrically heated source that is maintained at constant power. The temperature of the source depends upon the thermal conductivities of the surrounding gases. The source is usually a thin wire made of platinum or gold . The resistance within the wire depends upon temperature, which is dependent upon the thermal conductivity of the gas. TCDs usually employ two detectors, one of which is used as the reference for the carrier gas and the other which monitors the thermal conductivity of the carrier gas and sample mixture. Carrier gases such as helium and hydrogen has very high thermal conductivities so the addition of even a small amount of sample is readily detected. The advantages of TCDs are the ease and simplicity of use, the devices' broad application to inorganic and organic compounds, and the ability of the analyte to be collected after separation and detection. The greatest drawback of the TCD is the low sensitivity of the instrument in relation to other detection methods, in addition to flow rate and concentration dependency.

What are spacers? Why are they used in developing affinity matrices? (2) A ligand is directly attached to activated groups of the support. The macromolecules might encounter steric restrictions due to which they might not become adsorbed to the matrix. To obviate difficulty, it is usual to introduce a spacer between activated groups of the support and the ligand. This spacer is

known is the arm. Due to this, the ligand is projected at a distance from the matrix and the desired macromolecules will bind to the ligand without facing any steric hinderence. The spacer arms must possess two functional groups, one to react with the functional group of the matrix and the other to which the ligand could be attached. For e.g., hexamethylene diamine, 3, 3’ – diaminoprophylamine, 1,6 – diaminohexane are used in developing affinity matrices. What is RF value of compound? What is its significance? (2) {2006} The identification of a given compound may be made on the basis of the distance traversed by the solute relative to the distance moved by the solvent front. This ratio,which reflects the distribution coefficient of the given solute, is known as retardation factor (also known as relative flow), Rf and is constant for a given compound under standard conditions.

Significance: This value can be exploited to detect the known compound by matching its retardation factor value to those of the known compounds. What is adsorption isotherm? What is its significance in adsorption chromatography? (2) {2008} Adsorption is usually described through isotherms, that is, the amount of adsorbate on the adsorbent as a function of its pressure (if gas) or concentration (if liquid) at constant temperature. The quantity adsorbed is nearly always normalized by the mass of the adsorbent to allow comparison of different materials. Adsorption isotherms are classified into 5 types as discussed below Type I isotherms is that adsorption is limited to the completion of a single monolayer of adsorbate at the adsorbent surface. Type II isotherms do not exhibit a saturation limit as did Type I. This type of isotherm indicates an indefinite multi-layer formation after completion of the monolayer and is found in adsorbents with a wide distribution of pore sizes Type III isotherm is obtained when the amount of gas adsorbed increases without limit as its relative saturation approaches unity. Type IV isotherm is a variation of Type II, but with a finite multi-layer formation corresponding to complete filling of the capillaries Type V isotherm is similar variation of Type III obtained when water vapour is adsorbed on activated carbon at 100 0C. Significance: Adsorption isotherms are most commonly used to select the adsorbent or even the adsorption process as a unit operation for the adsorptive separation of gases. If the adsorption isotherm shape is Type I, II or IV, adsorption can be used to separate the adsorbate from the carrier gas. If it is Type III or V, adsorption will probably not be economical for the separation. Eg. Langmuir Isotherm is used for the adsorption of Acetic Acid on the surface of Norit A Activated Charcoal. The surface area of the Activated Charcoal available for adsorption of Acetic Acid will be determined from the Isotherm data and other assumptions.

What do you mean by isocratic elution (2) {2010} The process of continuous passage of suitable eluent (mobile phase) through packedcolumn separate the components of sample applied to the column is known as elution. Types of elution: Simple or isocratic elution. Stepwise or batch elution. Gradient elution. Isocratic elution: When a single solvent is used as an eluent during development, the process is known as isocratic elution. In this case the resolution is not satisfactorily obtained and the PH, ionic strength or polarity of phase is changed with respect to time. Affinity chromatography? (4) {2004, 2011} Introduction: Affinity chromatography is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Affinity chromatography combines the size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. Principle:

The molecule of interest will have a well known and defined property which can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in solution will not become trapped as they do not possess this property. The solid medium can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Properties of matrix:

The matrix should be inert. It should possess good flow properties. It should be chemically and mechanically stable at varies pH, ionic strength, and denaturating conditions employed for binding and elution. It should have minimum interaction with other molecule Ligand selection: The selected ligand should meet two most imported requirements. The ligand should interact strongly with desired macromolecule. The interaction must not be a very strong and if shoe it might to be damaged the desired macromolecules. The ligand to be bound should possess functional group that can be modified to form covalent linkage with the supporting matrix. Ligand attachment:

Activation of functional group of the matrix. Binding of the ligand to activated functional group of the matrix. For eg, CNBr activated polysaccharide supports which are freeze dried are commercially available. The arm: A ligand is directly attached to activated groups of the support the macromolecules might encounter steric restrictions due to which they might not become adsorbed to the matrix. To obviate difficulty, it is usual to introduce a spacer between activated groups of the support and the ligand. This spacer is known is the arm (68 carbon atoms). Due to this, the ligand is projected at a distance from the matrix and the desired macromolecules will bind to the ligand without facing any steric hindrance. These spacers have two functional groups such as, One to react with the functional groups of the matrix And other to which the ligand could be be attach easily Procedure: The procedure for affinity chromatography has similarities to other forms of liquid chromatography. The gel beads are swollen much in the same way as gel permeation before loading onto a column. The buffer chosen must be supplemented with any with any cofactors (metal ions) required for ligand –macromolecules interaction. The buffer can also possess a high ionic strength so as to minimize non-specific polyelectrolyte adsorption onto charged groups in the ligand. The sample is applied at the top of the column and the buffer flow started.

Once the macromolecule is bound, the column is eluted with more buffers to removenon-specifically bound unwanted molecules. The purified, bound component may now be eluted by taking recourse to either specific or non-specific elution. Specific elution: Specific or affinity elution is carried out by addition of compounds for which the ligand more affinity than it has for the desired macromolecule. Non-specific elution: Non-specific elution is carried out by changing either the pH or the ionic strength ofthe buffer. Change in any of these two parameters causes destabilization of the ligand – macromolecule link and causes the macromolecule to separate and elute of the column. Drawbacks: It is exceedingly costly. The other problem is that affinity chromatography cannot to be used as a first procedure. Applications: Affinity chromatography is not limited to proteins only; it is currently used to purify nucleic acids, and even whole cells and cell fragments. Isolation of messenger RNA from a crude RNA preparation is one of its often used special applications, In short, it may be said that all such biomolecules involved in specific interaction with other molecules can be purified with the help of affinity chromatography. Use of gel beads is another extension of affinity chromatography Immobilized enzymes is another extension of affinity chromatography they are known to be more thermally stable, less prone to protease digestion and easily stored as compared to the free enzyme. Explain the principle of operation of counter current distribution technique. Give a neat diagram of a single CCD unit? (4) {2004, 2005, 2010, 2012} Counter current distribution: a method for the separation and purification of (usually organic) substances that depends on the repetitive distribution of solute between two immiscible liquid phases in a series of vessels in which the phases are in contact. The process is continued until homogeneous substances are distributed in various sets of vessels in accordance with their distribution coefficient between the two liquid phases. The principle of counter current distribution is similar to that of chromatography; both procedures are used for analysis and purification of mixtures of similar compounds. Craig apparatus: the most widely used type of apparatus for carrying out counter current distribution experiments. The earlier version (1944) was constructed of metal and the later version (1949) was of modular design and constructed of glass. Procedure: CCC can be thought of as occurring in three stages: mixing, settling, and separation. Mixing of the phases is necessary so that the interface between them has a large area, and the analyte can move between the phases according to its partition coefficient.

A Craig apparatus consists of a series of glass tubes that are designed as 0 1 2 3… These tubes are arranged such that the lighter liquid phase can be transferred from one distribution tube to the next. The liquid-liquid extractions are taking place simultaneously in all tubes of the apparatus, which is usually driven electromechanically. The lower (heavier) phase of the two-phase solvent system (e.g. water) is the "stationary phase". The upper (lighter) phase (e.g. hexane) is the "mobile phase". In the beginning, tube 0 contains the mixture of substances to be separated in the heavier solvent and all the other tubes contain equal volumes of the lighter solvent phase. Next, the lighter solvent is added to tube 0, extraction (equilibration) takes place, and the phases are allowed to separate. The upper phase of tube 0 is then transferred to tube 1, fresh solvent is added to tube 0, and the phases are equilibrated again. The upper layers of tubes 0 and 1 are simultaneously transferred to tubes 1 and 2 respectively. This cycle is repeated to carry on the process through the other CCD elements. In this system, substances with a higher distribution ratio move faster compared than those with a lower distribution ratio.

Compare and contrast HPLC and FPLC with reference to applications in biochemical studies. (4) {2006, 2007} High Performance Liquid Chromatography or High Pressure Liquid Chromatography (HPLC) is a highly improved form of column chromatography. Components of HPLC: A solvent reservoir to store the mobile phase. High pressure pumps to push the mobile phase through the column. A device (injector) to inject the sample into the mobile phase. A column in which separation will take place. A detector used in detecting the concentration of the sample. Recorder to produce the chromatogram In Laboratory use Can be used to isolated and purify compounds for further use.

Can be used to identify the presence of specific compounds in a sample. Can be used to determine the concentration of a specific compound in a sample. Can be used to perform chemical separations. Enantiomers Biomolecules Other uses include: Forensics: analysis of explosives, drugs, fibers, etc. Proteomics: can be used to separate and purify protein samples Can separate & purify other biomolecules such as: carbohydrates, lipids, nucleic acids, pigments, proteins, steroids Study of disease: can be used to measure the presence & abundance of specific bimolecules correlating to disease manifestation. Pharmaceutical Research: all areas including early identification of clinically relevant molecules to large-scale processing and purification. Disadvantages: The high resolution and impressive versatility of HPLC have made it a widespread technique in biochemistry, analytical, and organic chemistry and many other areas of scientific research. However the technique itself does not lend itself readily available for the purification of proteins.This is because of two reasons Some modes of HPLC, commonly performed RPC require the use of organic solvents which will denature the proteins. Although large capacity of HPLC are available, they are very expensive and small capacity analytical columns are much available. FPLC: In 1982 Pharmacia introduced a new chromatographic method called Fast Protein Liquid Chromatography (FPLC) Its Stainless steel components of HPLC system are replaced with glass and plastic since; Stainless steel was thought to denature proteins And also since many ion-exchange separations of proteins involve salt gradients; thought that these conditions could results in attack of stainless steel systems. FPLC can also be used to separate other biologically active molecules, such as nucleic acid. FPLC is an intermediate between classical column chromatography and HPLC. FPLC pump delivers a solvent flow rate in the range of 1-499ml/hr HPLC pump= 0.010-10ml/min FPLC operating pressure: 0-40 bar. HPLC= 1-400bar Since lower pressures are used in FPLC than in HPLC, a wider range of column supports are possible. FPLC components: A standard FPLC consist of one or two high-precision pumps, a control unit, a column, a detection system.

Application: 1. The major use of FPLC is in the field of proteomics 2. FPLC carried as ion exchange chromatography: protein can be sequenced by determining the amino acids based on their charge on side chains (pKa) by altering the pH. 3. FPLC carried as affinity chromatography: different proteins can be separated in the form of hormones and receptors, enzymes using substrates. 4. FPLC carried as gel chromatography: protein mixtures can be separated based on their molecular size and molecular weight. 5. It can be used to study protein structure and functions. 6. Used to study post-translational modifications. Also used in studying protein-protein interactions. Paper chromatography (4) {2005, 2008} The first detailed description of chromatography is generally credited to Michael Tswett, a Russian biochemist, who separation chlorophyll from a mixture of plant pigments in 1906. Paper chromatography is a common type of plane chromatography which has 2 phases. One of these phases is stationary, while the other is mobile: the mobile phase either moves over surface or percolates through the interstices of the stationary phase. The sample mixture, introduced into mobile phase undergoes repeated interactions (partitions) between the stationary and mobile phases while being carried through then mobile phase. The partition principle: If two phases are in contact with one another and if both the phases contain solute,the solute will distribute itself in a definite ratio between the 2 phases. This is called as partition. The concentration of compound in the stationary phase to that of the concentration of the compound in mobile phase is called as partition co-efficient. Nature of the paper: The paper commonly used consists of highly purified cellulose. Cellulose, a homo polysaccharide of glucose, contains several thousand an hydro-glucose units linked through oxygen atoms. The paper also contains impurities through inorganic substances, adsorbed salts and mineral matter, which gets deposited on the paper while it is being processed. These impurities may be removed by washing the paper with 0.1HCL and drying it before chromatography is carried out. Cellulose fiber in the paper holds moisture tightly through formation of hydrogen bonds. Apparatus and paper development:

The apparatus required for paper chromatography consists of a support for paper, a solvent trough, and an airtight chamber in which the chromatogram is developed. The sample is applied to the paper as a small spot. This is done before dipping the paper into the eluting solvent. Device used for sampling are platinum loop, capillary tube, or a micropipette. Types of development: Ascending chromatography Descending chromatography Radial chromatography 2-dimensional chromatography Choice of solvent system: The mobile phase is usually a mixture of various solvents such as alcohols, acids, esters, ketones, phenols, amines and hydrocarbons etc… The solvents are selected in such a way that the resolution of sample components is satisfactory. Ideally, the solvent should be chosen that the 2 phases are immiscible. The sample components should have differing solubilities in the 2 phases. Such a choice would lead to maximum separation. Desirable characteristics that a solvent should possess are listed below Partition co-efficient of substances to be analyzed should range from 1-100 in favourof the aqueous phase. The solvent should be removable from the paper and to this effect its boiling point should ideally be less than 200cls. The solvent should be stable. It should not become oxidized when spread over the paper. Some solvents for example phenolic solvents, react with small quantities of copper present in the paper and get oxidized to produce brown or black tarry material. This material then migrates with the solvent front and distorts the chromatogram. Detection: There are various methods of detection available. If the sample components are colored, the analysis becomes simple as the distinctive color itself identifies the component. When the components are color less, they can be imparted by spraying the paper with the color producing reagents. Eg. Ninhydrin reagent spread on the paper reacts amines and amino acids to form a blue or purple color. Other methods of detection are UV and Infra red absorption Fluorescence and Radio activity

Retardation factor: The identification of the given compound may be made on the basis of the distance traversed by the solute related to the distance moved by the solvent front. This ratio which reflects distribution co-efficient of the given solute is known as the retardation factor (Rf) and is constant for a given compound under standard condition. Applications: The technique of paper chromatography has revolutionized biochemistry where difficult analyses with vanishingly small sample volumes are legion. The control of purity of pharmaceuticals, the detection of adulterants and contaminates in fruits and drinks, the study of ripening and fermentation, the detection of drugs and dopes in animals and humans, the analysis cosmetics and to top it all, the analyses of the reaction mixtures in biochemical labs are all performed routinely with paper chromatography technique. Use of affinity chromatography in isolation of mRNA: In molecular biologists frequently have to isolate mRNA from a total RNA preparation which contains other types of RNA, viz., rRNA and tRNA. Affinity chromatography is routinely used for this purpose. Oligo dt (many units of deoxythymidyl acid) is mobilized on an agarose matrix. When total RNa preparation is allowed to percolate through this column, only mRNA molecules have a poly A tail at their 3’-ends. These tails possibly bind to the oligo dT molecules immobilized on the matrix. Other RNA molecules do not have such poly A tails. After other RNA molecule eluted out, mRNA molecules bound to the matrix are eluted by changing the wash condition. A few years back a company called stratagene came up with what is known as a push column. The oligo dT attached matrix comes packed in a 1 ml syringe. This syringe is the column. The kit provides the eluting buffers. Total RNA preparation is layered at the top of the syringe column. After this one fixes the plunger at the top of the syringe column and pushes. The liquid is coming out of the syringe contains the RNA molecules other than mRNA. The column is washed twice again by filling syringe with buffer and pushing the plunger. After this eluting buffer is dispensed at the top of the syringe and the plunger is pushed again. The eluting liquid contains mRNA which can be collected. The entire procedure takes barely 10 minutes. All DNA binding proteins which show sequence specificity can be purified by this technique. Combine cytoplasmic RNAs and oligo (dT) matrix under hybridization conditions

Poly-A tail of mRNA binds to oligo (dT) matrix

rRNA and tRNA is washed away

Purified mRNA is eluted from the oligo (dT) matrix in water or low-salt buffer

What is chromatofocussing? Explain the operational procedure of this technique?(6) {2003, 2008, 2010, 2011} • Chromatofocusing is a versatile, high-resolution chromatographic technique suitable for separating protein that allows resolution of single proteins and other

ampholytes from a complex mixture according to differences in their isoelectric point. • Chromatofocusing utilizes ion exchange resins and is typically performed on fast protein liquid chromatography (FPLC) or similar equipment capable of producing continuous buffer gradients. • In contrast to typical ion exchange chromatography, where bound molecules are eluted from the resin by increasing the ionic strength of the buffer environment, chromate focusing elutes bound species by altering the pH of the buffer. • This changes the net surface charge of bound molecules, altering their avidity for the resin. • As the changing pH of the buffer system traverses the pI of a given molecule, that molecule will elute from the resin as it will no longer possess a net surface charge. • Proteins elute from a CF column in descending order of iso electric point. • Chromatofocusing is a powerful purification technique with respect to proteins as it can resolve very similar species only differing by 0.02 pH units. • The first step is selecting a medium, start buffer, and elution buffer for chromatofocusing which is dependent on appropriate pH gradient range on the basis of the iso electric point of the target protein. • After the pH gradient range has been selected, the appropriate CF gel medium and buffers are selected. • Finally the amount of buffer required is determined and the amount of gel required for the application is calculated. • If the iso electric point of the protein is known, the pH range of the CF experiment is chosen so that the target protein will elute after one-third to one-half of the pH gradient. • If the isoelectric point of the target protein is unknown, it may be determined by isoelectric focusing or by the test tube method. As bicarbonate ions and atmospheric carbon dioxide can cause fluctuations in the pH gradient, all buffers must be degassed before use. • The amount of gel required for a chromatofocusing run depends on the amount of sample, the nature of the sample and contaminants, and the degree of resolution required. Separation of proteins by chromatofocusing 1. In this procedure, the CF column is equilibrated in CF start buffer at a pH higher than that of the CF elution buffer. 2. As the lower-pH CF elution buffer moves through the column, the ion-exchange groups of the medium in the column are titrated by the CF elution buffer, forming a descending linear pH gradient in the column. 3. In a case where the isoelectric point (pI) of a protein is below the pH at which the column is equilibrated, the protein will have a net negative charge at the beginning of the experiment and therefore will bind to the anion-exchange medium in the CF column. 4. As the descending pH gradient slowly forms in the column, the pH at the position where the protein is bound will eventually drop below the pI of the protein, and the net charge on the protein will become positive. 5. The positively charged protein will then desorb from the anion-exchange medium and be carried through the column in the mobile phase.

6. As the desorbed and now positively charged protein advances through the column in the mobile phase, it will pass through the gradient, in which the pH in the column is increasing. 7. When the pH in the column finally exceeds the pI of the protein, the net charge of the protein again becomes negative. 8. Hence the protein is again adsorbed to the anion-exchange medium. 9. The net effect, as the protein adsorbs and desorbs many times, is a focusing effect which results in zone sharpening, sample concentration, and very high resolution. Advantages: Decreased expense due to the use of common buffer components. Ease of adjusting the slope of the pH gradient. Disadvantages: Some proteins will aggregate when present at relatively high concentrations and carry no net surface charge. This can cause blockage of the resin, which is highly problematic when using sealed columns of ion exchange resin on FPLC equipment, resulting in pressure buildup and possible equipment failure. Discuss the principle of ion-exchange chromatography & explain how proteins are fractionated on an ion exchange column? (6) {2003, 2004} Ion exchange chromatography is defined as the reversible exchange of ions in solution with ions electro statically bound to inert support medium. The governing factor in ion exchange reactions is the electrostatic force of attraction, which in turn depends mainly on the relative charge, the radius of hydrated ions and the degree of non bonding interactions. Ion exchange separations are carried out usually in columns packed with an ion exchanger. Ion exchanger is divided into two groups: Anion exchangers Cation exchangers Ions bound electrostatically to the exchanger are referred to as counter ions. Principle: The value of this technique lies in the fact that some compounds are electrostatically bound to ion exchanger where as the others are not. The exchanger is prepared in a way that it is fully charged. The sample containing the ionic species to be separated is allowed to percolate through the exchanger for such a length of time as will be sufficient for the following equilibrium to be achieved. E- Y+ +X+ ↔ E-X+ + Y+ where E- is the charged cation exchanger and Y+ is the counter ion of the opposite charge associated with the exchanger matrix. X+ is the charged molecule (bearing charge similar to the counter ion) in the sample to be separated. This molecule can now exchange sites with the counter ion as shown in the above relationship. The neutral and anionic molecules will not bind at all. Once the exchange of counter ion with the sample ion has been achieved, the rest of the uncharged and like charged species can be washed out of the column. Bound ions, X+ can now be eluted either by percolating the medium with increasing concentrations of Y+ (this

increases the possibility that Y+ will replace X+ in the above stated equilibrium because the former is more in concentration), or by increasing the pH of the solvent and hence converting X+ to an uncharged specie. Concentration of Y+ required to elute X+ will depend upon the quantity of charge possessed by X+. The greater the charge possessed by X+, the higher the concentration of Y+ required eluting it. In the second case, when the pH is being changed, the higher the pK of X+, the higher will be the pH required to elute it.

Procedure: The principles discussed above also apply to macromolecules such as proteins and nucleic acids, which are capable of possessing both positive and negative charges. The amphoteric nature of macromolecules, particularly proteins, can be exploited for purification purposes by using ion exchange chromatography. Here the protein mixture given is taken as the sample (protein A pI -4.0, protein B pI -5.5, protein C pI -6.7. This mixture is mixed with a suitable buffer with pH 5, at which the protein A will possess a net negative charge and protein B and C gains a net positive charge. When this is runned through a cationic exchanger, protein B and C gets bound to the column where as protein A gets eluted and it is collected. The bound B and C are eluted by using stronger cation containing mobile phase. Now the change the pH of the solution to 6 by using a suitable buffer, the protein B will gain a net negative charge and protein C will gain a net positive charge When this is runned through an anionic exchange column, the protein B will bind to the column where as the protein C will get eluted with the buffer and is collected. The bound protein B can be removed from the anionic exchanger by using stronger anion containing mobile phase. Thus the 3 proteins are separated using ion exchange chromatography.

Give the principle of elution of protein from cation exchangers? Two main groups of materials used to prepare ion exchange resin in ion exchange chromatography are polystyrene and cellulose. Selection of one or the other type of resin is done on the basis of compounds being separated. Polystyrene resin is prepared by polymerization reaction of styrene and divinely benzene. A higher concentration of divinely benzene produces higher cross-linkages. Increasing the cross-linkages increases the rigidity, reduces swelling, reduces porosity and reduces the solubility of the polymeric structure. Acidic cationic exchangers: sulphonic acid cationicexchangers are strong and

carboxylate cationic exchangers are weak. Strong anion exchangers are with tertiary amine and weak are those with secondary amines. Cellulose obtained in a highly pure state from common raw materials as cotton, softwood and hardwood. Carboxymethylcellulose is weakly acidic cationic exchanger. Diethylaminoethyl celluloseis is weakly basic anionic exchanger. Sulphoethyl is a strong cationic cellulose exchanger. Guanidoethyl is a strong anionic cellulose exchanger. Proteins of low molecular weight can be fractionized using polystyrene resins and those of high molecular weight using cellulose resins. Principle of elution: The elution of the bound sample is eluted by gradient elution processes where the eluting solvent is changed with time, either by gradually mixing a second solvent of greater eluting power with the first, less powerful solvent, or by a gradual change in pH or other property. Technique: For instance the sample of interest to be separated from the mixture is positively charged protein. Then a cationic exchanger like diethylaminoethyl is used as column. The sample is mixed with a suitable buffer such that the protein of interest alone binds to the column. Now this protein of interest can be eluted out by 2 methods. The first method is by changing the pH of the buffer (mobile phase) i.e pH greater than the protein pI value, so that the protein is now negatively charged and no longer binds to the column and thus elutes out. In the second method the mobile phase with stronger cation are added which replaces the bound positive protein of interest thus eluting it out. Explain the working of RP- HPLC and comment on the detection systems employed in HPLC (6) {2010} Reverse phase Chromatography (RP-HPLC) is useful for separating non polar solutes, and even weakly ionic substances. Here the polarity of the compounds used, in stationary phase and mobile phase is opposite to that of the normal phase. Water, an extremely polar solvent becomes the weakest eluent here. Methanol and acetonitrile are stronger eluent than water. Since, the non-polar substances are squeezed out of the polar phase. Therefore, the driving force for retention of a component is not its interaction with the stationary phase but the effect of the mobile phase in forcing the component onto the hydrocarbon bonded stationary phase. A gradient elution is carried out for sample containing many mixtures of variable polarity Stationary phase: Non-polar or hydrophobic Non-polar organic groups are covalently attached to the silica stationary particles. Most common attachment is a long-chain n-C18 hydrocarbon Octadecyl silyl group

(ODS) Mobile phase: Polar liquid or mixture of liquids Polar analytes will spend more time in the polar mobile phase. Will elute quicker than non-polar analytes HPLC detection system Major HPLC detectors: a. Refractive Index (RI) Detector b. Evaporative Light Scattering Detector (ELSD) c. UV/VIS Absorption Detectors d. The Fluorescence Detector a. Refractive index(RI) detector Results in the bending of light as it passes to another material of different density. Refractive index = how much the light is bent The presence of analyte molecules in the mobile phase will generally change its RI by an amount almost linearly proportional to its concentrations. RI = general Responds to the presence of all solutes in the mobile phase. Reference= mobile phase Sample= column effluent Detector measures the differences between the RI of the reference and the sample.

b. Evaporative Light Scattering Detector (ELSD) Analyte particles don’t scatter light when dissolved in a liquid mobile phase. Three steps: 1) Nebulize the mobile phase effluent into droplets. Passes through a needle and mixes with hydrogen gas. 2) Evaporate each of these droplets. Leaves behind a small particle of nonvolatile analyte 3) Light scattering Sample particles pass through a cell and scatter light from a laser beam which is detected and generates a signal.

c. UV/VIS Absorption detectors Different compounds will absorb different amounts of light in the UV and visible regions.A beam of UV light is shined through the analyte after it is eluted from the column.A detector is positioned on the opposite side which can measure how much light is absorbed and transmitted. The amount of light absorbed will depend on the amount of the compound that is passing through the beam.

d. Fluorescence detector Measure the ability of a compound to absorb then re-emit light at given wavelengths Some compounds will absorb specific wavelengths of light which, raising it to a higher energy state. When the compound returns to its ground state, it will release a specific wavelength of light which can be detected. Not all compounds can fluorescence / more selective than UV/VIS detection.

Explain how you would use the molecular sieve chromatography to determine the sub-unit composition of a protein? (6) {2007, 2009} Molecular sieve chromatography is a type of column chromatography in which separation is dependent upon molecular size. This method is also known as gel filtration, gel permeation or molecular exclusion chromatography. Principle: A column of gel beads or porous glass granules is allowed to attain equilibrium with a solvent suitable for molecules to be separated. On placing a mixture of molecule with different size from the top of column, the larger molecule pass through the interstitial space between the gels and comes out first without entering the pores and takes longer time due to retardation to elute out.

For a given type of gel, the distribution of a solute particle between the inner and

outer solvent is defined by distribution coefficient Kd.

Kd = 0, for large molecule (solute), Kd = 1 for smaller molecule and Kd value for intermediate molecule lies between 0 to 1. The effluent volume Ve is dependent on three variables Ve = Vo + Kd.Vi Vo = volume of the outer solvent or void volume Vi = volume of solvent inside gel or inner solvent Gel selection and characteristics The selection of gel depends upon the size and properties of sample and molecule of interest. Required characteristics of gel are It should be chemically inert It should have least ionic group It should have wide choice of pore and particle size It should have uniform particle and pore size It should have high mechanical rigidity Types of gels: Some of the commonly used gel are Dextran: It is polysaccharide of glucose produced by fermentation of sucrose by leuconostoc mesenteroids. Trade name – sephadex Polyacrylamide: It is produced by polymerizing acrylamide into bead form. Trade name – biogel. Agarose: It is made up of D-galactose and 3,6 – anhydro, 1-galactose. Trade name –sepharose Separation of protein: eg. Hemoglobin Hemoglobin – each hemoglobin tetramer consist of 2 alpha and 2 beta globin subunit. Each alpha globin subunit consist of 141 amino acids and each beta globin subunit consist of 146 amino acid and both slightly differ in their molecular weight Technique: Gel permeation chromatography can be performed by two technique Column chromatographic technique Thin layer gel chromatography The column is filled with gel filtration matrix (white gel) and is equilibrated in the mobile phase (phosphate buffered saline).

The sample hemoglobin first denatured using a suitable denaturing agents or chemical like 6M urea, 6M guanidine hydrochloride, beta-mercaptoethanol, etc. Now the sample contains two different subunits denatured with a slight difference in molecular weight. On running through the column the slightly larger beta subunit with molecular weight 15,867 D comes out first. Alpha subunit with molecular weight comparatively less comes later and eluted out and measured. The elution volume is calculated using the formula Ve=Vo+KdVt The elution volume of a number of proteins like gamma-globulin, transferring, serum albumin, ovalbumin, myoglobin and cytochrome C of known molecular weights is obtained by similar procedure. Using this known values the calibration curves are plotted taking log molecular weight vs. elution volume. The position of elution volume of denatured hemoglobin on such a plot gives the molecular weight of 2 subunits alpha and beta.

Similarly proteins like urease having three subunits i.e., alpha (65 KD), beta (21 KD) and gamma (12 KD). Here the alpha subunit (large) will elute out first the beta subunit (intermediate) will take longer time to elute out and the gamma subunit (small) will take the longest time to elute out