Analytical Techniques in Biochemistry and Molecular Biology

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The various chromatographic techniques have also been classified into the following types depending upon the forces or the interacting phenomenon between the solute molecules and the stationary phase: a b c d e Partition chromatography Adsorption chromatography Ion-exchange chromatography Molecular sieve chromatography Affinity chromatography The different chromatographic techniques are described in detail. This filter paper is then kept in an atmosphere already saturated with water vapours to form a thin film of water around cellulose fibres of the paper, which then acts as a stationary phase.

An appropriate solvent system, which functions as a mobile phase, is then allowed to flow over the sample spot. On coming in contact with mobile phase, the various components of the sample get partitioned between the stationary and the mobile phases. Those constituents having a higher affinity for the stationary phase move less rapidly as compared with those having higher affinity for the mobile phase. Evidently the components will get well separated from each other if their Kd values are sufficiently different.

Satisfactory separation of components of interest can hence be achieved by judicious selection of the mobile phase. Depending upon the direction of flow of mobile phase, the two commonly used systems are 1. Ascending paper chromatography 2. Descending paper chromatography In the ascending method, the solvent is kept at the base of the chamber and the edge of the paper where the sample has been applied is immersed in the solvent taking care that the sample spots do not get dipped in the solvent but remain just above the surface of the solvent.

The solvent moves up or ascends the paper by capillary action and the separation of different components occurs on the basis of differences in their partition coefficients. In the descending method, the paper is hung in such a way that the side where the sample has been spotted dips in a trough, which is fitted at the top of the chamber, and contains the mobile phase and this solvent travels down the paper under the force of gravity. The location of the compounds under investigation is carried out by making use of their specific chemical, physical or biological property.

For instance, if these components form coloured complex or product with a particular reagent, they can then be conveniently located by spraying the chromatogram with that reagent. In case the compounds absorb UV light or show UV fluorescence, the paper can be examined under strong UV light in darkness. The UV absorbing compounds would appear as dark spots while UV fluorescent compounds would show a characteristic fluorescence under UV light.

If in metabolic studies, a radioactive precursor has been used, then the products derived from it can be detected from the radioactive zones or spots on the chromatogram Fig. Modifications for Development of Chromatograms Sometimes a single run with one solvent system in a single dimension is not sufficient to obtain satisfactory separation of the components. A number of modifications has been employed to achieve better resolution of such overlapping or very closely located compounds. After air drying, the paper is again developed in a second solvent system in a direction perpendicular to the previous run.

The compounds having similar Rf values in first solvent system might have different mobilities in the second solvent system and hence they get separated. However, the limitation of bidimensional chromatography is that only one sample can be spotted or applied for analysis on each filter paper. Thus, the stationary phase is hydrophilic, whereas the mobile phase is hydrophobic in nature. For separation of hydrophobic substances like fatty acids, the filter paper is treated with lipophilic compounds like silicon grease, kerosene oil, paraffin, Vaseline, etc.

The chromatogram is air-dried between the successive developments. This mode of development of chromatogram can be used advantageously when the sample contains mixture of components some of which migrate quite fast and get well separated from each other and a group of other components which remain 46 4 Techniques in Biochemical Evaluation clustered near origin due to their low and closely similar Rf values.

These slow-moving components get further apart with each successive run resulting in their better separation. In this case the chromatogram is developed in the same solvent system in the same direction continuously for a long time even after the solvent has started dripping down from the other end of paper. Care, however, must be taken that the fastest moving compound does not get eluted out of the paper. This approach is useful for achieving better separation of compounds having very low and similar mobilities.

Evidently Rf value cannot be calculated as the solvent front cannot be calculated and determined due to overflow of the solvent. A thin layer of the stationary phase is laid over this inert support. The layer may be as thin as mm for analytical separations and as thick as 2—5 mm for preparative separations. A binding agent such as calcium sulfate or gypsum may be incorporated into the chromatographic media to facilitate firm adhesion of adsorbent to the plate.

For development, the plate on which the sample spots have been applied is placed in an air tight glass jar containing the solvent. The location and identification of separated components is carried out in the same manner as in the case of paper chromatography. Also like paper chromatography, TLC is amenable to two-dimensional mode of development. In TLC, depending upon the nature of the chromatographic media used, the separation can be achieved by partitioning, adsorption, ion exchange or molecular sieving phenomenon and the separation can be achieved within an hour.

This technique is relatively more sensitive since lower concentrations of compounds in the mixture can be successfully separated and detected. Corrosive agents like H2SO4 and high temperatures can be used to locate the separated compound which is not possible in paper chromatography Fig. Usually, commercially available columns have a porous sintered plate fused at their base which prevents the stationary phase from flowing out of the column.

This sintered base is positioned as near the base as possible in order to minimize the dead space to reduce the chances of post-column mixing of the separated compounds. Alternatively, a simple glass burette with a plug of glass wool at the base can be used as a column. At the base there is a small capillary tubing through which the effluent from the column flows into the test tubes in which fractions are collected. At the top of the column a solvent reservoir with a delivery system is fitted. The stationary phases used in column chromatography are water insoluble, porous, solid particles, and the resolution of sample components occurs depending upon the principle underlying the separation phenomenon.

The flow rate and resolution characteristics are influenced by the size and shape of the stationary phase. Large and coarse particles have higher flow rate but give comparatively poor resolution while finer particles with large surface to volume ratio have slower flow rate but greater resolution efficiency. Generally, particles of — mesh are used for routine analysis but for high resolution the smaller particles of — mesh are used.

For fractionation and separation of components, the sample is loaded on top of the column and eluted with an appropriate buffer. The effluent emerging from base of 48 4 Techniques in Biochemical Evaluation the column is collected in the form of fractions of fixed volume or fixed time in individual test tubes either using an automatic fraction collector or manually. The collected fractions are then analysed for the presence of the desired substance. The detection technique depends on physical, chemical or biological property of the compound.

Presence of coloured compounds can be identified simply from visual observation but for colourless compounds, alternative methods of detection are employed. They can be either the colour reactions or may be based on its unique physical property such as ultraviolet absorption, fluorescence, refractive index, etc. Column chromatographic techniques have been classified on the basis of the nature of the interactions occurring between solutes and the stationary phase which ultimately results in their separation.

Various types of column chromatography are given below. Different compounds bind with varying strengths and hence can selectively be desorbed. For good resolution, selection of right type of the adsorbent and the eluent or mobile phase is essential. Some of the commonly used adsorbents include charcoal, silica, alumina, hydroxyaptite, etc.

Eluent influences quality of separation since polarity of the mobile phase influences the adsorption considerably. Non-polar solvents favour maximum adsorption which decreases with increase in polarity of the solvent. In general, the polar solvents are preferred for the substances having polar or hydrophilic groups and nonpolar solvents for substances having hydrophobic or non-polar groups.

For example, alcoholic solvents containing —OH group substances ; acetone or ether for substances with carbonyl groups and hydrocarbons such as toluene or hexane for non-polar substances. For gradient elution, mixture of the polar and non-polar solvents of different ratios can be used to obtain eluent of varying polarities. Gel Filtration Size Exclusion Column Chromatography The chromatographic media used in this technique are porous, polymeric organic compounds with molecular sieving properties.

These are cross-linked polymers 4. The size of the pore is determined by degree of cross-linking of polymeric chains. Different solutes in a mixture get separated on the basis of their molecular size and shape during their passage through a column packed with the swollen gel particles. The large molecules in sample are unable to penetrate through the pores into the gel and thus remain excluded while the small molecules enter into the gel beads. Obviously the volume of solvent accessible to large molecules is very much less Vo , whereas small molecules, which can freely penetrate into the gel have access to solvent inside Vo the spherical beads.

This can effectively be achieved by passing preparation through a column of Sephadex G or G The high-molecular weight macromolecules like proteins and nucleic acids remain excluded from the gel particles and are recovered from the column immediately after void volume, while the movement of low-molecular weight compounds, such as salts, is considerably impeded due to their entry into the beads.

This method of desalting is relatively faster than the process of dialysis and hence is particularly suitable for desalting of labile compounds.

The high-molecular weight substances, however, remain in solution due to their complete exclusion. Because of reduced volume, the concentration of the higher-molecular weight substances in solution increases without any effect on pH and ionic strength. The added beads are then removed from the solution by centrifugation. Generally, for purification or fractionation purposes, the gel selected is such that the compound to be purified falls within the fraction range of the gel.

Larger the molecular weight of a compound, lesser will be its elution volume. In fact, this method has been exploited for determination of molecular weight of 50 4 Techniques in Biochemical Evaluation 1. A plot of log molecular weight vs. Hence, by using proteins of known molecular weights, a calibration curve can first be prepared and form the elution volume or Kd value, the molecular weight of protein of interest can readily be obtained. This method of molecular weight determination is simple, inexpensive and does not require homogeneously purified preparation of the protein or compound of interest and is non-destructive Figs.

Ion Exchange Chromatography Ion exchange chromatography is a type of adsorption chromatography in which retention of a solute occurs due to its reversible electrostatic interaction with the oppositely charged groups on an ion exchanger. Hence, this technique is useful for separation of compounds which bear a net electric charge such as 4. Ion exchangers are prepared from either certain synthetic resins which are insoluble porous organic molecules or naturally occurring biopolymers such as cellulose to which various groups known as fixed ions are covalently attached.

These fixed ions are balanced by equal and oppositely charged ions from the solution referred to as counter ions. Depending upon the nature of the counter ions, these ion exchangers are of two types: cation exchangers in which the counter ions are cationic or positively charged and anion exchangers which have negatively charged counter ions. Counter ions are mobile and can be easily exchanged by other similarly charged molecules in the sample. Nature of the resin matrix remains unchanged during this exchange process. Generally, resin-based ion exchangers are used for separation of low-molecular weight biomolecules and cellulosic ion exchangers are more suitable for isolation of marcromolecules such as proteins and nucleic acids.

Ion exchangers have to be precycled to get an appropriate charge and their complete swelling. In case of anion exchanger, it is treated first with alkali and then with acid and finally washed with water till it is neutral. Conversely, cation exchangers is first given the acid treatment and then alkali treatment and finally washed with water till neutral. After precycling, the ion exchanger is packed into a column and is equilibrated with the counter ion by passing 2—3 bed volumes of the starting buffer of a required pH.

Affinity Chromatography Purification by affinity chromatography is different from all other forms of chromatography in the sense that this technique does not make use of the differences in the physical properties like solubility, adsorption, molecular weight and ionic properties of the molecules to be separated, rather it exploits one of the unique and fundamental properties of biopolymers, i. Therefore, affinity chromatography is a type of adsorption chromatography in which the substance to be isolated is specifically and reversibly bound to a complementary binding substance ligand immobilised on an insoluble chromatographic bed material matrix.

The other substances in the mixture remain unbound and are washed away while the substance of interest the bound substance is subsequently recovered by displacement from the ligand either by specific affinity elution or by non-specific change in pH or ionic concentration elution. Purification by affinity chromatography is often of the order of several thousandfold recoveries of the material which are generally very high. The ligand must exhibit specific and reversible binding with the substance to be purified.

It must have chemically reactive functional groups which allow it to be attached to the matrix without destroying its binding activity with the substance of interest. It is possible to select a ligand which displays absolute specificity and binds exclusively with one particular compound only.

It is also possible to select a ligand which displays group specificity, e. Antigen—antibody interactions can be exploited for the purification of either of these. Similarly, for nucleic acid purification a complementary base sequence or histones and for lectins, cell surface receptors or polysaccharides can be successfully employed as ligands. An ideal chromatographic bed material matrix to which the ligand is covalently bound must possess the following attributes: 1.

It must possess suitable groups to which ligand can be covalently coupled. Many groups may be introduced into matrix to couple ligands. They may be nucleophilic as NH2, SH, OH or electrophilic such as activated acid chlorides, carbonyls activated by carbodiimide, iothiocyanate or diazonium salts. It must remain unchanged under the conditions of attachment of ligand. During the binding of the macromolecule and its subsequent displacement from ligand, it must retain its physical and chemical stability. It must not exhibit non-specific adsorption.

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It should have an open pore structure. The most commonly used matrices are cross-linked dextrans e. Sephacryl , agarose e. Sepharose , polyacrylamide gel Bio gel P , polystyrene, cellulose, porous glass and silica. Sepharose is a bead-form of agarose gel which displays virtually all features required of a successful matrix for immobilizing biologically active molecules.

The hydroxyl groups on the sugar residues can be easily derivatized for covalent attachment of a ligand. Using Spacer Arm During Affinity Separations Generally, it is observed that the active site of the biological substance, e. The length of the spacer arm is one of the important factors. If it is too short, the arm is ineffective and the ligand fails to bind the substance in the sample.

If it is too long, non-specific effects become pronounced and reduce the selectivity of separation. The optimum length of the spacer arm is generally 6—10 C-atoms or their equivalent. Chemical nature of the spacer arm e. There are two approaches which are generally followed for coupling of the ligand. In one case, spacer arm is first linked to the matrix followed by the coupling of the ligand, whereas the second approach involves the binding of the spacer arm to the ligand which is then linked to the matrix.

It is the first approach which is more convenient and is preferred over the second one Fig. For attachment of the ligand with the matrix, the matrix is given preliminary treatment with cyanogens bromide at pH This causes activation of the matrix and the molecules containing primary amino groups could then easily be coupled to CNBr activated matrices. Different spacer arms including 1,6-diamino hexane, 6-amino hexanoic acid, and 1,4-bis epoxy-propoxy butane have been used to which the ligand can be attached by conventional organo synthetic procedures involving the use of succinic anhydride and a water soluble carbodiimide.

A number of supports of agarose, dextran and polyacrylamide type are commercially available with a variety of spacer arms and ligands. Applications of Affinity Chromatography Affinity chromatography occupies a unique place in separation technology since it is the only technique which enables purification of almost any biomolecule on the basis of its biological function. The principle of affinity chromatography has been extended to purify a large number of enzymes, other proteins including immunoglobulins and receptor proteins and nucleic acids and so has contributed 54 4 Techniques in Biochemical Evaluation considerably to recent developments in the field of molecular biology.

For the purification of proteins involved in nucleic acid metabolism, immobilized nucleotides are quite useful. The technique of affinity chromatography has also been successfully employed for the separation of a mixture of cells into homogenous populations where it relies either on the antigenic properties of the cell surface or on the chemical nature of exposed carbohydrate residue on the cell surface on a specific membrane receptor—ligand interactions.

Useful modifications or methods of affinity chromatography technique have also been developed. Gas Chromatography Principle Gas chromatography GC as the name suggests, is particularly suited for the separation of gases and volatile liquids or solids in their gaseous state. The compounds of low polarity are best separated by GC.

The technique is highly sensitive, reproducible and has high speed of resolution. When the stationary phase is an active solid such as silica, the method is referred to as gas solid chromatography GSC.

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However, if the stationary phase is a liquid such as polymers of silicone coated onto the surface of an inert granular solid then the technique is known as gas-liquid chromatography GLC. The stationary phase, whether a solid or a liquid coated as thin film on surface of a solid support, is packed in a glass or stainless steel column which is narrow, coiled, 1—3-m long and with 2—4 mm internal diameter. An inert carrier gas mobile phase such as nitrogen, helium or argon is made to flow through the column. The temperature of the column is maintained high in an oven to keep the compound to be separated in their volatile state.

These volatilized compounds get partitioned between the liquid or solid stationary phase and the gaseous mobile phase and hence get separated because of differences in their partition coefficients. After leaving the column, the separated compounds pass through the detector, sensed and recorded by the recorder. Capillary columns of internal diameter 0. Two types of capillary columns viz. In WCOT, as the name suggests, walls of the capillary column are coated with the stationary phase.

Since the stationary phase is a liquid and is directly coated on the walls of the capillary column, only a small amount of the stationary phase is present in this system. Accordingly, only a small amount of the sample can be applied on to the WCOT column. In SCOT, the stationary phase is in the form of a thin layer on surface of a solid support which in turn is packed into the capillary column.

Hence, the capacity 4. Solid Support and Stationary Phase The purpose of the solid support is to provide a large uniform, inert surface area for holding a thin layer of the liquid stationary phase. The support should be inert, and should have high mechanical strength, large surface area, regular shape and uniform size. The most commonly used support is celite, the OH groups of which are modified by silanization with hexamethyl disilazane to minimize interaction with the sample. The correct choice of the stationary phase is perhaps the most important parameter in GC. Ideally, the stationary phase must be non-volatile and thermostable at temperature used for analysis.

It should be chemically inert towards the solutes of interest at the column temperature. The high boiling point organic compounds such as polyethylene glycols, methyl-phenyl and methyl-vinyl silicon gums, esters of adipic, succinic and phthalic acid, polyesters, polyethylene glycols are used as liquid stationary phases. The operating temperature must be compatible with the phase chosen. At very high temperature, the phase may get volatilized and cause excessive column bleeding which may contaminate the detector.

Pre-packed columns are also available but are costly. After packing, the column is kept in an oven for 24—48 h at temperature near the upper working limit. This is done to condition the column. While conditioning, the carrier gas is passed through the column at normal flow rates but the column is not connected to the detector, otherwise the detector may become gel contaminated.

Sample Preparation The sample should be prepared in such a way that it does not get retained on the column for excessive period of time. This will lead to poor resolution and peak tailing. The polar groups such as NH2, COOH and OH are derivatized by methylation, silanization and trifluoromethyl silanization to increase the volatile character and distribution coefficients of the compounds.

Solvents such as ether, heptane or methanol are used to dissolve the sample which is then injected with the help of a microsyringe onto the column through a rubber septum in the injection port. The temperature of the injection port is generally maintained higher than the temperature of the column to ensure rapid and complete volatilization of the sample. Too high temperature of the injection port may decompose the sample. Therefore, the temperature of the injection port should be such that it causes rapid vapourization of the sample without decomposing it.

Carrier Gas The primary function of the carrier gas is to carry the volatile components through the column. The gas used should be inert and should not react either with sample or with stationary phase. Its secondary purpose is to carry the separated components to the detector so that it is suitable for detector use. It should be readily available in extra pure form and be inexpensive.

Normally, nitrogen, helium and argon are the three most commonly used carrier gases. The column temperature must be high enough so that analysis can be accomplished in a reasonable length of time. Lower the temperature better is the resolution and longer is the analysis time. Therefore, a balance has to be struck between the peak retention time and resolution.

Chromatographic separation can be achieved isothermically where a constant temperature is employed or by temperature programming where the temperature is increased gradually. Detectors Characteristics such as selectivity, sensitivity, response, noise, minimum detectable quantity and linear range, should be given consideration while making choice of 4. The detector should be simple to operate, inexpensive and as far as possible insensitive to changes in flow rate and temperature. It has a wide linear response range and can detect as low a concentration as 1 ng. The detector consists of two electrodes.

One of the electrodes is the jet of the flame which is produced by introducing a mixture of hydrogen and air into the detector, while the other electrode is made of brass or platinum wire which is mounted near the tip of the flame. When the carrier gas carrying the sample components emerges from the column, the sample signal is recorded by the recorder.

It shows poor response for the compounds which possess neither of these elements. NPD is widely used in the analysis of organophosphorus pesticides. Here a radioactive source 63Ni ionizes the column gas and produces electrons which give a current across the electrodes to which suitable voltage is applied. When the carrier gas carrying the electron capturing substance emerges from the column, it captures the ionized electrons.

This results in the drop of the current which is traced on a chart paper by the recorder. The detector is best suited for the halogen-containing compounds such as pesticides DDT, dieldrin and aldrin. Amplifiers and Recorders When components leave the column and pass through the detector discussed above, small and weak electrical signals are produced which are amplified by an amplifier before they are fed to the recorder.

Recorders generally consist of two basic parts viz. High Performance Pressure Liquid Chromatography High performance liquid chramotography or high pressure liquid chromatography HPLC also works on the basis of partitioning, adsorption, ion exchange or 58 4 Techniques in Biochemical Evaluation molecular sieving phenomena. The conventional column chromatography suffers from two major flaws as it is generally a time consuming process and quality of resolution is poor.

This is mainly because of the fact that in conventional column chromatography the mobile phase percolates through the column under the force of gravity or by small pressure applied by peristaltic pump. This accounts for the slow flow rate, which in addition to extending the time required for elution of the sample creates the problem of peak broadening through diffusion phenomenon resulting in poor resolution.

In general, resolution of individual components can be improved by decreasing the particle size of stationary phase. However, in conventional column chromatography, this is not feasible because the use of finer gel material will further lower the permeability of the column contributing to the resistance to flow of mobile phase, which can be overcome by use of high pressure.

Therefore, the stationary phases of smaller particle size, which can withstand high pressures, have been developed and have facilitated the development of a new chromatographic technique called HPLC, which gives faster and superior resolution with sharp peaks. Components of HPLC The basic HPLC equipment consists of the following components: i solvent reservoir, ii pump, iii damping device, iv pressure gauge, v sampling device, vi column, vii detector, viii fraction collector and ix recorder.

The pump delivers the solvent from the reservoir at a constant flow which is smoothed out by means of damping device. The inlet pressure of the column is monitored with a manometer. After leaving the column, the sample components are monitored by the detector and their tracings drawn by the recorder. Solvent reservoir: The solvent used in HPLC must be of high purity as any traces of impurities or suspended material can seriously affect the column efficiency and can interfere with the detection system.

Solvents, as supplied commercially, contain substantial amount of dissolved air. Formation of air bubbles can seriously interfere with satisfactory separation by HPLC because the air bubbles affect the column efficiency and also the solute detection. Thus, some of the conventional solvent reservoirs are equipped with degassifier or the solvent can also be degassed by heating, stirring, subjecting it to vacuum, ultrasonic vibrations or bubbling helium gas before pouring it into the reservoir.

Highly pure solvents are available commercially, but even with such solvents it is advisible to introduce a 1—5-mm microfilter prior to the pump to prevent any particulate impurities from entering into the column. There is high resistance to flow of solvent and high pressures are, therefore, required to achieve satisfactory and constant flow rates. All materials in the pump should be chemically resistant to all the solvents used in HPLC.

Various pumping systems operate on the principle of either constant pressure or constant displacement. Constant pressure pumps facilitate delivery of the solvent at a constant pressure. A gas at high pressure is introduced into the pump which forces, in turn, the solvent from pump into the column. Constant pressure is maintained throughout, which causes a decrease in permeability of the column with time which in turn results in decreased flow rates. Such pumps do not compensate for this decrease in flow rate and so provide uniform and pulseless solvent flow.

The second type of pumps are the constant displacement pumps, which displaces a constant amount of the solvent from the pump into the column and so maintains a constant flow rate irrespective of the changing conditions within the column. These pumps produce small pulse of flow between two displacements and so pulse dampners are usually introduced between the pump and the column to smoothen the flow and to minimize the pulsing effect.

Two commonly used constant displacement pumps are: i Motor driven syringe type pump.

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Sample injection: Sample can be introduced into the column either by a syringe injection through a septum of an injection port or by a sample loop from which it is swept into the column by the eluent. The sample is loaded directly on top of the column to avoid appreciable mixing of the sample with the eluent. In the syringe 60 4 Techniques in Biochemical Evaluation injection mode, the sample is injected with help of a microsyringe which can withstand high pressures directly onto the column bed. While loading the sample, the system should not be under pressure.

Hence, before applying the sample, the pump is turned off and when the pressure is dropped near atmospheric pressure, the sample is introduced. After the sample has been injected, the pump is switched on again. This procedure is known as stop flow injection. The second type of system is loop injection. Here the sample is introduced with the help of a metal loop of fixed small volume. The loop is filled with the sample and by appropriately adjusting the sample valve, the solvent from the pump is channelled through the loop.

The sample is thus flushed by the solvent from the loop whose outlet opens directly at top of the column bed. HPLC columns: Since glass tubing cannot withstand pressures in excess of 70 atm, stainless steel precision bored columns with an internal mirror finish for efficient packing, are normally used.

These straight columns of 15—50 cm length and 1—4 mm diameter can withstand very high pressures of up to 5. At the end of the column, homogenously porous plugs of stainless steel or Teflon are used to retain the packing material and to ensure the uniform flow of the solvent through the column. At times, repeated application of impure samples may result in clogging and the loss of resolving power of the column. To prevent this, a short column of length 1—2 cm and internal diameter equal to that of analytical column is generally introduced between the injector and the analytical column.

This short column is called guard column and is packed with material with which analytical column is packed. The guard column retains the solid particles in the sample before it enters the main column. The guard columns can be replaced at regular intervals. Stationary phases: One of the basic requirements for HPLC is that the packing material which serves as stationary phase or support for stationary phase should be pressure stable, and withstand the operating pressure applied during separation Table 4.

Three forms of column packing materials are available based on the nature of the rigid solid structure: i Totally porous materials or microporous supports: In these supports the micropores ramify through particles which are generally 5—10 mm in diameter. The thickness of the porous layer is generally 1—3 mm. The size of glass beads used is between 25 and 50 mm.

The type of particular stationary phase, with their commercial names, which can be used for different types of chromatographic separations are listed in Table 4. Detectors: Detectors are the devices which continuously monitor changes in the composition of the eluent coming out of the column. Most commonly used 4. Refractive index detector RID : Refractive index RI of dilute solutions changes proportionally with solute concentration.

This relationship is exploited for quantitative detection of solutes in the column eluate. The relationship between the change in RI and solute concentration is only moderately dependent on the type of solute, making this a quite universal, yet not very sensitive detection principle, RID can, therefore, be applied to general purpose. This detector suffers from many defects including low sensitivity, tendency to be affected by temperature or flow speeds and incompatibility for being used in gradient elution unless chosen solvents are identical RI.

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It measures the bulk RI of sample eluent system. Hence, any substance whose RI differs sufficiently from that of eluent can be detected. Variation in flow rates also interfere with response of differential refractometer. Hence, very good damping is essential for the pumps producing pulsating flow.

Fixed wavelength detectors utilize lamps which emit light of a few discrete wavelengths. Lower wavelength lamps such as zinc lamps nm and cadmium lamps nm are available. The combination of the lamp and a filter determines the fixed operating wavelength of the detector. Variable wavelength VW detectors use a light source 62 4 Techniques in Biochemical Evaluation with a continuous emission spectrum and a continuously adjustable narrow band filter, called monochromator.

The most common light source for these detectors is the deuterium lamp whose usable emission spectrum ranges from about nm to about nm, with an intensity maximum between and nm. Above nm, the output intensity is low, therefore, some VW at wavelength above nm. Specially designed VW detectors have an additional or optional tungsten lamp, which can be used as detectors for HPLC have been introduced which allow automatic rapid change of wavelength setting within 1—2 s or less across their entire wavelength span, which typically ranges from about nm to about nm.

Electrochemical detectors: Methods used in HPLC based on electroanalysis can be classified as bulk property and solute property electrochemical detectors. Bulk property electrochemical detectors respond to a change in an electrochemical property of the bulk liquid flowing through the measuring cell , whereas the solute property electrochemical detectors respond to a change in voltage potentiomety or current voltammetry or coulometry when an analyte passes through the cell.

Fluorescence detectors: The quantity of fluorescent light emitted from excited molecules in dilute solutions is proportional to the intensity of excitation source, illuminated volume of the sample solution, quantum efficiency of fluorescence of sample and the concentration of the solute to be detected. Ideally, fluorescence radiation, as a result of suitable excitation of the sample molecules, is measured against a dark background.

Therefore, the main source of detector noise is dark current noise of the photodetector, which is mainly determined by temperature. It is defined as the gravitational force acting on a 1 g mass at distance r cm from the axis of rotation. The ultracentrifuge subjects a small volume of solution less than 1 mL contained in a quartz cell to a carefully controlled centrifugal force, and records, by means of optical and photographic systems, the movement of the macromolecules in the centrifugal field.

The solute molecules, which are initially uniformly distributed throughout the solution in the cell, are forced toward the bottom of the cell by the centrifugal field. This migration leaves a region at the top of the cell that is devoid of solute and contains only solvent molecules. The migration also leaves a region in the cell where the solute concentration is uniform.

A boundary is set up in the cell between solvent and solution in which concentration varies with distance from the axis of rotation. By the data obtained, namely, the sedimentation rate, the Svedberg unit S can be calculated. A Svedberg unit, named in honour of T. Typical S values are 4. With a knowledge of the diffusion coefficient, molecular weights can be readily calculated.

To determine the number of components in a solution, a simple centrifugation can be readily made and the number of boundaries based on concentration gradient peaks can be determined. Diffusion coefficient measurements need not be made. The most useful radioisotopes, 14C, 35S, 32P and 3H, are b-ray emitters; that is, when the nuclei of these atoms disintegrate, one of the product is an electron which moves with energies characteristic of the disintegrating nucleus. The b rays interact with the molecules through which they traverse, causing dissociation, excitation or ionization of the molecules.

It is the resultant ionization property which is used to measure quantitatively the amount of radioisotope present. A curies is the amount of emitter which exhibits 3. Specific activity. Dilutions factor. The factor is defined as Specific activity of precursor fed : Specific activity of compound isolated This factor is used frequently to express the precursor relation of a compound in the biosynthesis of a second compound.

Thus, in the sequence A! D, the dilution factor for C! D would be small, whereas for A it would be large. Therefore, a small dilution factor would indicate that compound C fed to a tissue has better precursor relationship to the final product that compound A with a large dilution factor. Percentage of incorporation. This is also useful to compare the proximity of a precursor in the biosynthesis of a second compound. Stable isotopes. As an example, deuterium, the hydrogen atom with mass of 2, is present in most H2O to the extent of 0.

The remainder of the hydrogen 4. This concentration of 0. It is possible to obtain heavy water in which Thus, the two stable isotopes of nitrogen are 14 15 7N and 7N , which have a normal abundance of If a sample of nitrogen gas contain 4. Other stable isotopes that are 7N available in enriched concentrations and therefore may be used as tracers in biochemistry are 8O17, 8O18, 6C13, 16S33 and 16S34; the normal abundance of these isotopes can be found in any chemical handbook.

The principles underlying the use of stable isotopes are similar to those employed with radioisotopes Table 4. Liquid scintillation counting is the most popular technique for measuring radioisotopes. The technique is based on the use of a scintillation solution containing fluors and a multiplier phototube.

The scintillation solution converts the energy of the radioactive particle into light; the multiplier phototube responds to the light by producing a charge which can be amplified and counted by a scaling circuit. In liquid scintillation counting, the radioactive substance is usually dissolved in a suitable organic solvent containing the fluor. Alternately, the radioactive sample, which can consist of filter paper containing the sample, which can consist of filter paper containing the sample, is suspended or immersed in the scintillation fluid.

Under these conditions the energy of the radioactive particle is first transferred to the solvent molecules, which may then ionize or become excited. It is the electronic excitation energy of the solvent which is transferred to the fluor solute. When the excited molecules of the solute return to their ground state, they emit quanta of light that are detected by the phototube. One problem associated with this technique is the quenching of the light output by coloured substances in the sample.

In addition, the fluor molecules may be quenched if foreign substances absorb their excitation energy before it is released as light. Methods are available for determining the amount of quenching exhibited by the radioactive sample. Scintillation counting is particularly useful for determining the weak b particles of tritium 3H and carbon14 14C. Chapter 5 Carbohydrate Estimations Carbohydrates defined as aldehydes or ketones of polyhydric alcohols, which also include those biopolymers, yield these compounds on hydrolysis.

They occur in animals, plants as well as microorganisms and serve diverse structural and metabolic roles. Sugars such as glucose are among the major sources of energy whereas starch and glycogen function as storage polysaccharides in plants and animals, respectively. In addition, carbohydrates are structural components of cell walls, connective tissues in animals and exoskeletons of invertebrates. Monosaccharides These carbohydrates are a single unit of polyhydroxy aldehydes or ketones and cannot be hydrolysed into simpler compounds.

Simple sugars like glucose, arabinose, ribose, fructose, galactose, etc. Oligosaccharides They contain 2—10 U of monosaccharides monomers linked to each other via. Polysaccharides Polysaccharides are composed of ten or more monosaccharide units linked to each other with glycosidic linkages, e. H2SO4 hydrolyses glycosidic bonds to yield monosaccharides which in the presence of an acid get dehydrated to form furfural and its derivatives.

These products react with sulphonated a-naphthol to give a purple complex. Polysaccharides and glycoproteins also give a positive reaction Fig. Procedure Add 2—3 drops of a-naphthol solution to 2 mL of the test solution. Taking precautions, gently pipette 1 mL conc. H2SO4 along the side of the test tube so that the two distinct layers are formed.

Observe any colour change at the junction of two layers. Appearance of purple colour indicates the presence of carbohydrates. Precautions 1. H2SO4 should be added along the sides of the test tubes causing minimal disturbance to the contents in the tube. In this the furfural produced reacts with anthrone to give bluish green coloured complex Fig.

Materials and Reagents 1. Boiling water bath. Observe whether the colour changes to bluish green. If not, examine the tubes again after keeping them in boiling water bath for 10 min. Starch gives blue colour with iodine, while glycogen reacts to form reddish brown complex.

Hence, it is a useful, convenient and rapid test for detection of amylase, amylopectin and glycogen. Reagents 1. Iodine solution: Prepare 0. Procedure Take 1 mL of the sample extract or test solution in a test tube. Add 4—5 drops of iodine solution to it and mix the contents gently. Observe if any coloured product is formed. Note the colour of the product. Monosaccharides usually react in about 1—2 min while the reducing disaccharides take much longer time, between 7 and 12 min, to get hydrolysed and then react with the reagent.

Brick red colour is obtained in this test, which is due to the formation of cuprous oxide Fig. Material and Reagents 1. Keep the test tubes in a boiling water bath. A briskly boiling water bath should be used for obtaining reliable results. Look for the formation of brick red colour and also note the time taken for its appearance. Ketoses undergo dehydration to give furfural derivatives which then condense with resorcinol to form a red complex. Prolonged heating will hydrolyse disaccharides and other monosaccharides which also eventually give colour Fig.

Note for the appearance of a deep red colour. This would indicate that the sample solution contains a keto sugar. Formation of yellow or red ppt of cuprous oxide denotes the presence of reducing sugars. Rochelle salt acts as the chelating agent in this reaction Fig. These solutions must be mixed immediately prior to use.

Mix thoroughly and place the test tubes in vigorously boiling water bath. Look out for the formation of red ppt of cuprous oxide which would indicate the presence of reducing sugars in the test solution. In this method sodium citrate functions as a chelating agent. Presence of reducing sugars results in the formation of red ppt of cuprous oxide.

Dilute to mL with water. Dissolve Cool and dilute to mL. Add Reagent No. Make the final volume to 1 L. Procedure Add 0. Keep the test tubes in a vigorously boiling water bath. Observe for the formation of red precipitates whose appearance would suggest the presence of reducing sugars in the given solution. The reducing sugars react with picric acid to form a red coloured picramic acid Fig. Boiling water both. Saturated picric acid: Dissolve 13 g of picric acid in distilled water, boil and cool.

Procedure Add 1 mL of saturated picric acid to 1 mL of sample solution followed by 0. Heat the test tubes in a boiling water bath. Appearance of red colour indicates the presence of reducing sugars in the sample solution. This sugar can be distinguished from other monosaccharides by its reaction with conc.

Oxidation of other monosaccharides yields soluble dicarboxylic acids whereas galactose produces insoluble mucic acid. Boiling water bath Soild galactose Solid glucose Conc. HNO3 Procedure Take about 50 mg galactose and 50 mg glucose separately in different test tubes. Add 1 mL of water and 1 mL conc. HNO3 to each tube. Heat the tubes in boiling 5. Add 5 mL water and keep them over night. Insoluble mucic acid will be formed in the case of galactose but not glucose or other sugars. Reaction is due to the formation of furfural in the acid medium which condenses with orcinol in the presence of ferric ions to give a blue—green coloured complex, which is soluble in butyl alcohol Fig.

Dissolve 1. Heat in a boiling water bath. Observe for the formation of blue—green coloured complex. For accurate determination of a particular sugar, it may be necessary to separate out the various carbohydrates in a mixture or a tissue extract by chromatographic techniques and then estimate them individually. Some of the methods which are employed for determination of different groups or types of sugars are: 76 Method Anthrone method Ferricyanide method Dubois method Somogyi-Nelson method Picric acid method High pressure liquid chromatography HPLC Enzyme methods 5 Carbohydrate Estimations Applicable For total soluble sugars For total soluble sugars For total soluble sugars For reducing sugars For reducing sugars For separation and determination of monosaccharides, disaccharides, and oligosaccharides For estimation of specific sugars In HPLC method, sugars in an appropriately processed sample are first separated from each other by HPLC and their presence as well as the amount in the column effluent is continuously monitored and recorded by a suitable detector such as RI refractive index detector.

The identity of sugars in individual peaks is established by comparison with elution profiles of authentic samples of different sugars. Amount of some of the sugars in a sample can also be determined enzymatically. One main advantage of this technique is that prior separation of individual sugars is not essential since some of the enzymes exhibit absolute substrate specificity amount of glucose can conveniently and accurately be determined, even if the sample contains other closely related sugars like galactose, mannose etc.

Chapter 6 Estimation of Lipids Lipids are non-polar organic biomolecules which are totally or nearly insoluble in water but are quite soluble in non-polar organic solvents like ether, chloroform or benzene. They serve as major structural components of the membranes and also form a protective coating on many organisms. Some of the vitamins and hormones are lipids. There are several different classes of lipids but all of them derive their distinct properties due to the hydrocarbon nature of their structure. Lipids have been classified in several ways but the most acceptable classification is the one based on the structure of their backbone.

Based on this, they are divided into two groups: 1. Complex lipids. Simple lipids. Complex lipids are esters of fatty acids. Among various forms of complex lipids such as acylglycerides, phospholipids, sphingolipids and waxes, fatty acids are covalently joined by an ester linkage to a trihydroxy alcohol, glycerol, or its derivative. Since the complex lipids yield soap on alkaline hydrolysis, they are also called saponifiable lipids. Simple lipids, on the other hand, do not contain fatty acids and are therefore called non-saponifiable lipids. They include compounds such as terpenes, sterols, etc.

This property of specific solubility in non-polar solvents is used for extracting lipids from tissues. In biological materials, the lipids are generally bound to proteins and they are, therefore, extracted with either R. Inclusion of methanol or ethanol in the extraction medium helps in breaking the bonds between the lipids and proteins. Take 1 g of the oil seed and grind it in the presence of 5 g of anhydrous sodium sulphate in a pestle and mortar. A small amount of acid washed sand may be used as an abrasive if the seed material is tough.

Add 20 mL of chloroform—methanol mixture to it and transfer it to an air tight glass stoppered iodometric flask. Shake the content of the flask on a mechanical shaker for 1 h and then filter it through a glass-sintered funnel. Repeat the extraction of the residue twice and pool the filtrates.

Remove the solvent from the residue by distilling under vacuum. Take the pooled fractions in a separatory funnel, shake it thoroughly and allow it to stand for 5 min. The lipids will be recovered in the lower chloroform layer while soap, glycerol and other water insoluble impurities move into the upper layer. Drain out the lower layer and treat the upper layer again 3—4 times with 5—10 mL of chloroform—methanol mixture to extract any residual lipid from it. Collect the lipid containing fractions in a pre-weighed beaker. Record the weight of the beaker and determine the amount of crude lipids in the sample by subtracting the weight of empty beaker.

This process is called saponification. From the amount of potassium hydroxide utilized during hydrolysis, the saponification value of a given fat sample can be calculated. The saponifiaction value is defined as mg of KOH required to saponify 1 g of the given fat Fig. It may be recalled that three molecules of KOH are consumed for saponification of each molecule of tracylglycerol irrespective of chain length of fatty acid.

Each gram of a triacylglycerol with shorter chain fatty acids will contain larger number of molecules of the triacylglycerol and will thus require much more KOH. The saponification value is therefore an indication of average molecular weight of the fatty acids in an acylglyceride. The procedure involves refluxing of known amount of fat or oil with a fixed but an excess of alcoholic KOH. The amount of KOH remaining after hydrolysis is determined by back titrating with standardized 0.

Weigh accurately 1 g of the fat sample in a conical flask and dissolve it in about 3 mL of the fat solvent Reagent No. Add 25 mL of 0. Cool to room temperature and add a few drops of phenolphthanlein into the flask. Titrate the contents of the flask with 0. Similarly, run a blank by refluxing 25 mL of 0. Calculation 0.

As alcohol is highly inflammable therefore precaution is required during heating. During refluxing, effective cooling of condenser is required so that alcohol does not get evaporated during saponification. In addition, the fats often become rancid during storage and this rancidity is caused by chemical or enzymatic hydrolysis of fats into free acids and glycerol. The amount of free fatty acids can be determined volumetrically by titrating the sample with potassium hydroxide. The acidity of fats and oils is expressed as its acid value or number which is defined as mg KOH required for neutralizing the free fatty acids present in 1 g of fat and oil.

The amount of free fatty acids present or acid value of fat is a useful parameter which gives an indication of the age and extent of its deterioration. Conical flasks. Test compounds olive oil, butter, margarine, etc. Standardize this solution by titrating known volume of 0. Procedure 1. Take 5 g of fat sample in a conical flask and add 25 mL of fat solvent Reagent No. Shake well and add a few drops of phenolphthalein solution and again mix the contents thoroughly.

Titrate the above solution with 0. Note the volume of KOH used. Repeat the steps 1—3 with a blank which does not contain any fat sample. Calculations 0. Hence, factor of The generally accepted parameter for expressing the degree of carbon to carbon unsaturation of fat, oil or their derivatives is iodine value. Iodine value or 82 Fig.

It is a useful parameter in studying oxidative rancidity of triacylglycerols since, higher the unsaturated, greater is the possibility of rancidity. Estimation of iodine number is based on the treatment of a known weight of fat or oil with a known volume of standard solution of iodine monochloride, and then determining the amount of unused iodine monochloride from iodine liberated, on addition of excess of KI.

The released iodine is titrated against 0. Stoppered bottles. Burette 25 mL. Mix both the solution and make the volume upto 1 L. To check its normality, take 20 mL of 0. Dilute to mL with water and titrate with thiosulphate solution till the yellow colour appears. Now add a few drops of starch solution Reagent No. Shake thoroughly and allow it to stand in dark for 1 h. Similarly, prepare a blank in which fat solution is replaced by chloroform. After the reaction time of 1 h in dark, rinse the stopper and neck of the bottle with 50 mL of water and add 10 mL of potassium iodide solution.

Titrate the liberated iodine with standard sodium thiosulphate solution till the content of the flask becomes pale yellow in colour. Add a few drops of starch solution and continue to titrate it further with sodium thiosulphate solution till the blue colour disappears. Calculations The difference between the blank and test readings gives the amount of 0.

One litre of 0. The iodine number can thus be calculated as follow: Volume of 0. The bottles must be shaken thoroughly throughout the titration to ensure that all the iodine is expelled from the chloroform layer. All triacylglycerols are soluble in diethyl either, chloroform and benzene. They are slightly soluble in cold methanol, ethanol and acetone but their solubility increases on warming.

Understanding of their solubility characteristics is helpful in developing efficient procedures for extraction of various lipids from the biological materials. Fatty acids butyric, palmitic and oleic acids 2. Fats and oils butter, olive oil, cod liver oil, phospholipids, etc.

Solvents water, acetone, ethyl alcohol, chloroform, diethyl either, etc. Take small amount of different lipids in various test tubes and add water. Shake well and check their solubility. Repeat the solubility test using different solvents.


Record the observation regarding solubility of these lipids and conclude about their solubility characteristics. Friedrich Lottspeich Editor , Joachim W. Engels Editor. Request permission to reuse content from this site. Undetected country. NO YES. Selected type: E-Book. Added to Your Shopping Cart. Evaluation Copy Request an Evaluation Copy. Analytical methods are the essential enabling tools of the modern biosciences.

This book presents a comprehensive introduction into these analytical methods, including their physical and chemical backgrounds, as well as a discussion of the strengths and weakness of each method. It covers all major techniques for the determination and experimental analysis of biological macromolecules, including proteins, carbohydrates, lipids and nucleic acids.

The presentation includes frequent cross-references in order to highlight the many connections between different techniques. The book provides a bird's eye view of the entire subject and enables the reader to select the most appropriate method for any given bioanalytical challenge.