RADIOIMMUNE ASSAY “+ Introduction * Principle RIA Procedure Separation techniques ** Components of radioimmunoassay “* Analysis by competitive antibody binding “> Instrumentation «+ Advantages of RIA * Disadvantages of RIA “+ Applications of RIASAY LU | INTRODUCTION. Radio hnmuno Assay (RIA) is an elegant tech. in analytical chemistry. If substance to be analysed is in very low quantities, in the orders of micrograms, nanograms, conventional methods like gravimetric and colorimetric method fail. RIA finds extensive application in the assay of many substances which are present in trace amount in blood. Specifically RIA measures the actual effect of changing concentrations of a particular substance present in a biological fluid (eg. blood, plasma, urine) based on an in vitro system consisting of radioactive standards of the same substance and a specific antibody. In a true sense, RIA is nothing but an indirect method of analysis because it does not make use of either the radioactive standard or the antibody present in the original sample. History Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay. In 1977 Yalow received the Nobel Prize for her and Berson's development of RIA Skelley et al (1973) listed a number of substances that may be determined quantitatively by the help of the RIA method namely: nucleic acids, proteins, enzymes, prostaglandin, steroidal hormones, antibodies, cancer and viral antigens, vitamins.QO) PRINCIPLE. * The radioimmunoassay technique is based on the isotope dilution principle, along with the use of a specific antibody to bind to a portion of the substance to be measured. If an antigen (for example, a hormone) is mixed with a specific antibody to that substance, an interaction will occur, forming an antigen/antibody complex that is chemically different from either the antigen or the antibody. | If there is insufficient antibody to complex all the antigen present, mixing — of the antibody with a known amount of labeled antigen along with an | unknown amount of labeled antigen allows quantitation of the unlabeled antigen. The specificity of an immunoassay depends upon antibodies produced in immunization. Antigen + Antibody = Complex + Leftover antigen Antigen + Antibody = Complex* + Leftover antigen Then Antigen + Antigen*+ Antibody Complex + Complex + Left Over antigen + Left Over antigen* | * Competitive binding of radiolabelled antigen and unlabelled antigen to a | high affinity antibody. The labelled antigen is mixed with the antibody at a concentration that saturates the antigen-binding sites of the antibody. As the concentration of the unlabelled antigen increases more labelled | antigen will be replaced from the binding site. |The decrease in the amount of radiolabelled antigen bound specific antibody in the presence of the test samples is measured to determine the | amount of antigen Present in the test sample. In std Condition, amount of labelled antigen bound to the antibody | decreases as the amount of unlabelled antigen increases in sample. Radioimmunoassay (RIA) Antibody (~Z Antigen Radiolabelled ote ee om “ — | Uae Hy ¢ Free fraction Bound fraction \ Count radioactivity The general procedure of RIA consists of adding suitable quantities of standards, unknown, labeled antigen and antibodies to a buffer solution and allowing the reaction to reach equilibrium. Most of the competitive binding assays are of equilibrium type, in which all the reagents are added at the same time and the reaction is allowed to proceed until equilibrium is established. In some cases it may be necessary to add tracer at a later stage to improve the sensitivity. This type of assay is known as sequential saturation assay.At the end of the incubation period the free and bound fractions are separated using a suitable technique. The distribution of radioactivity in each sample is determined by counting either free or bound or both with the use of suitable counting equipment. The estimation of antigen concentration is made by comparing the | inhibition observed in the unknown with that produced by standard solutions of known antigen concentration. For this purpose, the dose response curves are plotted using the data from the standards, and the unknown antigen concentration is read directly from the graph.SEPARATION TECHNIQUES. 1 Once the primary reaction is complete it is necessary to determine the distribution of the tracer between the free and the bound form. Usually this requires the bound fraction be physically separated from the free fraction. A variety of techniques described for this purpose exploit physicochemical differences between the tracer in its free and bound form. The operative criteria for a separation procedure are efficiency and practicality. The efficiency of a procedure can be defined as the completeness with which the bound and free phases are separated. The practicality includes speed, simplicity, applicability and cost. Various methods include electrophoresis, gel filtration, solid-phase adsorption of antigen, solid-phase absorption of antibody, immune- precipitation and fractional precipitation. QO COMPONENTS OF RADIOIMMUNOASSAY (RIA) Radioimmunoassay involves three components: 1) Pure antigen 2) Radio-labeled antigen, and 3) Antiserum (antibody) * In addition a separation technique is essential to estimate the distribution of radioactivity in the free and bound fractions. The sensitivity of an assay depends to a large extent on the quality of these components and choice of a suitable separation technique.> Pure antigen The preparation of standards and tracer and the production of | antibodies depend on the availability of pure antigen. Several procedures, such as electrophoresis, chromato electrophoresis, gel filtration, and ion exchange chromatography are available for the extraction and purification of hormones from biologic samples. A pure synthetic preparation, if available, can be substituted for the | natural preparation. | Anumber of hormones produced synthetically are now available with a | purity to match the best materials isolated from natural sources. In any case, before the antigen can be used as a standard, the specificity between this antigen and the antigen in the test sample toward the antibody binding sites must be clearly established. Radio labeled of anti The labeled antigen used as a tracer must generally be present in low concentrations because the Quantity of substance to be measured is usually small. The concentration of Ag* must not be greater than the least quantity of Ag to be measured. It is thus necessary to have labeled antigen of high specific activity, and once labeled, it must maintain the same characteristics of the unlabeled antigen to react qualitatively and quantitatively with the antibody. C-14 and H-3 have the principal drawback of long half lives (5740 and 12.3 years respectively) and they are also more difficult to measure, | requiring cumbersome liquid scintillation counting.They are almost exclusively used in those systems in which the addition of a larger molecule may alter the immune-reactivity of the system. Radioiodine generally fulfills the label requirements; I'25 is preferred over I'31 because of its longer half life, ease of handling, and higher counting efficiency. | | activity was described by Hunter and Greenwood 9-10 using | A method of producing radioiodinated proteins with high specific chloramine-T. Other methods have been developed, such as the use of iodine monochloride and Electrolyte labeling. Lactoperoxidase polyacrylamide- coupled lacto-peroxidase, conjugation method, but chloramine-T continues | to be the most commonly used technique. Antibody The sensitivity and specificity of RIA depend on the affinity of the antigen-antibody reaction and the highly specific binding sites on the antibodies used. The production of a good antiserum is essential for a satisfactory assay. In order to produce a highly specific antiserum, the antigen should have a unique antigenic determinant. | Production of antiserum invloves rather a complex procedure. Antibodies are relatively easily raised against protein compounds of | molecular weight in excess of 4,000 to 6,000. | For smaller proteins and non- immunogenic substances such as thyroid and | steroid hormones, cyclic nucleotides and drugs, it is necessary to conjugate the compound to a larger polypeptide or a protein such as albumin, thyroglobulin or polysine prior to immunization.It is customary to couple the hapten in such a way as to expose any functional groups characteristic of the molecule, so that the likelihood of production of specific antibodies is enhanced. * Antibody formation is generally augmented by adding an adjuvant to the antigen, which retards its absorption and increase the antigenic stimuli. These substances include aluminium hydroxide, gelatin, mineral oil and Freund's adjuvant. * The latter is most commonly used and consists of a neutral detergent, paraffin oil, and killed bacteria. * The general method of inducing antibody formation is to inject into a number of animals the pure antigen mixed with Freund's adjuvant. * Based on the view that impurities may have an adjuvant effect, some investigators prefer to immunize with relatively impure material, rather than a highly purified or synthetic equivalents. Standards In reference standards are necessary in order to interpolate values of samples to be measured. A material intended as a standard should have certain characteristics, 1. It should be available in large quantities. It should best able. It should not contain substances which can interfere with assays. It should be highly purified. It should be available in a form which allows convenient and accurate aoe preparation for radioimmunoassay. There are several types of RIA standard: 1. International standards distributed by WHO.|| 2. Reference preparations distributed by National Institute of Arthritis and Metabolic Disease and laboratory preparations of restricted use prepared by the investigator ora manufacturer and not having international validity. | ANALYSIS BY COMPETITIVE ANTIBODY BINDING OR | ISOTOPICALLY LABELLED COMPOUNDS | Radioimmunoassay is nothing but a competitive binding assay employing the principle of reversible binding of a labelled antigen to its specific antibody; and the ability of unlabelled antigen not only to compete in the reaction but also to displace labelled antigen from antibody. Nevertheless, the antibody and labelled antigen are always present as limiting factors and the concentration of unlabelled antigen (present continually. It has been observed that the percentage of antibody-bound labelled antigen declines progressively as a consequence of saturation of the combining sites on the antibody molecule. ‘Aitigen ‘Antibody Bound Antibody Freeentigen Key @ = Labelled Antigen O =Unlabelled Antigen e 4 = Anligen with tree binding uy either as standard solution or as sample under examination) is increased |An ideal behaviour has been assumed in Fig. whereby most radioimmunoassay | very closely approach this condition. In order to fulfill the requirements of an | ideal behaviour the following criteria must be accomplished, namely: i) The non-radioactive antigen (A) and radioactive antigen (A*) are | indistinguishable chemically i.e. both of them are identical chemically. _| | ii) The two reactions ultimately go to completion i.e., the equilibrium constants of the binding of labelled acid unlabelled antigen to antibody are not only equal but also are so huge in number that may be regarded as infinite. The antigen and antibody react in 1:1 ratio. There is no cross reactions observed in the medium i.e. the antibody being specific only for the single antigen indicated in the reactions or being determined. Q INSTRUMENTATION The two most vital equipments essentially required for radioimmunoassay (RIA) are, namely: (i) Centrifuge, and (ii) Radioactive Counters. These two equipments shall now be discussed briefly as follows: > Centrifuge * Acentrifuge which is capable of generating 1200-2500 rpm using swing- bucket-rotor or 3500 to 4000 rpm using a fixed-angle-head rotor can | be employed effectively. However, the former type is preferred because of the fact that here the pellet is formed at the bottom of the test tube and the supernatant | layer is more easily removed in comparison to the latter type where thepellet is formed at an angle. | In case, a centrifuge having relatively less gravitational force is employed then it is absolutely necessary to enhance the centrifugation time until suitable pellets are formed duly. | “+ Radioactive counters | In usual practice, two types of radioactive counters are mainly employed depending on the type of radioactive substance used. Namely (a) Gamma Counters, | (b) Scintillation Counters. > Gamma Counters: The gamma well counter consists of a scintillation detector with a hole in the center, for a sample to be placed inside for increasing the geometric efficiency and hence the counting efficiency of the counter, and other associated electronics. Well counters are used namely for in vitro measurements of different samples. They are usually available with automatic sample changers and | are mostly programmable with computers. Their major advantage is high detection efficiency which is from 50% to 70% for 140 keV gamma photons. These are used invariably for the gamma-energy emitting isotopes, for instance I731is the most common iodine-isotope. Examples of use of sample counterLead shield = PM-tube Scintillation Counters: It consists of a scintillator which generates photons in response to incident radiation. A sensitive photomultiplier tube (PMT) which converts the light to an electrical signal and electronics to process this signal. Sciontillator consists of a transparent crystal, usually a phosphor, plastic or organic liquid. Principle: When high energy atomic radiations are incident on a surface coated with some fluorescent material, then flashes of lights are produced. The scintillations are detected with the help of a photomultiplier tube that gives rise to an equivalent electric pulse. Working: When an ionizing particle passes into the scintillator material, atoms are ionized along a track. The photon from the scintillation strikes a photocathode and emits electrons which accelerated by a pulse and produce a voltage across the external resistance This voltage is amplified and recorded by an electronic counter.* These are mostly used for counting beta-energy-emitting isotopes, such as: tritium H, or C,, and (Carbon-14) isotopes. * First and foremost, radio-immunoassays were universally based on the H? | or Ci, isotope labelling technique, but this has the main disadvantage of using liquid-scintillation counting. Therefore, the comparatively much simpler technique of gamma-ray counting ruler by labelling . Photo-sensitve compounds with 17+, surface 125, or [131 is now being Scintiiation increasingly _ utilized mategel wherever such labelling Radiation is practically feasible. ADVANTAGES OF RIA Radio immuno assay is very sensitive technique used to measure concentrations of antigen without the need to use a bioassay. 2. Itcan measure one trillionth (10-12) of a gram of material per milliliter of blood. 3. It is structurally specific as antigen: antibody reactions are highly specific. 4. Itisindirect method of analysis. 5. It is a saturation analysis as active reagent added in smaller quantity than that of analyte.Q DISADVANTAGES OF RIA 1, Non-specificity of the technique and Prolonged reaction time (in days) as a consequence highly diluted reagent is used. | I | | 2. Radioactive lodine used in is not a cheap reagent. 3. Possible health hazards due to handling of radioisotopes. 4. All the reagents must be added precisely. 5. Non-sensitivity of the method and Limited assay range. 6. Lack of direct linear relationship between analyte concentration and signal response. 7. Difficulty of automation. Involvement of the processes of extraction, purification and concentration of the specimen under investigation, 9. Heat treatment of the specimen resulted invariably in degradation and destruction of the substances, and 10. Many processes involved ultimately make the analysis rigorous and unnecessarily sluggish. QO APPLICATIONS OF RIA | * Narcotics (drug) detection. *° The test can be used to determine very small quantities (eg. nanogram) of antigens and antibodies in the serum. * The test is used for quantitation of hormones, drugs, HBsAg, and other | viral antigens. | * Early cancer detection. * Measurement of growth hormone levels. * Analyze nanomolar and picomolar concentrations of hormones in _ | | biological fluids.Blood bank screening for the hepatitis (a highly contagious condition) virus. Tracking of the leukemia virus. Diagnosis and treatment of peptic ulcers. Research with brain chemicals called neurotransmitters.“- Introduction “+ Solid-liquid extraction >» Maceration Decoction Percolation Continuous extraction by soxhlet apparatus “* Liquid-liquid extraction > Batch extraction > Continuous extractionQ INTRODUCTION. Importance of separation techniques has been recognized since the ancient | times. Separation of active principle from a given material or a formulation received very much attention both from isolation and | analysis point of view. Ever since ancient times analyst or pharmacist have been confronted with task of separation of complex mixtures. The separation of active | principles from the preparation like tincture, extracts, pills or separation and isolation of vitamins, enzymes or hormones from their natural and always been tough task. It is thus necessary to separate the constituents of given mixture or formulation before an appropriate analytical method is applied. In order to achieve this one of the two methods are often adopted. These are; 1. To remove interfering substance 2. To remove or isolate the active substance Now depending on type of preparation, nature and amount of active material either of above method is adopted. In order to achieve separation various methods are adopted depending upon nature and amount of material. These methods are based on principles of chemistry such as Filtration Sublimation Distillation ExtractionExtraction Solubalising the active constituents and removing it with suitable extracting solvent is the most common technique followed in the field of analysis. Extraction methods can be classified as Follows. 1) Solid-liquid extraction 2) Liquid-liquid extraction These methods are represented in schematic diagram given below; A solid is extracted by usinga suitable solvent in three different ways. 1. Maceration 2. Percolation 3. Decoction 4. Conti ction by using Soxhlet apparatusQ Maceration * In this method of extraction Maceration involved soaking plant materials (coarse or powdered) in a stoppered container with a suitable solvent | such as water, methanol, ethanol etc. and allowed to stand at room | temperature for a period of minimum 3 days with frequent agitation. * The processed intended to soften and break the plant's cell wall to release the soluble phytochemicals. After 3 days, the mixture is pressed or strained by filtration. * In this conventional method, heat is transferred through convection and conduction and the choice of solvents will determine the type of| compound extracted from the samples. * However, the maceration period for infusion is shorter and the sample is boiled in specified volume of water (eg. 1:4 or 1:16) fora defined time for decoction. aan i i ecoction. Schematic illustration of Maceration Nia Ve toury ushed or cut small or Moderatel. Placed ina closed vessels Whole of the selected solvent (Menstruum) added Allowed to stand for seven days shaking occasionally PMR Eia Pee hiGcco Cem ence tris)Clarified by subsidence or filtration (Sera se PLC CT Tete) ) Drea Te Co) Advantages 1) This technique is the easiest and simple method. 2) Maceration is a cold extraction method, wherein the sample is not exposed to heat. Hence it is especially useful for extracting heat labile | substance. | 3) However, organic waste come into an issue as large volume of solvents is | used and proper management of the waste is needed. 4) Alteration in temperature and choice of solvents enhance the | extraction process, reduce the volume needed for extraction and can be introduced in the maceration technique, when such alteration is not objectionable. Limitations 1) Maceration may not extract the sample completely in some cases because of cold condition. 2) In this method, solvents used in the soaking process play a critical role. QO Decoction * Decoction is only suitable for extracting heat-stable compounds, hard plants materials (e.g. roots and barks) and usually resulted in more oil- soluble compounds compared to maceration and infusion.Decoction is also the name for the resulting liquid. Although this method of extraction differs from infusion and percolation, the resultant liquids can sometimes be similar in their effects, or general appearance and taste. Applications | * In brewing, decoction mashing is the traditional method where a | portion of the mash is removed to a separate vessel, boiled for a time and then returned to the main mash, raising the mash to the next temperature step. * In herbalism, decoctions are usually made to extract fluids from hard plant materials such as roots and bark. To achieve this, the plant material is usually boiled for 1-2 hours in 1-5 liters of water. It is then strained. Ayurveda also utilizes this method to create Kashayam type of herbal medicines. ° For teas, decoction involves boiling the same amount of the herb and water that would be used for an infusion (one teaspoon per cup) for about five to ten minutes. Q Percolation © Unique equipmentcalled percolator is used in percolation. * Apercolator is a cone shaped vessel having cock at the bottom. On small scale copper percolators were originally used but now a day these are largely replaced with percolators made up of Glass or stainless steel. * Dried powdered samples are packed in the percolator; a solvent is added from the top of the percolator while keeping the cock should remain open.. When solvent is about leaving percolator, the cock is closed. The additional volume of solvent is added so as to get its level above the sample to be extracted. The percolator is then covered from top and the sample is allowed to | macerate for 24 hours. The percolation process is usually done at moderate rate (e.g. 6 drops/min) until the extraction is completed before evaporation to get a concentrated extracts. At the end of that period, the cock is opened and percolate (extract) of the sample is allowed to fall slowly from the bottom. The mass in percolator is then pressed and the percolate is collected. The sufficient volume of sample is then added so as to get the required volume. The percolate is then filtered to get the clear extract.Q Continuous extraction by soxhlet apparatus * A Soxhlet extractor is a piece of laboratory apparatus invented in 1879 by Franz von Soxhlet. Originally designed for the extraction of a lipid from asolid material. However, a Soxhlet extractor is not limited to the extraction of lipids. Typically, a Soxhlet extraction is only required where the desired | compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired compound has a significant solubility in a solvent then a | simple filtration can be used to separate the compound from the insoluble substance. Consist of three parts as follows; 1. Around bottom flask with solvent 2. Athimble containing sample to be extracted 3. Condenser| * Normally a solid material containing some of the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet extractor. The Soxhlet extractor is placed onto a flask containing the extraction solvent. The Soxhlet is then equipped with a condenser. The solvent is heated to reflux. The solvent vapour travels up a distillation arm and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material. * The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound will then dissolve in the warm solvent. * When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times, over hours or days. During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. * After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. * The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded.Extraction is the process of removing a constituent from one phase by | bringing this into contact with a second, immiscible liquid phase. Principle * Liquid-liquid extraction, also known as partitioning, is a separation process consisting of the transfer of a solute from one solvent to another, the two solvents being immiscible or partially miscible with each other. Frequently, one of the solvents is water or an aqueous mixture and the | other is a non-polar organic liqui As in all extraction processes, liquid- liquid extraction comprises a step of mixing (contacting) followed by a step of phase separation. It is important to consider both steps in the selection of solvents and modes of operation. Thus, while vigorous mixing is favorable to the transfer of the extractable from one solvent to the other, it may also impair the ease of phase separation by forming emulsions. Liquid-liquid extraction which is the transfer of a solute from one liquid phase to another liquid by contact. This process is also called | Partitioning or distribution. Feed solution +solute(s) Extract+solute (s) | | Solvent Raffinate Solvent ExtractionLiquid-liquid extraction by using two immiscible liquids can be carried out by using two techniques: Batch extraction and continuous extraction Q Batch extraction The batch extraction carried out by usinga set of separating funnels. A solution from which a substance is to be extracted and an immiscible extracting solvent are introduced in a separating funnel. Two phases are mixed thoroughly in order to extract the substance from one phase to the other. If the extracting solvent is heavier, it would form a lower layer in the separating funnel. This heavier layer, containing the extracted material, is removed and stored in a separated container. For more effective and complete separation, more of fresh extracting solvent is added to the lighter layer which is retained in the separating funnel and the extraction is continued. This procedure is repeated till the extraction is almost complete. If the extracting solvent is lighter, it would form an upper layer in the separating funnel. In such a case, The lower layer is removed and transferred to another separating funnel, containing fresh extracting solvent. The extraction is completed by is method is carried out when the partition coefficient of a solute is high. Method is simple and quick and hence, is widely used for the extraction ona small scale; a continuous extraction process is applicable.Q Continuous extraction Extracting solvent lighter than the sample solution: The continuous extraction method is carried out when the partition coefficient of solutes is low. An immiscible extracting liquid is kept flowing continuously through the solution the solution from which a solute is to be extracted. Although there is not enough time for the equilibrium to reach, a solute is extracted continuously in this extraction method. | This method requires a special kind of extractor, depend on whether the | solvent used for extraction is lighter or heavier than the sample | solution. It is similar to that of the Soxhlet apparatus but instead of one solvent, two solvents are used in this case. The extracting solvent (e.g. non-aqueous solvent), which is lighter than the sample solution (e.g. aqueous solvent), is placed in a container (A). It is connected to a thimble (B) holding the sample solution to half its capacity. A glass tube (C), having a funnel shaped opening at one end and a glass | bulb with holes at another end, is placed inside the thimble. 1 This body is then connected to a condenser (D), which is attached at the upper end of the thimble. The extracting solvent in the container (A) is heated. Vapor of the | extracting solvent passes through the thimble into the condenser and falls as droplets into the funnel type glass tube (C). The droplets of extracting liquid enter the glass tube and then escape through the holes in the glass bulb.The extracting solvent being lighter than the solution to be extracted, passes through the sample solution to be extracted, passes through the sample solution. In this process, it extracts the solute and gets accumulated on the top of the sample solution. When sufficient quantity of the extracting liquid gets collected in the thimble, it overflows back to the flask (A) from the side are (S). The process is continued till ee oie | Extracting solvent heavier than the sample solution: * The extracting solvent is placed in a round bottom flask (A) and is heated. The sample solution to be extracted is present at the centre of the thimble (B). The vapor of the solvent passes from the upper side tube (U) of the flask into the thimble and then to the condenser (D) attached at the top of the thimble. On cooling, the droplets of the heavier extracting solvent drop down in the glass tube (C) and pass through the sample solution.In this process, it extracts the solute and gets accumulated at the bottom of the thimble, from where it overflows into the flask (A) through the lower side tube (L). The rate of heating of the extracting solvent in the flask (A) is adjusted so as to maintain the level of the sample solution between two side tubes (U) and (L) of the flask, so that the sample solution does not overflow at any time during the extraction process. This is continued till the complete extraction is achieves. This method of extraction is used when the material to be extracted has low partition coefficient for a pair of solvents. SO if APPLICATIONS OF LIQUID-LIQUID EXTRACTION Determination of iron as 8-hydroxy quinolate: Ferric iron can | be extracted from aqueous solution with 1% 8-hydroxy quinoline in | chloroform by double extraction. The pH of aqueous solution must be lie between 2 to 10.* Determination of uranium as 8-hydroxy quinolate: Uranium may be determined by as 8-hydroxy quinolate in presence of some EDTA, | provided the pH is 8.8 The presence of interfering elements e.g. Fe, Al etc. | may be masked by increasing the amount of EDTA in solution. Crude plant extracts can be purified by liquid-liquid extraction. The counter current extraction is used for the effective separation of | compounds from each other, when the number of components to be separated in more and components possess partition coefficients closer to each other. Sample extracted from biological fluids can be characterized for the metabolites, after separating the components using counter-current extraction.