CHROMATOGRAPHY.pptx by hemant yadav m pharm

chromatofraphy

1. Chromatography ( Paper , Thin layer and Ion exchange chromatography) SUBMITTED BY: AMAN , M.PHARM, 1 ST SEM SUBMITTED TO: MRS ANJU GOYAL (ASSISTANT PROFESSOR)

2. Chromatography Chromatography is defined as a method of separating a mixture of components into individual components through equilibrium distribution between two phases. The technique of chromatography is based on the differences in the rate at which the components of a mixture move through a porous medium ( called stationary phase ) under the influence of some solvent or gas ( called moving phase). The chromatographic method of separation involves the following steps: Adsorption or retention of a substance or substances on the stationary phase. Separation of the adsorbed substances by mobile phase. Recovery of the separated substances by a continuous flow of the mobile phase, the method being called elution. Qualitative and quantitative analysis of the eluted substances . Types of Chromatography Techniques: Stationary phase may be solid or liquid. Mobile phase may be liquid or a gas.

3. Technique Stationary Mobile Phase Column/ Adsorption chromatography Soild Liquid Partition chromatography Liquid Liquid Paper chromatography Liquid Liquid Thin layer chromatography Liquid/Soild Liquid Gas- Liquid chromatography Liquid Gas Gas- Soild chromatography Soild Gas Ion Exchange chromatography Soild Liquid PAPER CHROMATOGRAPHY Introduction: It is one of the oldest and simplest techniques for qualitative analysis but can also be used for quantitative determination. Paper chromatography is a variant of liquid chromatography where the components of the mixture are separated by the unidirectional flow of the liquid mobile solvent over the filter paper to which the spot of the mixture is applied. Cellulose filter paper is often used as the stationary phase in paper chromatography.

4. Principle of Paper Chromatography This technique is a type of partition chromatography in which the substances are distributed between two liquids, one is stationary liquid ( usually water) which is held in the fibers of the paper and called stationary phase, the other is the moving liquid or developing solvent and called the moving phase. The components of the mixture to be separated migrate at different rates and appear as spots at different points on the paper. The movements of substances relative to the solvent is expressed in terms of Rf values i.e. migration parameters : Rf : R is related to migration of solute from relative to the solvent front as: Rf = Distance travelled by the solute from the origin line Distance travelled by the solvent from the origin line Rf value depends upon a number of factors which are: The solvent employed Medium used for separation i.e. quality of paper Nature of mixture Temperature Size of the vessels in which operation is carried out.

5. Methodology/Technique: Developing the Chromatogram involves three stages: 1.) Sample Preparation: Samples can come either as liquids or solids. Liquids can be spotted without any pretreatment. Solid samples need to be dissolved in a suitable solvent as only liquids can be applied to the chromatography paper. 2.) Spotting and Developing: Spotting of the sample is done with the help of a capillary tube or automated applicator. The sample is applied as a neat spot on a horizontal line drawn with a pencil close to one edge. Allow the spot to dry and then immerse the paper in the developing chamber as per the selected technique with the marked spot above the solvent level. The solvent begins to move and draws the sample components differentially along with it. At the end of the development take out the paper and mark the solvent front with another line . Allow the paper to dry in drying cabinets with the provision of electrical heating before visualization.

6. 3.) Visualization of Spots : Coloured spots are easily observed on developed chromatograms. However, different approaches need to be adopted when colourless components are to be observed. It is convenient to classify such methods as specific or non-specific. Non specific method: Iodine chamber and UV viewing cabinet Specific method: For amino acids ,primary and secondary amines –A 0.2% ninhydrin solution in water saturated with butanol is sprayed on briefly heated paper chromatogram when deep blue or purple spots begin to appear. b) For alkaloids – Dragendroff’s agent gives orange or orange – red precipitates in presence of alkaloids c) For Phenols – Ferric chloride solution spray results in appearance of different coloured spots – read, blue, green or purple when phenols are present. d) Aldehydes and Ketones – 2,4 – dinitro phenyl hydrazine in a mixture of methanol and sulphuric acid (Brady’s reagent) spray results in bright orange or yellow colour spots in presence of aldehydes and ketones.

7. Types of Paper Chromatography Ascending Paper Chromatography Descending Paper Chromatography Ascending- Descending Chromatography Circular/Radial Chromatography Two- Dimensional Chromatography Ascending Paper Chromatography: When the development of paper is done by allowing the solvent to travel up the paper, it is known as ascending technique. In this , mobile phase is placed in a suitable container at the bottom of the chamber or in the chamber itself. The samples are applied a few centimeters from the bottom edge of the paper suspended from a hook . Paper may be rolled into a cylinder, held together by staples.

8. 2. Descending Chromatography: When the development of the paper is done by allowing the solvent to travel down the paper, it is known as descending technique. The apparatus consists of a well- sealed glass tank of suitable size and shape which is provided with a through for the mobile phase in the upper portion. The paper with sample spotted is inserted with the upper end in the trough containing the mobile phase, the jar itself having been equilibrated with the mobile phase prior to elution. 3. Ascending-Descending Chromatography: It is the hybrid of ascending and descending chromatography. In this technique, the upper part of the ascending chromatography can be folded over a glass rod allowing the ascending development to change over into the descending after crossing the glass rod . 4. Circular/Radial Chromatography: Circular paper chromatography permits separation of sample components in the form of concentric circular zones through radial movement of liquid phase.

9. 5. Two- dimensional chromatography: Chromatogram development occurs in two direction at right angles. In this mode, the samples are spotted to one corner of rectangular paper and allowed for first development. The paper is again immersed in the mobile phase at a right angle to the pervious development for the second chromatogram. Applications Paper chromatography has been applied to the separation of many organic and biochemical products. For example- it has been utilized in the determination of indoles in whole urine and in the study of barbiturates, antibiotics, hormones, and amino acids. It has also been used in the study of inorganic metal salts and complex ions. Paper chromatogram of some 2,4- dinitrophenylhydrazone. Solvent is mixture (2:1) of isooctane and a light mineral oil. Separating colored pigments Reaction monitoring Isolation and Purification Pathology and Forensic science Analyzing complex mixtures(certain organic compounds such as carbohydrates and amino acids are identified or detected from a complex mixture of organic compounds with the help of paper chromatography.

10. Advantages of Paper chromatography: Simple and Rapid Paper Chromatography requires very less quantitative material. Paper Chromatography is cheaper compared to other chromatography methods. Both unknown inorganic as well as organic compounds can be identified by paper chromatography method. Paper chromatography does not occupy much space compared to other analytical methods or equipments. Good resolving power Limitations/Disadvantages of Paper Chromatography Large quantity of sample cannot be applied on paper chromatography. In quantitative analysis paper chromatography is not much effective. Complex mixture cannot be separated by paper chromatography. Less Accurate compared to HPLC or HPTLC

11. THIN LAYER CHROMATOGRAPHY Introduction: TLC is one of the simplest, fastest, easiest, and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds. TLC is a form of liquid chromatography consisting of : A mobile phase (developing solvent) A stationary phase (a plate or strip coated with a form of silica gel) Analysis is performed on a flat surface under atmospheric pressure and room temperature) Definition: Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent soild / ( liquid) spread over a glass plate and liquid as a mobile phase. It is similar to paper chromatography, expect that a thin (0.25mm) layer of some inert material, such as Aluminium oxide, Magnesium oxide, Silicon dioxide. A layer of any one of these oxides is made from a slurry of powder in a suitable inert solvents. The slurry is spread evenly over a flat surface (glass) and dried. It may be spread manually or mechanically.

12. PRINCIPLE OF TLC TLC is based on the principle of separation. And separation depends on the relative affinity of compounds towards stationary and mobile phase. The compounds under the influence of mobile phase travel over the surface of the stationary phase, During this movement, the compounds with higher affinity to stationary phase travel slowly while others travel faster. Thus, separation of components in the mixture is achieved. Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character are identified by means of suitable detection techniques. VERSATILITY OF TLC Simple equipments : TLC mostly requires very simple equipments, such as : micro-slides ; specimen jars with lid ; glass-sprayers ; strips of glass sheet ; small chromatank etc. Short development time : In TLC, the separation is very rapid i.e., the development time is of short duration (say 1 hour) for reasonably good separation on inorganic adsorbent layers. Wide choice of stationary phase : TLC may be used for adsorption, partition (including reversed phase) or ion-exchange chromatography,

13. Quick recovery of separated constituents : TLC permits the possibility of removal of the adsorbent coating on the plates by scraping with a spatula. In other words, a spot or a zone can be removed quantitatively, and the separated constituent dissolved in an appropriate solvent is estimated either by suitable spectrophotometric or colorimetric analysis. Separation effects : The separation effects obtained by TLC are more distinctive and superior than those of paper chromatography. Easy visualization of separated components : Detection of fluorescence components when exposed to UV light is much easier than on paper by virture of the fact that inorganic material (i.e., adsorbent) has intrinsic fluorescence. Variable thickness of think layers : The method employed in TLC may be further extended to preparative separations by using thicker layers and also to meet separations by column chromatography. Trace analysis : TLC method is suitable as micro method in trace analysis. EXPERIMENTAL TECHNIQUES OF TLC The various techniques with regard to thin layer chromatography (TLC) are as stated below, namely : 1.) PREPARATION OF THIN LAYERS ON PLATES. The paramount importance with regard to the preparation of thin layer is that it must be uniform and consistent throughout. (a) Pouring of Layers : In order to obtain layers of equal thickness, a measured amount of the suspension or slurry is placed on a given-size plate that is rested on an absolutely leveled surface. The plate is subsequently tipped backward and forward to permit the slurry (or suspension) to spread uniformly on the surface of the plate.

14. (b) Dipping : Two plates at a time back-to-back are dipped together in a slurry of the adsorbent in either chloroform or chloroform-methanol. However, this particular methods is not much in use now-a-days. (c) Spraying : This makes use of a small paint-sprayer for the distribution of the suspension or slurry onto the surface of the glass-plate. Disadvantages : There are mainly two major disadvantages of this technique, namely : Non-uniformity of layers on a single-plate. Variation observed from one plate to the other was significant. (d) Spreading : In this particular case, the suspension or slurry is put in an ‘applicator’, which is subsequently moved either over the stationary glass-plate or vice-versa i.e., it is held stationary while the glass plate is pulled or pushed through. This technique termed as ‘spreading’ usually yields uniform thin layers on the glass plates. (e) ‘TLC-Plates ready-for Use’ (or Pre-coated Plates). precoated TLC-plates essentially have : first, a special abrasive-resistant layer containing no gypsum ; and secondly, the layer contains a reliable fluorescent indicator that is excited to emit a fluorescence under either a short-wave or a long-wave UV light. CHOICE OF ADSORBENTS: It is chiefly based on certain crucial informations like : (i) Solubility of the substance e.g., hydrophilic and lipophilic (ii) Nature of the compound i.e., whether it is acidic/basic/neutral/amphoteric (iii) Reactivity of compound with either the solvent or the adsorbent iv) Chemical reactivity of compounds with the binders.

15. Inorganic adsorbents Aluminium Oxide Aluminium Silicate Bauxite(Aluminium oxide ore) Bentonites Calcium Carbonate Calcium Hydroxide Calcium Oxalate Calcium Silicate Calcium Sulphate Dicalcium Phosphate Fuller’s Earth Hydroxyl- Apatite Kieselguhr(Diatomaceous Earth) Magnesium Silicate Silica Gel Tri- Calcium Phosphate Zinc Carbonate Organic adsorbents Cellulose and Acetylated Cellulose Charcoal and Activated Carbon Dextran Gels Ion- Exchange Resins Polyamide Polyethylene Powder Sucrose

16. CHOICE OF SOLVENT SYSTEM IN TLC The choice of solvent or a mixture of solvents used in TLC is solely guided by two important factors : The nature of the constituent to be separated i.e., whether it is polar or non-polar The nature of the process involved i.e., whether it is a case of ‘adsorption’ or ‘partition chromatography’. ACTIVATION OF ADSORBENT In fact, it is extremely important to eliminate as completely as possible the solvent imbedded into the thin layer of coated adsorbent. It is achieved conveniently first by air-drying the TLC plates for a duration of 30 minutes and then in a hot-air oven maintained at 110 °C for another 30 minutes and subsequently cooling them in a dessicator. This drying process helps a great extent in rendering the adsorbent layer active. SPOTTING OF THE COMPONENTS The following points may be strictly adhered to while spotting the component or mixture of components on a TLC plate, namely : (i) The sample is normally applied as a solution in a ‘non-polar solvent’ as far as possible, since the use of a polar solvent may cause: (a) spreading out of the starting spot (b) affect directly the Rf value of components.

17. (ii) The solvent employed for dissolving the sample must be easily volatile-in nature so that it should be removed from the TLC plate before development commences (iii) The ‘area of application’ should be smallest as far as possible so as to achieve a sharper resolution. (iv) To maintain the size of the spot ‘small’ repeated applications is made by allowing the solvent to evaporate after each application. It can be easily achieved by : (a) Pre-warming the TLC-plate (b) Passing a stream of hot-air right below the sample spot (from a hair-drier). (v) For exclusively ‘preparative work’ the sample is applied in a narrow-band the width of which must be kept as narrow as possible. DEVELOPMENT OF THIN LAYERS The spotted TLC plates, after evaporation of the sample solvent, is placed in a closed chamber saturated with vapours of the developing solvent(s). One end of the plate is then wetted with the developer by means of ascending-technique After the developer has traversed one-half to two-thirds the total length of the TLC plate, the latter is removed from the chamber, air-dried and the positions of the components are located by any of several methods. There are three major factors which essentially govern the ‘development of thin layers’, namely : (i) Equilibration of the chamber (or chamber-saturation) (ii) Protection against oxidation (temperature and light) (iii) Visualization.

18. Equilibration of the Chamber The equilibration of the chamber or chamber-saturation is a vital factor to obtain reproducible Rf values. It may be achieved by allowing the solvent system to remain in the chamber for at least 1 to 2 hours so that the vapours of the solvent(s) would pre-saturate the latter adequately. This is done to obtain distinct separation of constituents, uniform solvent from and prevent evaporation of the solvent on TLC-plates. Two-Dimensional Chromatography : It is also termed as two-dimensional planar chromatography. Here, the sample is spotted in one corner of a square TLC plate (size : 20 cm × 20 cm). The development is first carried out in the ascending direction using solvent-1. The solvent is then eliminated by evaporation and the plate is rotated through 90°, following which ascending with the second solvent is accomplished. After removal of the solvent the spots of separated constituents are located by spraying with specific reagents. Example : Mixture of amino acids obtained from protein hydrolysates are separated by this method and spots located by using Ninhydrin Reagent that forms a pink to purple product with amino acids.

19. Centrifugal Chromatography : It essentially makes use of the ‘centrifugal force’ so as to accelerate the flow of solvent through the thin-layer of the chromatogram. The layers of plaster-of- paris bound alumina or silica gel were directly applied to either circular glass or aluminium plates with a hole in the centre to enable it to fit into the centrifuge. As usual, the sample mixture is applied 2.5 cm from the centre hole and the solvent system is set to allow a constant flow, with the centrifuge rotating at 500-700 RPM. In this manner, the usual developing time of 35 minutes is drastically reduced to mere 10 minutes by acceleration. DETECTION OF COMPONENTS After development of TLC plates, the next important step is to detect the separated components so as to determine their respective Rf values. Example : (i) Coloured Substances : e.g., Xanthophylls, Chlorophylls, Carotenes, etc., may be located visually. (ii) Colourless Substances : e.g., alkaloids, steroids, amino acids and the like may be detected under short-wave UV-light or a long-wave UV-light. These substances may also be detected as brown/dark brown spots when exposed to I2-vapours in a closed dessicator. (iii) Specific Detecting Reagents : A few specific detecting reagents are normally used for a particular class of compounds e.g., Aniline-phthalate reagent : for carbohydrates Ninhydrin reagent : for amino-acids Dragendorff’s reagent : for alkaloids

20. (iv) Chromic acid/conc. H2SO4 : These corrosive reagents usually char the organic material on TLC plates and may be seen as dark brown spots. EVALUATION OF THE CHROMATOGRAM After completing the detection procedure the various separated solutes on the TLC plate are marked with the help of a sharp needle; subsequently, their evaluation may be carried out either qualitatively or quantitatively. Qualitative Evaluation : The Rf value (Retention Factor) various separated solutes is determined accurately. The Rf value represents the differences in rate of movement of the components duly caused by their various partition coefficients i.e., their different solubility in the mobile and stationary phases. Rf = Distance travelled by a solute Distance travelled by solvent Important Points : Due to the always longer path of the solvent front, the Rf value is invariably lesser than 1. Rf value is always constant for each component only under identical experimental parameters Rf value depends upon a number of governing factors, such as : quality of the layer material ; activation grade of the layer ; thickness of layer ; quality of solvent ; equilibration of chamber ; chromatographic technique employed (e.g., ascending, descending) ; presence of impurities ; and conc. of sample applied.

21. Quantitative Analysis: The quantitative analysis of chromatographically separated constituents may be carried out with high degree of accuracy and precision in two manners, namely : (i) Direct Method : i.e., the quantitative determinations is performed directly on the adsorbent layer. (ii) Indirect Method : i.e., the separated constituents are quantitatively removed from, the adsorbent and subsequently estimated after elution. Direct Methods : Measurement of Spot-areas : This method is solely based on a mathematical relationship existing between the prevailing spot area and the amount of component present. It is not quite accurate due to high random errors. (ii) Densitometry : The intensity of the colour of a component is measured on the chromatogram using a densitometer. (iii) Spectrophotometry : Characterization of the separated spots by reading the absorption or fluorescence curves directly from TLC plates is carried out with the help of Chromatogram Spectrophotometer. Besides, IR-spectroscopy, reflectance spectroscopy, spark chamber method etc., may also be employed for the direct evaluation of chromatograms. Indirect Methods : These methods are based on elution techniques, followed by micro-analysis of the resultant eluate by adopting one or more of the undermentioned known methods, namely : Colorimetry ; Fluorimetry ; Radiometry ; Flame-photometry ; UV Spectrophotometry ; Gravimetry ; Polarography.

22. APPLICATIONS OF TLC To identify the presence of undesirable specific organic compounds present as impurities in a number of pharmaceutical substances, namely : morphine in apomorphine hydrochloride ; hydrazine in carbidopa ; 3-aminopropanol in dexampanthenol ; etc Related substances present in official drugs, namely : related substances present in a wide number of potent pharmaceutical substances e.g., aminophylline ; baclofen ; chloramphenicol ; carbamazepine etc Foreign alkaloids present in alkaloidal drugs, for instance : atropine sulphate ; codeine . Foreign steroids present in steroidal drugs, for example : betamethasone valerate Ninhydrin positive substances in official amino acids e.g., glutamic acid ; leucine TLC helps in selecting the best combination of solvent and adsorbent for a given column separation.

23. ION EXCHANGE CHROMATOGRAPHY Introduction : Ion exchange chromatography is type of adsorption chromatography. There is a reversible exchange of similar charged ions. Mostly similar charged ions like cations and anions can be conveniently separated by this technique Definition : Ion exchange chromatography is a process by which a mixture of similar charged ions can be separated by using an ion exchange resin which exchanges ions according to their relative affinities . PRINCIPLE OF ION EXCHANGE CHROMATOGRAPHY This is by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin. CATION EXCHANGE: The separation of cations using cation exchange resin. The cations to be separated are present in solution and exchanges for similar ions present in cation exchange resin, a soild matrix. The exchange can be represented by the following equation:

24. The cations retained by the solid matrix of ion exchange resin can be eluted by using buffers of different strength and hence separation of cations can be effected. ANION EXCHANGE: Separation of anion using anion exchange resin can be carried out. The anions to be separated are present in solution and exchange for similar ions present in anion exchange resin, a solid matrix the exchange can be represented by the following equation: The anions retained by the solid matrix of ion exchange resin can be eluted by using buffers of different strength and hence separation of anions can be effected.

25. The fundamental requirements of a useful resin are : The resin must be sufficiently cross-linked to have only a negligible solubility The resin must be sufficiently hydrophilic to permit diffusion of ions through the structure at a finite and usable rate The resin must contain a sufficient number of accessible ionic exchange groups and it must be chemically stable; The swollen resin must be denser than water. Factors Affecting ion exchange resin: 1 . Nature of exchanging ions: ( a ) At low aqueous concentrations and at ordinary temperatures the extent of exchange increases with increasing charge of the exchanging ion, i.e. (b) Under similar conditions and constant charge, for singly charged ions the extent of exchange increases with decrease in size of the hydrated cation while for doubly charged ions the ionic size is an important factor but the incomplete dissociation of salts of such cations also plays a part (c) With strongly basic anion exchange resins, the extent of exchange for singly charged anions varies with the size of the hydrated ion in a similar manner to that indicated for cations. In dilute solution multicharged anions are generally absorbed preferentially.

26. 2. Nature of ion exchange resin : The absorption* of ions will depend upon the nature of the functional groups in the resin. It will also depend upon the degree of cross-linking: as the degree of cross-linking is increased, resins become more selective towards ions of different sizes (the volume of the ion is assumed to include the water of hydration); the ion with the smaller hydrated volume will usually be absorbed preferentially. Exchange of organic ions : Although similar principles apply to the exchange of organic ions, the following features must also be taken into consideration. 1. The sizes of organic ions differ to a much greater extent than is the case for inorganic ions and may exceed 100-fold or even 1000-fold the average size of inorganic ions. 2. Many organic compounds are only slightly soluble in water so that non-aqueous ion exchange has an important role in operations with organic substance. Clearly the application of macroreticular ( macroporous ) ion exchange resins will be often advantageous in the separation of organic species. Ion exchange capacity: The total ion exchange capacity of a resin is dependent upon the total number of ion-active groups per unit weight of material, and the greater the number of ions, the greater will be the capacity. The total ion exchange capacity is usually expressed as millimoles per gram of exchanger. Values for the total exchange capacities, expressed as mmolg-1 of dry resin The total exchange capacity expressed as mmol ml-1 of the wet resin.

27. ION EXCHANGE CHROMATOGRAPHY Ion chromatography is relatively new technique which employs well established principles of ion exchange and allows electrical conductance to be used for detection and quantitative determination of ions in solution after their separation.

28. Separator column : The specific capacity of the separating column is kept small by using resins of low capacity. For example, low-capacity anion exchangers have been prepared by a surface agglomeration method in which finely divided anion exchange resin is contacted with surface sulphonated styrene-divinylbenzene copolymer; the small particles of anion exchanger are held persistently on the oppositely charged surface of the sulphonated beads. These resins are stable over a wide range of pH, in which respect they are superior to glass- or silica-based pellicular resins. Suppressor column : Where electrical conductance is used for detection of sample ions in the effluent from the columns, an eluant background of low conductivity is required. The function of the suppressor column is to convert eluant ions into species giving low or zero conductance, e.g. where NaHCO3 is used as eluant for anion analysis this is converted into a background of dilute carbonic acid by using a strong acid suppressor resin. Detectors : Although electrical conductance has been widely used for detecting ions in ion chromatography, the scope of the technique has been considerably extended by the use of other types of detector. It is convenient broadly to classify detectors into two series. 1. Detectors employing electrochemical principles: (a) Conductimetric detectors. Conductance is a fundamental property of ions in solution making it an ideal technique for monitoring ion exchange separations because of its universal and linear response. It is the optimum mode of detection for strong acid anions ( pKa < 7), providing high sensitivity in the absence of background electrolyte. (b) Amperometric detectors. This type of detector may be used to detect ions which are electrochemically active but not readily detected by conductance measurement, e.g. weak acid anions such as CN-, HS- ( pKa > 7). The detector commonly features interchangeable silver or platinum working electrodes and may be used alone (no suppressor then being required) or simultaneously with a conductivity detector.

29. 2. Detectors based on established optical absorption and emission techniques : Spectrophotometric detectors : The operation of spectrophotometric detectors is based on the absorbance of monochromatic light by the column effluent in accordance with the Beer Lambert law. As most organic species have significant absorption in the UV region of the spectrum, these detectors have wide application. Sensitivity clearly depends on how strongly the sample absorbs at the wavelength of maximum absorption, but detection limits in the low- (or even sub-) nanogram range may be achieved in favourable conditions. (b) Fluorescence detectors: Although only a small proportion of inorganic and organic compounds are naturally fluorescent, the inherent sensitivity and selectivity of fluorescence detection offers significant advantages. The development of appropriate pre-column and post column derivatisation procedures has furthered the application of fluorescence detection for the trace analysis of non-fluorescent or weakly fluorescing species.

30. APPLICATION OF ION EXCHANGE CHROMATOGRAPHY Separation of similar ions from one another, For example- mixture of Li ion , sodium ion and potassium ion can be separated by passing their solution through a cation exchanger. Removal of interfering radicals Softening of Hard Water Complete demineralization of water Separation of Lanthanides Separation of Actinides Purification of organic compounds extracted in water Separation of sugars Separation of amino acids Preparation of pure reagents Hydrometallurgy

31. REFRENCES Gurdeep. R Chatwal, Sham K. Anand; instrumental method of chemical analysis (Analytical chemistry)