Ion excghnge chromatography

brief discussion about ion exchange chromatography

1. Seminar on ion exchange chromatography PRESENTED BY MANOJ KUMAR . M H.T.NO.636217885003 M.Pharmacy 1 st year UNDER GUIDANCE OF DR.S.Y. MANJUNATH DEPARTMENT OF PHARMACEUTICAL ANALYSIS SRIKRUPA INSTITUTE OF PHARMACEUTICAL SCIENCES [AFFILIATED TO OSMANIA UNIVERSITY] [Approved by PCI;AICTE]

2. INTRODUCTION Ion exchange chromatography is a chromatographic process that separates ions & polar molecules based on their affinity to the ion exchangers i.e. cationic or anionic exchange resins. Ion exchange chromatography retains analyte molecule on the column based on ionic interactions. Essentially the molecule undergo electrostatic interactions with opposite charge on the stationary phase matrix. Columns used for ion exchange are characterized by the presence of charged groups covalently attached to the stationary phase. Anion exchangers contain bound positive groups, where as cation exchangers contain bound negative groups

3. Principle….. Reversible exchange of ions b/w ions present in the solution & ion exchange resin Cation exchange chromatography retains positively charged  cations because the stationary phase displays a negatively charged functional group. Anion exchange chromatography retains anions using positively charged functional group.

4. CLASSIFICATION OF RESINS According to the chemical nature they classified as- 1. Strong cation exchange resin 2. Weak cation exchange resin 3. Strong anion exchange resin 4. Weak anion exchange resin According to the Source they can - Natural : Cation - Zeolytes, Clay Anion - Dolomite Synthetic : Inorganic & Organic resins Organic resins are polymeric resin matrix The resin composed of – polystyrene ( sites for exchangeable functional groups ), divinyl benzene ( cross linking agent ) – offers stability

5. Structural types of ion exchange resins a ) Pellicular type with ion exchange resins: »30 - 40 μ with 1-2 μ film thickness »Very low exchange capacity b) Porous resin coated with exchanger beads » Size 5 - 10 μ » Porous & highly efficient c) Macroreticular resin bead » Not highly efficient & low exchange capacity d) Surface sulfonated & bonded electrostatically with anion exchanger ≫ less efficient & low exchange capacity

6. Physical properties of ion exchange resins Cross linking: It affects swelling & strength & solubility Swelling: When resin swells, polymer chain spreads apart Polar solvents → swelling Non-polar solvents → contraction Swelling also affected electrolyte conc . Particle size & Porosity ↑surface area & ↓particle size will ↑rate of ion exchange Particle size 50-100 mesh / 100-200 mesh Regeneration Cation exchange resin are regenerated by treatment Anion exchange resin are regenerated by treatment with NaOH,

7. Ion exchange resin should have following requirements: It must be chemically stable It should be insoluble in common solvents It should have a sufficient degree of cross linking The swollen resin must be denser than water It must contain sufficient no. of ion exchange groups Practical requirements: column Packing of column Application of sample Mobile phase elution Analysis of eluate

8. Schematic Diagram of Ion exchange chromatography

9. Column: Column used in the laboratories are made up of glass but those used in industries are made up of either high quality stainless steel or polymer, which are resistant to strong acids and alkalis. The separation is improved by increasing the length of the column but the length cannot be increased beyond a critical length. Uneven flow of liquid is possible in case of column too wide or too narrow in size . A Dimension of column is 20:1 to 100:1 for the higher efficiency can be used. Packing of the column : In this wet packing method is used. The resins is mixed with the mobile phases and packed in the column uniformly. The sample to be separated is dissolved in the mobile phases and introduced all at once into the column. Application of the sample : After packing the column, the solution to be analyzed is added to the top of the column and allowed to pass through the bed of ion exchanger. For this purpose the syringe or pipette is utilized 

10. Mobile phase : The organic solvents are less useful so they are not used these days. Only different strength of acids, alkalis and buffer are used as eluting solvent . E.g.: 0.1 N HCl , 1N NaOH, phosphate buffers, acetate buffers, borate buffers, phthalate buffers, etc. Developments of the chromatogram and elution: After introduction of the sample, development of the chromatogram is done by using different mobile phases. The aqueous salt solution is adjusted to a constant ionic strength. The choice of the mobile phase depends on the selectivity of the resin for the solute ions. Two types of elution techniques are used: a . Isocratic elution b . Gradient elution In Isocratic elution technique, the same solvent composition is used i.e., same strength of acids or alkalis or buffers are used .

11. In gradients elution techniques, initially less acidic or basic mobile phase is used. Then, acidity or basicity is increased at regular intervals. The different fraction of the elution is collected volume wise or time wise and analyzed Analysis of the elute or Detection: Different fractions are collected with respect to the volume or time is analyzed for their contents. Several methods of analysis can be used which depends upon the nature and quantity of the ionic species are: * Conductometric method * Amperometric methods * Flame photometric method * Polarography * UV. Spectroscopy * Radiochemical methods using Geiger Muller counter, ionization chamber method .

12. Conductivity detector is the most common and useful detector in ion exchange chromatography. Conductivity detection gives excellent sensitivity when the conductance of the eluted solute ion is measured in an eluent of low background conductance. Therefore when conductivity detection is used dilute eluents should be preferred and in order for such eluents, to act as effective competing ions, the ion exchange capacity of the column should be low. The results obtained are stored in computer .

13. Advantages : Detectability: useful for the detection of many inorganic salts and also for the detection of organic ions with poor uv absorptivity like alkyl amines or sulfonates . Preparative separations: usually preferred because of the availability of volatile buffers . volatile buffers makes the removal of mobile phase easier. Useful to resolve very complex samples, i.e. in the case of multi step separation Useful for separation of mixtures of biological origin, in organic salts and some organo- metallics Disadvantages: Column efficiency is less It is difficult to achieve control over selectivity and resolution Stability and reproducibility of the columns become questionable after repeated use

14. APPLICATIONS: 1.Separation of similar ions A mixture of sodium, hydrogen and potassium can be separated using cation exchanger resin. A mixture of Chloride, bromide, and iodide can be separated using basic anion exchange resin. Method: Mixture of chloride, bromide & iodide is passed through basic anion exchanger using 0.5M sodium nitrate as eluant. Chloride will first elute. Raise the concentration of Sodium Nitrate, Bromide will elute, raise the concentration of Sodium Nitrate further, iodide ion will elute . .

15. 2 .Softening of hard water: Hardness of water due to ca, mg and other divalent ions . This water is passed through cation exchanger charged with the sodium ions. Ca & Mg ions retained in the column while sodium is exchanged . 3 .Complete demineralization of water: Removal of both cations & anions . Step A) Hard water is first passed through an acidic cation exchanger- Ca, Mg & Na are exchanged by H+ions . Step B) This water is then passed thro a basic anion exchanger – Cl, NO2, SO4- are exchanged by OH- ions of the exchanger . 4.Separation of sugars: sugars-borate complexes. This complex is separated on Dewax . In this disaccharides separated from mono .

16. 5 .Separation of Amino Acids: protein after hydrolysis is introduced to a short column on special polystyrene sulphonic acid resin at pH 2 and eluted with 0.35N sodium citrate buffer of pH 5.25. acidic & neutral Amino acids first leave the column as unseparated then others . 6.Medicinal importance: Anionic resins are introduced in the treatment of ulcer while cation exchangers have been used to remove Na+ from body during the treatment of hypertension and edema. The resins are also used as a diagnostic aid in gastric acidity tests. The resins have been successfully used with other medicinal agents to achieve delayed action dosages

17. 7.Removal of interfering radicals: Phosphate ion is the interfering with the calcium & barium ions. Phosphate is removed using sulphonic acid cation exchanger. Calcium & barium ions exchanged with H+ ions while phosphate ion pass through the column. The process has to be repeated so that the phosphate ions are completely removed. Now, the calcium and Ba+ ions held by resin will be removed by using suitable eluent. Finally, these ions are estimated by the usual methods

18. 8.Other applications For the measurement of various active ingredients in medicinal formulations, For the measurement of drugs and their metabolites in serum and urine, for residue analysis in food raw materials, For the measurement of additives such as vitamins and preservatives in foods and beverages .

19. Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) Methods for the liquid chromatographic analysis of carbohydrates have often employed silica-based amino bonded or polymer-based, metal-loaded, cation-exchange columns, with refractive index (RI) or low-wavelength ultraviolet (UV) detection. These analytical methods require attention to sample solubility, sample concentration and, in the case of the metal loaded cation-exchange columns, also require column heating. In addition, RI and low-wavelength UV detection methods are sensitive to eluent and sample matrix components. This usually precludes the use of gradients and often requires stringent sample cleanup prior to injection. As a result, an improved chromatographic technique known as high-performance anion exchange (HPAE) was developed to separate carbohydrates.

20. Coupled with pulsed Amperometric detection (PAD), it permits direct quantification of nonderivatized carbohydrates at low - picomole levels with minimal sample preparation and cleanup . HPAE chromatography takes advantage of the weakly acidic nature of carbohydrates to give highly selective separations at high pH using a strong anion-exchange stationary phase. HPAE-PAD is extremely selective and specific for carbohydrates because: 1. Pulsed amperometry detects only those compounds that contain functional groups that are oxidizable at the detection voltage employed 2 . Neutral or cationic sample components in the matrix elute in, or close to, the void volume of the column . Therefore, even if such species are oxidizable, they do not usually interfere with analysis of the carbohydrate components of interest.

21. Anion-Exchange Chromatography Mechanism of Separation Although anion-exchange chromatography has been used extensively to analyze acidic carbohydrates and glycopeptides. I t has not been commonly used for analysis of neutral sugars. However , examination of the pKa values of the neutral monosaccharaides listed in below Table shows that carbohydrates are in fact weak acids. At high pH, they are at least partially ionized, and thus can be separated by anion-exchange mechanisms . This approach cannot be used with classical silica-based columns because these matrices dissolve at high pH. Anion exchange at high pH is, however, ideally suited

22. Table1: Dissociation constants of some common carbohydrates (in water at 25 °C) Sugar pka Fructose 12.03 Mannose 12.08 xylose 12.15 Glucose 12.28 Galactose 12.39 Ducitiol 13.43 Sorbitol 13.60 α -methyl glucoside 13.71

23. Thermo Scientific™ Dionex™ CarboPac™ Columns 1.Dionex CarboPac PA1 and PA-100 Columns Dionex , now part of Thermo Fisher Scientific, designed the Dionex CarboPac series of columns specifically for carbohydrate anion-exchange chromatography. These columns permit the separation and analysis of mono- , oligo-and polysaccharides . The Dionex CarboPac PA1 and Dionex CarboPac PA100 are packed with a unique polymeric , nonporous, Thermo Scientific™ Dionex ™ MicroBead ™ Pellicular resin. Dionex MicroBead resins exhibit rapid mass transfer, high pH stability (pH 0–14 ), and excellent mechanical stability that permits back pressures of more than 4000 psi (28 MPa). Column reequilibration after gradient analysis is fast, generally taking 10 min or less.

24. The Dionex CarboPac PA1 is particularly well-suited to the analysis of monosaccharides and the separation of linear homopolymers, while the Dionex CarboPac PA100 is optimized for oligosaccharide resolution and separation. 2 . Dionex CarboPac MA1 Column Reduced carbohydrates (also called sugar alcohols) have traditionally been a difficult class of carbohydrates to separate by liquid chromatography. They are weaker acids than their nonreduced counterparts (compare the pKas of glucose and sorbitol or galactose and dulcitol in Table 1 ), and are therefore poorly retained on the Dionex CarboPac PA1 and PA100 columns. The Dionex CarboPac MA1 was developed to address the challenge of retaining and separating extremely weak acids. This column is packed with a macroporous polymeric resin which has an ion-exchange capacity 45 times that of the Dionex CarboPac PA1

25. . As a result, weak anions bind more strongly to the column, requiring higher sodium hydroxide concentrations for elution. The increase in hydroxide ion concentration leads to greater ionization of the sugar alcohols, with greatly improved retention and resolution on the column. Nonreduced neutral oligosaccharides can also be analyzed on the Dionex CarboPac MA1 column, although their analysis times are longer than on the Dionex CarboPac PA1 and PA100 columns. Retention of carbohydrates on the Dionex CarboPac MA1 can be manipulated by altering the sodium hydroxide concentration of the eluent (Table 2). Note that the elution order of several of the compounds changes with the sodium hydroxide concentration .

26. Table 2. k´ Values of selected analytes on the Dionex CarboPac MA1 Column carbohydrate 0.05 0.14 0.25 0.38 0.50 Glucose 15.70 7.19 4.31 2.91 Mannose 13.55 6.15 3.72 2.53 Galactose 17.82 8.25 4.99 3.43 Glycerol 1.13 0.99 0.89 0.80 0.72 M-inositol 1.32 1.08 0.86 0.69 0.56 S-inositol 1.63 1.30 1.02 0.81 0.64 Glcnol 1.81 1.40 1.09 0.89 0.75 Flucitol 1.94 1.63 1.40 1.18 1.05 Sorbitol 6.43 4.72 3.33 2.55 2.06 Dulcitol 6.52 5.04 3.73 2.87 2.26 Mannitol 8.98 6.38 4.37 3.28 2.63 Fucose 10.34 4.72 2.52 1.69 1.25 lactitol 14.97 9.61 5.49 3.57 2.43 Eluent concentration (M Naoh )

27. Comparison of the Dionex CarboPac MA1, PA1, and PA100 characteristic Dionex CarboPac MA1 Dionex carbo PA1 Dionex CarboPac PA100 Recommended applications Mono- and disaccharide alcohol analysis in food products, Monosaccharide compositional analysis, linear homopolymer separations, saccharide purification Oligosaccharide mapping and analysis Resin composition 8.5-μ m-diameter vinylbenzylchloride divinylbenzene macroporous substrate fully functionalized with an alkyl quaternary ammonium group 10-μ m-diameter polystyrene/ divinylbenzene substrate agglomerated with 350-nm Dionex MicroBead quaternary amine functionalized latex 10-μ m-diameter ethylvinylbenzene/ agglomerated with 350-nm Dionex MicroBead quaternary amine functionalized latex

28. Dionex MicroBead latex cross-linking N/A, no latex 5% cross-linked 6% cross-linked Anion-exchange capacity 4500 μeq per 4 × 250-mm column 100 μeq per 4 × 250-mm column 90 μeq per 4 × 250-mm column Recommended flow rate 0.4 mL /min (4 × 250-mm column) 1 mL /min (4 × 250-mm column) 1 mL /min (4 × 250-mm column Organic solvent compatibility 0% 0-2% 0-100% pH compatibility pH 0–14 pH 0–14 pH 0–14 Maximum back pressure 2000 psi (14 MPa) 4000 psi (28 MPa) 4000 psi (28 MPa)

29. Pulsed Amperometric Detection I. Theory of Operation Pulsed amperometry permits detection of carbohydrates with excellent signal-to-noise ratios down to approximatel 10 picomoles without requiring derivatization. Carbohydrates are detected by measuring the electrical current generated by their oxidation at the surface of a gold electrode. The products of this oxidation reaction also poison the surface of the electrode, which means that it has to be cleaned between measurements. This is accomplished by first raising the potential to a level sufficient to oxidize the gold surface. This causes desorption of the carbohydrate oxidation products. The electrode potential is then lowered to reduce the electrode surface back to gold.

30. A.Monosaccharides—Neutral and Amino Sugars These sugars can be successfully separated on the Dionex CarboPac PA1 column using isocratic conditions with 16 mM sodium hydroxide as the eluent. Because the concentration of sodium hydroxide used for the separation is only 16 mM, the column should be regenerated after each run. Otherwise, carbonate will start to contaminate the column, irrespective of the care taken to eliminate it from eluents and samples. Regenerate the column by washing it with 200 mM sodium hydroxide for 10 min at a flow rate of 1.0 mL/min. This procedure will also remove other strongly bound contaminants such as peptides and amino acids. Separation of neutral and amino monosaccharides derived from glycoproteins

31. B. Sugar Alcohols Mono- and oligosaccharide sugar alcohols can be separated using the Dionex CarboPac MA1 column with sodium hydroxide eluents. Examples of isocratic separations are shown in Figures 1 and 2. Gradients can be used to improve separations (Figure 3) or to accelerate the elution of late-eluting components (Figure 4). Table 2 shows that the elution order of certain carbohydrates may be altered by changing the sodium hydroxide concentration Figure 1. Isocratic separation of a group of alditols plus glucose and fructose on the Dionex CarboPac MA1 column Figure 2. Separation of reducing and nonreducing carbohydrates. Food alditols and aldoses are separable under isocratic conditions on the Dionex CarboPac MA1.

32. Figure 3. Separation of alditols found in biological fluids. The NaOH gradient improves the separation of sorbitol and dulcitol, which are poorly resolved at NaOH concentrations that permit resolution of glycerol from inositol Figure 4. Separation of monosaccharide alditols released by direct s-elimination from glycoproteins. The hydroxide gradient following the isocratic separation of the first three components accelerates the elution of mannitol as well as any oligosaccharide alcohols that may have been released during the s-elimination process.

33. REFERENCE: B.K. Sharma; Instrumental Methods of Chemical Analysis; page. No: 123-160 .  G. Vidya Sagar; A Text Book of Pharmaceutical Analysis; volume-II ; page. No: 13 – 18 . Gurdeep R. Chatwal, Sham K. Anand; Instrumental Methods of Chemical Analysis; page. No: 2.662-2.672.