ION EXCLUSION CHROMATOGRAPHY

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1. ION EXCLUSION CHROMATOGRAPHY PREPARED BY- SOBHI GABA

2. Name : SOBHI GABA Designation: STUDENT Department: M. PHARMA (PHARMACEUTICAL CHEMISTRY) E-mail: [email protected]

3. Ion-exclusion chromatography was first introduc e d by Wheaton and Bauman in 1953. It involves the use of strong anion- or cation -exchange resins for the separation of ionic solutes from weakly ionized or neutral solutes. INTRODUCTION

4. Principle of ion exclusion chromatography

5. In this mode of chromatography , the charge sign on the ion-exchange resin used is the same as that of the weakly ionized solutes. That is, solutes with a partial negative charge (such as carboxylic acids) are separated on a cation - exchange resin having anionic sulfonate functional groups, whereas solutes with a partial positive charge (such as weak bases) are separated on an anion exchange resin having cationic quaternary ammonium functional groups. This is the opposite situation to that occuring in ion-exchange chromatography. Principle continued…

6. The principle of this type of chromtography is based on Donnan Exclusion Mechanism Non ionic or partially ionized molecules are held up the stationery phase whereas the strongly ionised molecules experiences repulsion from the stationery phase due to which they are eluted first. The principles of ion-exclusion chromatography can be illustrated in a schematic manner by considering the chromatographic system to be comprised of three distinct phases. The first of these is the flowing eluent, which passes between the beads of ion exchange resin (i.e. through the interstitial volume). The second zone is the polymeric network of the resin material itself, together with its bound ionic functionalities, the third zone is liquid occluded inside the pores of the resin bead. The polymeric resin can be considered as a semi-permeable, ion-exchange membrane which separates the flowing eluent from the stationary occluded liquid inside the resin

7. Figure presenting the 3 phases of the chromatographic system in ion exclusion chromatography RESIN – CATION EXCHANGE RESIN acidic solutes, such as acetic acid and HCI, and (b) basic solutes, such as NH 3 and NaOH .

8. Ionic solutes of similar charge to the stationary phase (generally sulfonated cation exchangers are used for weak acid analytes ), experience repulsion from the resin surface, where as neutral species can penetrate the resins pores and stationary occluded phase, thus experiencing retention.

9. When the column is filled with water, which is pumped through as mobile phase, the water soluble molecules build up hydration spheres around the dissociated functional groups of the support. W ater contained in the pores of the support and in the hydration spheres is immobilized thus forming the stationary phase The basic mechanism is that the neutral and uncharged molecules can penetrate the resin, whereas similarly charged co-ions are repelled owing to the presence of dissociated functional group immobilized in the stationary phase . RETENTION MECHANISM

10. Thus, analytes are separated by : Exclusion or repulsion if they are ionized and have the same charge as the resin. ii. Adsorption if they are non-polar or partially ionized.

11. The eluent is water or aqueous acid or base with little or no organic modifier, which offers environmental and economic benefits in addition to compatibility with aqueous sample matrices . Difficult separations such as simple aliphatic carboxylic acids (e.g., formate , acetate, propionate and butyrate) are possible. It is compatible with a wide range of detection methods. The columns can be ion exchange columns, reversed-phase or normal-phase columns, or dynamically modified reversed phase with an ion pairing agent. It is stable for long-term analysis of complex samples such as wine or mustard. ADVANTAGES

12. I. STATIONERY PHASE 1 . COLUMN Glass, stainless steel or polymer Length: Diameter ratio 20:100 to 100:1 2. PACKING THE COLUMN Wet packing method II. APPLICATION OF THE SAMPLE After packing sample is added to the top of the column, use syringe or pipette III. MOBILE PHASE Acid, Alkali, Buffers IV. ELUTION Components of mixture separated & move down the column at different rates depending upon the affinity of the ion for ion exchanger. V. ANALYSIS OF THE ELUATE spectrophotometric ., Conductometric PRACTICAL REQUIREMENTS

13. INSTRUMENTATION

14. 3 types of eluents are used in ion exclusion chromatography. Water eluent Acid eluent ( dilute solutions of strong mineral acids to be employed for the elution of anionic solutes, or dilute solutions of strong bases to be employed for the elution of cationic solutes). Complexing eluent ( The retention and detection properties of some solutes can be enhanced if a complexing agent is added to the eluent. An example of this approach is the use of a mannitol eluent for the determination of boric acid, in which the mannitol serves to complex the boric acid to form a species which is more easily detectable by conductivity measurements than is boric acid alone). ELUENTS

15. Removing dissolved gases in mobile phases is an important step for ensuring proper function of pump check valves, and to prevent outgassing in the detector flow cell. II. Degassing unit

16. The analytical column consists of stationery phase (wet packing) The column packing consist of a reactive layer bonded to inert polymeric particles. Stationary phases must satisfy implicitly a number of requirements as narrow granulometric distribution (mono-disperse), large specific surface area, mechanical resistance , stability under acid and basic pH and rapid ion transfer . The most common resins used in ion exclusion chromatography are high capacity PS-DVB-based strongly acidic cation exchange resins of 5 μ particle size. Styrene/ divinylbenzene copolymers are the most widely used substrate materials. Since they are stable in the pH range between 0 to 14, eluents with extreme pH values may be used. The copolymerization of styrene with divinylbenzene is necessary in order to obtain the required stability of the resin. III. ANALYTICAL COLUMN

17. RESINS USED IN ION EXCLUSION CHROMATOGRAPHY

18. Suppression plays a key role in the analysis of anions and organic acids using ion chromatography and conductivity detection. Suppressor is a device placed between the column and the detector, and acts to reduce the background conductivity of the eluent and enhance the conductivity of the analytes . For anion analysis, the suppressor is a high capacity cation exchange membrane or resin in the acid form. It removes cations from the eluent and replaces them with H+ . Suppression decreases the background conductivity of the eluent minimizes baseline noise transforms analytes in free anions with protons as counter ions (which involves a remarkable increase in the conductivity signal) optimizes the signal-to-noise ratio increases the detection sensitivity of the measurement system IV. SUPPRESSOR

19. Analyte : Na + Cl – + RSO 3 – H + H + Cl – + RSO 3 – Na + This example of an anion analysis includes a sodium analyte counter ion . This ion is replaced with a proton with an equivalent conductivity that is five times higher. This significantly increases the conductivity of the sample solution and therefore also the signal strength. Salts from weakly dissociated acids (e.g., sodium carbonate/sodium hydrogen carbonate) are used as eluent . Eluent Na + HCO 3 – + RSO 3 – H + H 2 CO 3 + RSO 3 – Na + The eluent counter ions are also replaced with protons. The carbonic acid that is produced in this way is unstable and only weakly dissociated, meaning that lower background conductivity is measured. Depending on the eluent composition, background conductivity values of 10 to 20 µS/cm are typical for chemical suppression.

20. Detectors used in this type of chromatography are as follows:- Conductivity Detector Detectors with UV- Visible spectrophotometery Fluorescent based Detectors DETECTORS

21. In ion chromatographic practice, conductivity detectors are most commonly used. The conductivity of the solutions is an additive property, depending on the quality of the ions (mobility) and the number of ions (concentration). In principle, the conductivity detector can be used for some non-aqueous eluents. The sensitivity of these detectors depends on the temperature; during the separation and detection the temperature must be kept strictly constant. CONDUCTIVEITY DETECTORS

22. Detectors with UV-Visible spectrophotometry are often used for detection. This is used in cases where the component is absorbed in the UV-Visible range . Examples include iodide, nitrite, nitrate, iodate or chromate ions. The detector is photodiode and the cell is a quartz cuvette. Deuterium and tungsten lamps are used as a source of light . In addition, a diode array detector can be used, if the purpose is to simultaneously detect light absorption at different wavelengths. UV- VISIBLE DETECTORS

23. We can detect fluorescent materials with fluorescence-based detectors. The principle of detection is that the components of the sample are excited by a given wavelength light and the components emit light and we can detect this light. For biological samples, this type of detection method is common. FLUORESCENT BASED DETECTORS

24. Separation of carboxylic acids Determination of Weak inorganic acids and bases Determination of strong inorganic acids . Determination of neutral molecules . D etermination of water in some organic solvents Determination of amino acids and its derivatives APPLICATIONS

25. The separation of carboxylic acids is the most common application of ion-exclusion chromatography Separation of carboxylic acids

26. Solutes such as fluoride , carbonate , cyanide, borate, sulfite, phosphates, nitrite, arsenite , arsenate and ammonium have been determined using this approach . Interference from strongly ionized species is minimal because these solutes are unretained and appear at the column void volume . Ion-exclusion chromatography can therefore readily separate weakly ionized solutes in samples containing high concentrations of ionic species, e.g. seawater and wastewater. Determination of Weak inorganic acids and bases

27. Determination of strong inorganic acids

28. Determination of neutral molecules .

29. Determination of water in some organic solvents

30. Determination of amino acids and its derivatives

31. THANKS For Further Detail/SUGGESTIONS Please Contact ISF COLLEGE OF PHARMACY, MOGA (An Autonomous College) Ghal Kalan , GT Road, Moga - 142001, ( Pb .) E- mail: [email protected] Website: www.isfcp.org