ion exclusion chromatography.pptx

this ppt involves detailed description of ion exclusion chromatography.

1. Presented By Chintu Lahkar M.pharm , Roll no- 02 Pharmaceutical chemistry NETES Institute Of Pharmaceutical Science Mirza, Kamrup-781125,Assam ION EXCLUSION CHROMATOGRAPHY

2. CONTENTS Introduction Principle Instrumentation Applications Reference

3. INTRODUCTION First introduced by Wheaton and Bauman in 1953. Useful technique for separation of ionic and non-ionic substances. IEC is opposite to ion-exchange chromatography. Ion exclusion chromatography (IEC) separates ionic from non-ionic compounds using an ion exchange stationary phase. Negatively charged ions are separated on cation-exchange resins, while positively charged species are separated on anion-exchange resins. The same column can be used for both ion exchange and ion exclusion chromatography. IEC is also referred to by several other names, including ion exclusion partition chromatography, ion chromatography- exclusion mode, and Donnan exclusion chromatography.

4. PRINCIPLE Ionic substances pass quickly through the column. Non-ionic (molecular) or partially ionized substances are held up and are eluted more slowly. Functions by Donnan exclusion mechanism. Mobile phase- liquid (water or aqueous acid or base) Stationary phase- ion exchange resins Cationic Anionic Chromatographic system consists of 3 phases. Mobile phase Resin phase Occluded liquid phase

5. Contd. The mobile phase passes through the interstitial volume existing between the beads of the ion exchange resin. The resin phase is the solid resin network and functionalized groups, which can be considered to be a semipermeable ion exchange membrane separating the flowing mobile phase from the stationary occluded liquid inside the resin. An occluded liquid phase is formed by mobile phase that becomes trapped within the pores of the resin phase. This trapped liquid acts as the stationary phase of the system

6. When the column is filled with water, pumped through as a mobile phase, the water molecules accumulate as hydration spheres around the dissociated functional groups of the support. Water contained in the pores of the support and in the hydration, spheres is immobilized, thus forming the stationary phase. Contd.

7. Contd. The commonly accepted retention mechanism of IEC involves the formation of a pseudo semipermeable membrane around the resin stationary phase 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, whereas neutral species can penetrate the resins pores and stationary occluded phase, thus experiencing retention Due to the high density of charged groups in the stationary phase a potential difference is generated which leads to the formation of a virtual membrane - the Donnan membrane. This charged membrane only permits neutral molecules to reach the stationary phase. Charged molecules can not pass the Donnan membrane

8. Stationary phase Column Glass, stainless steel or polymer Length: Diameter ratio 20:100 to 100:1 Sacking the column Wet packing method Application of the sample After packing sample is added to the top of the column, use syringe or pipette Mobile phase Acid, Alkali, Buffers INSTRUMENTATION

9. ELUENTS: 3 types of eluents are used in ion exclusion chromatography. Water eluent Acid eluent Complexing eluent DEGASSING UNIT: 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. ANALYTICAL COLUMN: The analytical column consists of stationary phase (wet packing) with reactive layer bonded to inert polymeric particles. Stationary phases must satisfy implicitly a number of requirements as narrow granulometric distribution (monodisperse), large specific surface area, mechanical resistance, stability under acid and basic pH and rapid ion transfer. Contd.

10. Contd. 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 co-polymerization of styrene with divinylbenzene is necessary in order to obtain the required stability of the resin.

11. Contd. SUPPRESSOR: 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+. 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

12. Contd. DETECTOR: Conductivity Detector: 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. Detectors with UV- Visible spectrophotometry: 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.

13. Contd. 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. Fluorescent based Detectors: 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

14. APPLICATIONS Determination of neutral molecules: Neutral compounds such as sugars and alcohols can be separated by IEC. Determination of strong inorganic acids: Sulphate, nitrate and chloride ions, and strong base cations such as sodium, ammonium, potassium. Magnesium and calcium ions commonly found in acid rainwater. Separation of carboxylic acids: - The separation of carboxylic acids is the most common application of ion-exclusion chromatography Determination of Weak inorganic acids and bases: - IEC has found increasing use for the determination of weakly ionized inorganic anions such as fluoride, nitrite, phosphate, sulphite , arsenite , arsenate, bicarbonate, borate and cyanide. Determination of water in some organic solvents. Determination of amino acids and its derivatives.

15. REFERENCE D. A. Skoog, F J. Holler, S. R. Crouch, “Principles of Instrumental Analysis”, 6 th edition, Thomson Brooks/Cole, page no: 839-847. S. Ahuja, M. W. Dong, “ Handbook of Pharmaceutical Analysis by HPLC”, Volume 6, ELSEVIER Academic Press, page no: 219- 253. G. D. Christian, P. K. Dasgupta, K. A. Schug , “ Analytical Chemistry”, 7 th edition, John Wiley & Sons, page no: 653-665. M. Mori, K. Tanaka, Q. Xu, M. Ikedu , H. Taoda , W. Hu, "Highly sensitive determination of hydrazine ion by ion-exclusion chromatography with ion-exchange enhancement of conductivity detection." Journal of Chromatography A 1039, no. 1-2 (2004): 135-139. B. K. Glód , "Ion exclusion chromatography: parameters influencing retention."  Neurochemical research  22 (1997), pageno:1237-1248.    

16. OPEN TO DISCUSSION

17. THANK YOU