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Ion Exchange Chromatography. + + ---- The stationary phase has an ionically charged surface opposite to that of the analyte Factors controlling retention:

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Presentation on theme: "Ion Exchange Chromatography. + + ---- The stationary phase has an ionically charged surface opposite to that of the analyte Factors controlling retention:"— Presentation transcript:

1 Ion Exchange Chromatography

2 The stationary phase has an ionically charged surface opposite to that of the analyte Factors controlling retention: Charge Dimension Retention time Principle

3 Cation exchange

4 Anion exchange

5 Competition between the analytes and the ions in the mobile phase Retention mechanism A Anion of the mobile phase interacts with cation in the stationary phase Progression of the analyte ion depends on its affinity for the cation affinity of the MP anion concentration of the MP anion pH

6 Influence of the couter-ion Mobile phase 1.Acetate 2.Lactate 3.Succinate 4.Nitrite 5.Malate 6.Chloride 7.Nitrate 8.Tartarate 9.Citrate 10.Sulfate column: Carbon IC BI-01 mobile phase: (a)0.35 mM salicylic acid–0.1 mM sodium salicylate (pH 3.5, flow rate 0.8 ml/min) (b)2.0 mM benzoic acid–1.2 mM tris aminomethane (pH 4.4, flow rate 1.0 ml/min) column temperature: 40 °C Yoshikawa et al., Talanta 72 (2007)

7 Influence of the pH Ionisation state of the stationary phase (for weak ion exchangers) Ionisation state of the solute Mobile phase Influence of the salt concentration

8 Use of an elution gradient Mobile phase The higher the net charge, the higher the salt concentration required for desorption The desorption curve is shifted to the right with increasing net charge

9 Use of an elution gradient Mobile phase The separation of four species with different negative net charges using salt gradient mode

10 Exchange capacity = concentration of interaction sites Nature of the ionic group = strength of the interaction Strong anion exchanger: -NR 3 + Weak anion exchanger: -CH 2 CH 2 -N(CH 2 CH 3 ) 2 H + Strong cation exchanger: -SO 3 - Weak cation exchanger: -CO 2 - Nature of the supporting phase (silica gel or polymer) = possible additional interactions Stationary phase

11 The simplest of all LC detectors Consisting of only two electrodes When ions enter the detector cell, the electrical resistance between the electrodes changes and a signal is recorded Can only detect those substances that ionize Electrical Conductivity Detectors Frequently used in the analysis of inorganic and organic acids, bases and salts

12 Example: inorganic anions F-F- Cl - PO 4 3- SO 4 2- Br - I-I- NO 3 - NO 2 - Dugo et al., Food Chemistry, 102 (2007) Standard inorganic anions Stationary phase: Metrosep Anion (RNH 3 + ) Dual 1 column (150 × 3.0 mm, 10 μm) Mobile phase: pH 8 buffer (CO 3 2- / HCO 3 - ) / 2% acetone; 0.5 mL/min Conductivity detector, 20°C

13 Example: inorganic anions F-F- Cl - Br - I-I- Dugo et al., Food Chemistry, 102 (2007) Standard inorganic anions Stationary phase: Metrosep Anion (RNH 3 + ) Dual 1 column (150 × 3.0 mm, 10 μm) Mobile phase: pH 8 buffer (CO 3 2- / HCO 3 - ) / 2% acetone; 0.5 mL/min Conductivity detector, 20°C Increasing size

14 Example: Cyanide in drinking water Drinking water with and without cyanide Dionex IonPac AS 15 (250 x 2 mm) 63 mM NaOH in degassed deionized water, flow rate 0.25 mL/min, column T 30°C Detection: conductivity Cl - Br - CN - Christison et al., J. Chromatogr. A, (2007) in press

15 Example: heavy metals Pb 2+ Cu 2+ Cd 2+ Co 2+ Zn 2+ Ni 2+ Ion chromatogram of a standard solution of heavy metals Eluent (E): 50 mM oxalic acid–95 mM lithium hydroxide; Detection: UV-visible spectrophotometry at 520 nm after post-column derivatisation with 0.2 mM PAR (pyridylazoresorcinol), 3 M ammonium hydroxide, and 1 M acetic acid. Santoyo et al., J. Chromatogr. A, 884 (2000)

16 Example: heavy metals Pb 2+ Cu 2+ Cd 2+ Co 2+ Zn 2+ Ni 2+ Ion chromatogram of groundwater sample Santoyo et al., J. Chromatogr. A, 884 (2000)

17 pH pK a2 2-4 pK a NH 3 + -R-CO 2 H NH 3 + -R-CO 2 - NH 2 -R-CO 2 - Cation exchangeAnion exchange Elution order pK a2 ▲ Dimension ▲ Hydrophobicity ▲ pH gradient ▲ pK a1 ▼ Dimension ▲ Hydrophobicity ▲ pH gradient ▼ Amino acids

18 Example: cation exchange amino acid analysis Column: TMR-A/75 low-capacity cation-exchange column mobile phase: (A) 25 mM H 3 PO 4 –CH 3 OH (30:70) (B) 25 mM Na 2 HPO 4 –CH 3 OH (30:70) temperature 40 °C detection: UV 210 nm Yokoyama et al., J. Chromatogr. A, 1085 (2005)

19 Example: amino acid analysis in urine samples Chromatogram for urine of patient with phenylketonuria. Chromatogram for urine of healthy newborn. Yokoyama et al., J. Chromatogr. A, 1085 (2005)

20 Charge properties of proteins and peptides The titration curve reflects how the net charge of a protein or peptide varies with pH Charged amino acids on the surface of a protein can bind to oppositely charged ligands of the ion exchanger Change the pH = change the ionisation state = change retention

21 Example: proteins and peptides IEC is another general method for protein purification or enrichment When pH>pI, the protein will have a negative net charge and will bind to a positively charged support or anion exchange medium When pH

22 Example: Carbohydrates Carbohydrates in coffee Dionex CarboPac PA 20 (150 x 3 mm, 6.5 μm) in degassed deionized water, flow rate 0.45 mL/min, column T 31°C Electrochemical detection Murkovoc et al., J. Biochem. Biophys. Methods, 69 (2006) 25-32

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