ion exchange chromatography. + + ---- the stationary phase has an ionically charged surface opposite...
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The stationary phase has an ionically charged surface opposite to that of the analyte
Factors controlling retention:
Charge
Dimension
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Retention time
Principle
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Cation exchange
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Anion exchange
Competition between the analytes and the ions in the mobile phase
Retention mechanism
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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
Influence of the couter-ion
Mobile phase
1. Acetate2. Lactate3. Succinate4. Nitrite5. Malate6. Chloride7. Nitrate8. Tartarate9. Citrate10.Sulfate
column: Carbon IC BI-01mobile 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) 305-309
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
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
Use of an elution gradient
Mobile phase
The separation of four species with different negative net charges using salt gradient mode
Exchange capacity = concentration of interaction sites
Nature of the ionic group = strength of the interaction
Strong anion exchanger: -NR3+
Weak anion exchanger: -CH2CH2-N(CH2CH3)2H+
Strong cation exchanger: -SO3-
Weak cation exchanger: -CO2-
Nature of the supporting phase (silica gel or polymer) = possible additional interactions
Stationary phase
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
Example: inorganic anions
F-
Cl-
PO43-
SO42-
Br-
I-
NO3-
NO2-
Dugo et al., Food Chemistry, 102 (2007) 599-605
Standard inorganic anions
Stationary phase: Metrosep Anion (RNH3+) Dual 1 column (150 × 3.0 mm, 10 μm)
Mobile phase: pH 8 buffer (CO32- / HCO3
-) / 2% acetone; 0.5 mL/minConductivity detector, 20°C
Example: inorganic anions
F-
Cl-
Br-
I-
Dugo et al., Food Chemistry, 102 (2007) 599-605
Standard inorganic anions
Stationary phase: Metrosep Anion (RNH3+) Dual 1 column (150 × 3.0 mm, 10 μm)
Mobile phase: pH 8 buffer (CO32- / HCO3
-) / 2% acetone; 0.5 mL/minConductivity detector, 20°C
Increasing size
Example: Cyanide in drinking water
Drinking water with and without cyanideDionex IonPac AS 15 (250 x 2 mm) 63 mM NaOH in degassed deionized water, flow rate 0.25 mL/min, column T 30°CDetection: conductivity
Cl-
Br-
CN-
Christison et al., J. Chromatogr. A, (2007) in press
Example: heavy metals
Pb2+
Cu2+
Cd2+
Co2+
Zn2+
Ni2+
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) 229-241
Example: heavy metals
Pb2+
Cu2+
Cd2+
Co2+
Zn2+
Ni2+
Ion chromatogram of groundwater sample
Santoyo et al., J. Chromatogr. A, 884 (2000) 229-241
pH
pKa2
2-4
pKa1
9-10
NH3+-R-CO2H NH3
+-R-CO2- NH2-R-CO2
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Cation exchange Anion exchange
Elution orderpKa2 ▲
Dimension ▲
Hydrophobicity ▲
pH gradient ▲
pKa1 ▼
Dimension ▲
Hydrophobicity ▲
pH gradient ▼
Amino acids
Example: cation exchange amino acid analysis
Column: TMR-A/75 low-capacity cation-exchange columnmobile phase: (A) 25 mM H3PO4–CH3OH (30:70) (B) 25 mM Na2HPO4–CH3OH (30:70) temperature 40 °Cdetection: UV 210 nm
Yokoyama et al., J. Chromatogr. A, 1085 (2005) 110-116
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) 110-116
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
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<pI0 the protein will have a positive net charge and will bind to a negatively charged support or cation exchange medium
Salting out will elute the protein from the columnThe salt solution will compete the protein in binding to the column
The column has a higher attraction for the charge of salts than for the charged protein and it will release the protein in favour of binding the
salts instead
Different proteins will elute at different salt concentrations, so an elution gradient can be used
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°CElectrochemical detection
Murkovoc et al., J. Biochem. Biophys. Methods, 69 (2006) 25-32