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

Ion Exchange Chromatography

+

+- -- -

The stationary phase has an ionically charged surface opposite to that of the analyte

Factors controlling retention:

Charge

Dimension

+ ++ +++

+ + +

Retention time

Principle

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Cation exchange

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+ ++

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Anion exchange

Competition between the analytes and the ions in the mobile phase

Retention mechanism

+

+

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A-

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A-

-- A-

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A--

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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

-

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