Lecture 18: Ion

Chromatography
 Ion Chromatography

Separation of ionic species by HPLC is not practical

since ionic solutes will have weak retention on
reverse-phase columns and interact strongly with the
polar mobile phase in normal phase chromatography.

Techniques have been developed to help separate

ions and ionizable species.
 Ion Chromatography

Techniques have been developed to help separate

ions and ionizable species.

• Ion Suppression
• Ion Interaction or Ion Pairing
• Ion Exchange
• Ion Exclusion
 Ion Chromatography

Group 1– traditional TLC + HPLC

1. normal phase chromatography
2. reversed phase chromatography

Group 2– Ion Chromatography

4. ion exchange chromatography
3. ion pair chromatography
5. ion exclusion chromatography
 Ion Exchange
Defined as the use of liquid chromatographic methods for the
separation of inorganic anions and cations and low molecular
weight water-soluble organic acids and bases.

Cations and anions form a weak ionic binding with the

stationary phase.
C: Stationary p. = polar (e.g. R-SO3–)
mobile p. = polar (e.g. HNO3 aq.)
or
A: Stationary p. = polar (e.g. R-NR3+)
mobile p. = polar (e.g. Na2CO3 aq.)
Ion-Exchange Chromatography

This technique has been well established for the separation of

ionic solutes.

In aqueous solutions, an ion-exchanger consist of anions,

cations, and water, where either the anion or cation are
chemically bound to an insoluble matrix.

The chemically bound ions are called fixed ions and the
opposite charges ions are called counter ions.
 Ion Exchange

Ion-exchange methods are divided into two main groups

• non-suppressed ion chromatography – comprises all methods
that use an ion exchange column to separate mixtures
• suppressed ion chromatography - uses a suppressor between
the ion exchange column and detector.
Ion Exchange
 Ion Exchange
The suppressor modifies the eluent and solute to improve
detection.

The suppressor requires a regenerant (or scavenger) solution to

enable operation for extended periods.

IC w/o suppression IC w/ suppression

- high bkg conductivity - low bkg conductivity
- high linearity for calibration - non linear calibration
- low cost setup - more expensive
- can choose many eluents - eluent must react to H2O
 Ion Exchange

Stationary Phases

Ion exchange stationary phases typically use low ion-exchange

capacities (range of 10-100 uEq/g). This is historically because
conductivity detectors were originally used in IC, which prefer
eluents of low background conductance to enhance
detectability.

Ion exchangers also tend to have high chromatographic

efficiency.
 Ion Exchange

Stationary Phases

Ion exchangers have functional groups confined to a thin shell

around the surface of the stationary phase particle to reduce the
number of functional groups (ion-exchange capacity) and
improve mass-transfer (chromatographic efficiency).
 Ion Exchange

Composition of the Stationary Phase

substrate / resin carrier

spacer group
(alkyl chain)

group that carries the

separating capacity
 Ion Exchange

Composition of the Stationary Phase

Substrates
• Polystyrene/divinylbenzene
• Polymethacrylate
• Polyalcohol
• Hydroxyethylmethacrylate (HEMA)
• Silicate

Cation Exchangers Anion Exchangers

• sulfonates • quaternary ammonium groups
• carboxylates • alkyl amines
• hydroxy-alkylamines
• alkyl amines with acrylate
type cross-linking
 Ion Chromatography

Ion-Exchange Chromatography

Stationary Phases
 Ion Chromatography

Ion-Exchange Chromatography

Stationary Phases

For cation-exchange resins

- Sulfonic acid exchangers are classified as strong acid types
(keeps negative charge on the fixed ion over a wide pH range)
- all other cation-exchange resins are weak acid types (need a
higher pH for use)
 Ion Chromatography

Ion-Exchange Chromatography

Stationary Phases

For anion-exchange resins

- quaternary amine exchangers are classified as strong base
types
- all other anion-exchange resins are weak base types (functions
only when the pH is low enough to protonate nitrogen)
 Ion-Exchange Chromatography

Stationary Phases

Ion-exchange capacity
• important property
• determined by # of functional groups per unit weight of resin
• most common units are
milliequivalents of charge/gram of dry resin
• measured by saturating a known weight of resin with an ion,
then seeing how much it takes to replace the ion during
washing
• polymeric #’s are 3-5 mEq/g
• silica- based #’s are much lower
 Ion-Exchange Chromatography

Mobile Phases

Aqueous solution with a suitable salt or mixture of salts.

Sometimes a small amount of organic solvent may be present.

Salt mixture may be a buffer, or a separate buffer can be added.

Main component is the competing ion.

 Ion Exchange
Mobile Phases
Must be compatible with the detector.
Dissolves and carries the sample

Anions Cations
• Phthalic acid • Nitric acid
• Salicylic acid • Tartaric acid
• p-Hydroxybenzoic acid • Tartaric acid/dipicolinic acid
• Benzoic acid • Tartaric acid/citric acid
• Borate • Sodium dihydrogene phosphate
• Borate/Gluconate • Oxalic acid/ethylene diamine/acetone
• Potassium hydroxide
• Carbonate/bicarbonate
 Ion Exchange
Cation Separation Mechanism
Stationary phase and mobile phase compete for the analyte
 Ion Exchange
Anion Separation Mechanism
Stationary phase and mobile phase compete for the analyte
 Ion-Exchange Chromatography

Mechanism

Example anion-exchange material:

M+E- + A- → M+A- + E-

where,
M+E- - represents the exchanger
M+ - denotes the insoluble matrix containing fixed + ion
A- - solution anion
 Ion-Exchange Chromatography

Factors affecting retention - contributions of the eluent

• eluent pH – affects the functional group, eluent, and solute ion

charge
• nature of competing ion – affects the selectivity coefficient
• concentration of competing ion – affects the equilibrium
equation
• eluent flow-rate – faster flow rates – less time for interactions
• temperature – higher temps generally increases rate of
diffusion leading to increase interactions
 Ion-Exchange Chromatography
General rules to predict affinity for ion-exchange
1. Increase in charge of the solute ion increase its affinity for the ion-
exchanger.
2. Smaller solvated solute ions have greater binding affinity than larger ions
3. Higher the degree of cross-linking of the ion-exchange resin (pores)
greater the preference for smaller solute ions
4. Increasing polarizability of the solute ion increases the binding power

5. Ion-exchange capacity of the ion-exchanger sometimes affects selectivity

coefficients but is difficult to predict
6. Functional group on the ion-exchanger also sometimes affect selectivity
coefficients but is again difficult to predict
7. Degree of interaction between solute ion and ion-exchange matrix are
difficult to predict and specific to individual ions
 Ion-Exchange Chromatography
General rules to predict affinity for ion-exchange

In general it seems that the effect on the water-structure around the

ions and solute seem to have an effect on its free energy. Well solvated
ions interact less with the stationary phase than ions that disrupt the
water-structure.
 Ion-Exchange Chromatography

Applications

Particularly helpful in the separation of amino acids and

proteins.
Ion-Exchange Chromatography
Ion Exchange
Ion-Interaction or Ion-Pairing Chromatography

For strong acids or bases, these are ionized at

operating pH’s (3-8) for reverse-phase
chromatography.

Cations and anions react to a non-ionic molecule by

adding a lipophilic counter ion.
The resulting non-polar molecules are then separated
in the RP-mode.
Stationary p. = non-polar (e.g. C18) or ion exchange
resin (Polystyrene divinyl benzene cross-linked
polymers)
mobile p. = polar (e.g. acetonitrile or methanol/water)
Ion-Interaction or Ion-Pairing Chromatography

Stationary and Mobile Phase

Reverse phase technique with similar stationary and

mobile phase with addition of an ion-interacting
reagent (IIR)

Stationary phases - Polystyrene divinylbenzene (PS-

DVB) polymers, C18, C8, phenyl and cyano groups.
Ion-Interaction or Ion-Pairing Chromatography

Stationary and Mobile Phase

IIR requirements:
-appropriate charge that is unaffected by eluent pH
-suitable lipophilicity to permit adsorption onto non-
polar stationary phase
-compatibility with other eluent components
-compatibility with detection system
Ion-Interaction or Ion-Pairing Chromatography

Stationary and Mobile Phase

Separation of anionic solutes - use strong base

cations as IIR at pH ~ 8, i.e. tetraalkylammonium ions

Separation of cationic solutes - use strong acid

anions as IIR at pH ~ 4, i. e. aliphatic sulfonate ions.
Ion-Interaction or Ion-Pairing Chromatography

Stationary and Mobile Phase

The IIR coats the stationary phase and is in

equilibrium with the column.
Ion-Interaction or Ion-Pairing Chromatography

Mechanism

When comparing the retention of a solute with and

without IIR added, several observations are made;

-The retention of neutral solutes is not altered significantly with

or without the IIR
- the retention of solutes with the same charge as the IIR
decrease with the IIR addition
- the retention of solutes with the opposite charge as the IIR
increase with the IIR addition
Ion-Interaction or Ion-Pairing Chromatography

Mechanism

Also when composition of eluent is altered;

-the retention of solutes having the opposite charge

to the IIR increases with increasing IIR concentration
- the retention of solutes having the opposite charge
to the IIR increases with increasing IIR lipophilicity
- Retention of all solutes decreases with increasing
percentage of modifier in the eluent
Ion-Interaction or Ion-Pairing Chromatography

Mechanism

Three mechanisms have been proposed to explain the changes

in retention:
• ion-pair
• dynamic ion-exchange
• ion-interaction
Ion-Interaction or Ion-Pairing Chromatography

Ion-Pair Mechanism

An ion-pair forms between the solute ion and the IIR. This
occurs in the mobile phase and the resultant ion-pair partitions
onto the lipophilic stationary phase.
As the percentage of organic increases the ion-pair retention
decreases.
Ion Chromatography
Ion-Interaction or Ion-Pairing Chromatography

Dynamic Ion-Exchange Mechanism

A dynamic equilibrium is set between IIR in the eluent and IIR

adsorbed on the stationary phase.
The adsorbed IIR imparts a charge to the stationary phase so
that it behaves as an ion-exchanger.
Total concentration of IIR adsorbed is dependent on percentage
organic solvent in the mobile phase.
As organic solvent concentration increases IIR on stationary
phase decreases.
Ion-Interaction or Ion-Pairing Chromatography

Dynamic Ion-Exchange Mechanism

Solutes having the same charge as the IIR are

repelled from the charged stationary phase and have
decrease retention times.
Ion Chromatography
Ion-Interaction or Ion-Pairing Chromatography

Ion-Interaction Mechanism

Intermediate between the other two models by incorporating

electrostatic effects and adsorption effects.

Adsorbed IIR ions make up a primary layer of charge, then a

secondary layer of opposite charge ions form producing a
double layer.
Transfer of solutes through the double layer to the stationary
phase is a function of electrostatic effects.
Ion Chromatography
Ion-Interaction or Ion-Pairing Chromatography

Applications

For separation of strong acids and bases especially

inorganic anions and cations.
Ion Chromatography
 Ion-Exclusion Chromatography

Other names for this technique:

• ion-chromatography exclusion (ICE)
• ion-exclusion partition chromatography
• donnan exclusion chromatography
• ion-moderated partition chromatography

By adding H+ ions the stationary phase is transformed into a

non ionic but polar Donnan membrane. Only non dissociated
molecules can enter this membrane. If they dissociate they are
excluded from the stationary phase. The separation depends on
the dissociation constant of the respective molecules.
mobile p. = polar (e.g. H2SO4 aq.)
 Ion-Exclusion Chromatography

Uses strong anion or cation exchange resins for the separation

of weakly ionized or neutral solutes.

In this mode the charge on the ion exchange resin is the same
as the solute (opposite of ion-exchange chromatography).
 Ion-Exclusion Chromatography

Mechanism

Complete anionic or cationic solutes cannot cross the ionized

resin and penetrate into the occluded liquid phase thus
remaining in the mobile phase.

While weakly ionized or neutral solutes can move into the

occluded liquid phase and be separated.
Ion-Exclusion Chromatography
 Ion-Exclusion Chromatography

Stationary Phase

Typically high capacity, fully functionalized PS-DVB polymeric

ion-exchange resins.

Particle sizes are 5-10 um with about 4-16% cross-linking.

Column sizes are larger (30 cm long with 7 mm internal
diameter) than conventional HPLC columns to accommodate the
larger volume of resin material (larger swelling for occluded
liquid phase).
 Ion-Exclusion Chromatography

Mobile Phase

Simple in composition – deionized water with the

solutes or dilute solutions of minerals with the
solutes.

Organic modifiers (i.e. methanol, acetonitrile,

acetone) are sometimes added to affect the
adsorption sites.
 Ion-Exclusion Chromatography

Factors affecting retention

1. As degree of ionization of the solute increases the retention
decreases (determined by pKa of solute, pH of eluent and organic
modifier content).
2. As hydrophobic interactions between the solute and the stationary
phase increases retention increases.
3. As molecular size of the solute increases retention decreases.
4. As degree of cross-linking of the stationary phase increases
retention increases.
5. As temperature increases generally retention increases.
 Ion-Exclusion Chromatography

Applications

Mostly used for a wide range of small, neutral or partially ionized

molecules, such as carboxylic acids, inorganic weak acid
anions, and weak organic bases.

Separation of carboxylic acids is the most common application

and gives excellent separation for complex sample matrices,
such as urine, plasma, food, beverages, and pharmaceuticals.
Ion Chromatography
 Ion Chromatography

Detectors

Conductivity
Amperometric
IC/MS
UV-vis (for UV active ions, e.g. nitrite, nitrate, thiosulfate)
 Assignment
• Read Chapter 6: Principles and Practice of Modern
Chromatographic Methods, Peter E. Jackson,
Academic Press.
• Thermo presentation on history and trends of IC –
on class website.