Ion Exchange ChromatographyPrinciples: Retention is based on electrostatic interaction between solute ions andfixed ion charge on the stationary phase (S.P)

Cations needs a cation exchanger and anions needs anion exchanger

How a stationary phase (S.P) is built?

Styrene divinylbenzene resin

AnionExchanger

CationExchanger

Crosslinkbetweenpolymers

(aka. backbone)

Ion-Exchange Groups (A group that carries a separation capacity)Strong Cation Exchanger Weak Cation Exchanger

Strong Anion Exchanger Weak Anion Exchanger

At low pH CM loses cation exchange capacity due to protonated S.P

At high pH DEAE losesanion exchangecapacity due to deprotonated S.P

Polymeric

Silica-based

COOH

COOH

COOH

COOH

pH <4

a) Undissociatedweak cation exchanger

COO-

COOH

COO-

COOH

4<pH<8

b) Partially dissociatedweak cation exchanger

COO-

COO-

COO-

COO-

pH>8

c) Dissociatedweak cation exchanger

What mobile phase characteristics will affect the retention of ionic compounds(e.g., cations) if weakly acidic cation exchanger is used?

*pH of the M.P. The M.P affects retention of cations. For example, at low pHthe ionogenic groups of the resin is protonated and cation exchanger loses its ion-exchange capacity -------- tR of cations decreases (case a)

Mechanism of Retention in Ion-Exchange Chromatography (IEC)

1. The column is equilibrated with the M.P and the counterions have occupied ion-exchangesites on the S.P. The sample ions are injected and are about to enter the first segment of S.P

Taken from Weston

2. As the sample cations enters the column they start displacing the M.P counterions from the ion-exchange sites on the S.P (process called adsorption of solute ions on the S.P).

3. Because this is a flowing system(M.P is continously being flushed into the column). Therefore,the M.P counterions will start displacing the sample ions from the IE sites.4. After all the sample ions are eluted off the column the S.P is regenerated (ie., flushed with M.P counterions) to clean off extra sample ions that have retained on the S.P

All M.P counterions except one has been displaced

frome the IE sites

Some sampleions havealready beingdisplaced by M.P counterions

Synthesis of polymeric-based ion-exchange columns

I. Copolymerization of styrene with divinylbenzene

II. Sulfonation or amination of copolymer (PSDVB)

Strongly acidic cation-exchange resin prepared from sulfonation of PSDVB

Type of Ion-Exchange packing material

Ion Exchange Resin

Strongly basic anion exchanger is prepared from amination of PSDVB

Crosslinking is a measure of polymer strength. --Heavily crosslink resin have more IE sites-----tR increases --Liqhtly crosslink resin have less IE sites ----tR decreases

Polymeric resins are made in 3-D networks by cross-linking hydrocarbon chains. The resulting resin is insoluble, inert and relatively rigid. Ionic functional groups are attached to this framework.

--Resin crosslinking varies from2—10%. Resin <2% crosslinkare soft gels

Lightly crosslinked rapid Equilibrium Between S.P and M.P, which Increase N but also tR

resins sells with organic solvents (if lightly crosslinked)

Heavily crosslinked- More rigid and less Porous. Slow equilibration of solutes. HigherIE capacity, tR increases, N increases)Resins do not swell with organic solventsdue to greater mechanical strength

Ion-Exchange Equilibria: Due to the competition between M.P counterions and sample ions anequilibrium is established between the counterions (C+) and the sample ions (S+)

“Ions of same charge are reversibly exchange between the two phases”

F-C+ + S+ F-S+ + C+

(S.P) (S.P)(M.P) (M.P)

S+ = sample ionsC+ = counter ions of the M.P

Ion-Exchange SelectivitySelectivity CoefficientsPreference for ions of particular resins is often expressed through an equilibrium relationship using the selectivity coefficient. The coefficient is described below.For the exchange of Li+ in solution for Na+ on the resin:

S.P Li+ NaNa+Li +

« + ++R- R-

S.P M.PM.P

[R-Na+]S.P [Li+]M.P

[R-Li+]S.P [Li+] S.P

=K

K describes the relative selectivity of resin for Li+ and Na+ ions.Greater the value of K means forward reactions is more favorable than reversereactions. Thus, selectivity of Na+ for the ion-exchange resin is higher thanfor Li+ but why?

Why Li+ has lower selectivity (retained less) compared to Na+ on ion-exchange columns?

Li+ ions is small cation compared to Na+. However, it has large hydrated radius.

The larger hydrated radius of the Li+ ion do not have much access to the negative

charge on the S.P, compared to Na+ cation.

Li+

H20

H20 H20H20

H20H20

H20H20 H20

Ionic Radius = 0.90 0AHydrated radius = 3.40 0A

Na+ H20

H20H20

H20H20H20

H20

Ionic radius = 1.16 0AHydrated radius = 2.760A

http://www.bbc.co.uk/dna/h2g2/A1002709

Effectively, these selectivity coefficients are a measurement of a resins preference for an ion. The greater the selectivity coefficient, the greater the preference for the ion. For example, a strong acid cation resin with 8% crosslink has a selectivity coefficient for sodium vs. hydrogen of 1.56, while the selectivity coefficient for calcium vs. hydrogen is 4.06. As a result, calcium is strongly retained by the ion exchange over the sodium

K value is measured withrespect to H+

--*Increasing the percentage of crosslinking of the resin usually increases the retention on polymer-based columns

Polymeric resins are made in 3-D networks by cross-linking hydrocarbon chains.

The resulting resin is insoluble, inert and relatively rigid. Ionic functional groups

are attached to this framework.

www.physioviva.com/movies/gibbs-donnan/index.html

Donnan Equilibrium in Ion Exchange Chromatography

Nice animation at --

When an ion-exchanger (S.P) is placed in an electrolyte solution: “the concentration of electrolyte is higher outside the resin than inside it.”

R+Cl-R+Cl-

R+Cl-

R+Cl- R+Cl-K+K+

K resin +

K+K+K+Cl-

K+Cl-

K+Cl- K+

K+

K+Cl-

K+

K+R+Cl-

The equilibrium between the M.P ions insolution and the M.P ions inside theresin (S.P) is called Donnan Equilibrium

Consider a quaternary ammonium [N(CH3)4]+,

anion-exchange resin = R+ in its chloride (Cl-)form immersed in a solution of KCl

It can be shown from the thermodynamic that the ion-product inside the resin is approximately equal to the ion product outside the resin: [K+]i [Cl-] i = [K+]0 [Cl-]0 -----------(1)

i= concentration of electrolyte (M.P) ions inside the resin o= concentration of electrolyte (M.P) ions outside the resin

--From consideration of charge balance we know that: [K+]0 = [Cl-]0 ---------------------(2)

--Inside the resin there are three charged species, and the charge balance is

[R+]i + [K+]i = [Cl-]i -----------------------------(3)

Substituting (eq.2) and (eq 3) into eq(1) we get :

[K+]i ([R+]i +[K+]i) = [K+]0 -----------------------------(4) 2

What does the above equation tell us?

The concentration of [K+]0 must be higher than [K+]i, which is true because K+

has the same charge as the R+ and the electrostatic repulsion will decrease itsconcentration inside the resin.

Example calculation showing the exclusion of cations by the anion exchanger

Suppose that the concentraton of cationic sites in the resin (in anion exchange resin) is 6.0 M. When the Cl- form of the resin is immersed in 0.050M KCl, what will be the ratio of [K+]0/[K+]i?

[K+]i ([R+]i +[K+]i) = [K+]0 -----------------------------(4)2We know that from the above equation:

[K+]i + [R+] [K+]i) = [0.050]0 2 2

[K+]i + (6.0 [K+]i - 0.025 = 0 2

ax2 + bx -c = 0

x = -b + b2 - 4ac 2a

[K+]i = -6.0 +/- (6)2 - 4 (1)(-0.0025) 2(1)

= -6.0 +/- 36.01 2

= -6.0 +/- 6.000833 2.0

[K+]i = 0.000833 = 0.0004166 ~ 0.00042 2.0

[K+]0 = 0.0500 ~ 1.2 x 102

[K+]I 0.0042

0.00042 X 100 = 0.84% 0.050conc of K+ inside the resinis less than 1%!

The counterion (e.g., Cl-) is not excluded from the resin. There is no electrostatic barrier to penetration of a solute anion into the anion exchange resin

“Cations with the same charge as the resin are excluded.”

Solute anions competes for the anion exchange sites with the counter ions

Silica-Based Ion Exchange Materials

H

+

Functional group is covalently attachedto the silica surface and fixed ions areformed as a part of this group

What are the advantages/disadvantages of silica-based over polymer based IEC?Silica-basedAdvantages: (1) Have lower capacity than polymer-based. Due to low IE capacityN is better. (2) Do not swell with organic solvents.

Disadvantages: (1)If not fully functionalized or bonded, unreacted silanols itselfcan act as a cation exchanger (peak tailing and adsorption becomes a problemsfor the separation of cations e.g., quaternary amines). (2) Limited pH range to work with

Therefore, it appears that polymeric ion-exchange columns are best for theseparation of small inorganic and organic anions and cations. For large organiccations/anions silica-based columns would provide better separation capabilitydue to limitation of the use of organic solvents on majority of polymeric based columns

Major Factor affecting retention in ion-exchange chromatography

A) Eluent or M.P pHB) Nature and Charge of the competing ions (i.e., M.P counter ions)C) Concentration of the competing ions

I. Influence of Solvent (M.P)

II. Influence of Solute (Injected sample ions)A) Charge on the solute ionB) Solvated size of the solute ionC) Polarizibility of the solute ion

III. Influence of Stationary PhaseA) The ion-exchange capacity of the ion-exchangerB) The functional group of the ion-exchanger

I. Influence of Solvent (M.P)

A) pH of the M.PIf we increase in pH of the mobile phase For example, use of sodium borate at pH 10 (instead of using HCl at pH 2.0) will make the weak anion exchange resin neutral at high pH and retention of anions will decrease

N(CH3)2

N(CH3)2

N(CH3)2

N(CH3)2

pH>9

c) Dissociatedweak anion exchanger

N(CH3)2

N(CH3)2

N(CH3)2

N(CH3)2

pH <4

a) Undissociatedweak anion exchanger

+

+

+

+

B) Nature and Charge of the mobile phase counterions

SO3-

SO3-

SO3-

SO3-

a) Strong cation exchanger

+N(CH3)3

+N(CH3)3

+N(CH3)3

+N(CH3)3

a) Strong anion exchanger

Ca2+ (CaCl2) will be a much strongermobile phase counter-cation, i.e., decrease the retention time for the separation of alkali metalcations (Li+, Na+, K+, Rb+, Cs+) compared to the use of H+ (HCl) as M.P

CO32- (Na2CO3) is a much stronger mobile

phase counter-anion for the separationanions (F-, Cl- Br-, I-, SO4

2-) compared tothe use of HCO3

- (NaHCO3)

Ba2+ (BaCl2) is a much stronger mobile phase compared to Ca2+(CaCl2)

NO3-(NaNO3) is stronger eluent anion

compared to Br-(NaBr)

(C) Concentration of the Competing Ions

Increasing the concentration of counter ions (e.g., H+) in the mobile phaseFor example, the use of 1M HCl is much stronger mobile phase comparedto 0.1 M HCl (because the former concentration have more mobile phasecounter cations (shown on the right). Hence the retention of solute cationswill decrease.

II. Influence of solute injectedA) Ions of weak acids (phenols) or bases (aniline) may loose charge and are retained less on ion-exchange columns.For example phenol (pKa =9.1) is weakly retained if the mobile phase pH isless than 9.0, but will be retained at high pH values between 10-12Similarly, aniline (pka = 3.2) is weakly retained on cation exchange columns atpH 7.0 mobile phase compared to pH 2.0 mobile phase

(B) The charge on the solute cations and anions.--inreasing the charge of the solute cation or anion increases its affinity for the IE sites on the column. The decreasing retention order will be: Pu4+>>La3+>>Ba2+>Tl+ PO4

3->SO42->Br-

(C ) The solvated size of the solute cation or anion. Ions with the smaller degree ofsolvation exhibit greater binding affinity and retained longer (e.g., Cs+>Rb+>K+>Na+> Li+) or (I->Br->Cl->F-)

Li+H20

H20 H20H20

H20H20

H20H20 H20

Cs+ H20

H20H20

H20H20H20

H20F-

H20

H20 H20H20

H20H20

H20H20 H20

I- H20

H20H20

H20H20H20

H20

D) Polarizibility of the solute anions--Ability of an ion’s electron cloud to be deformed by nearby charges. Deformation of the electron cloud induces a dipole in the solute ion. Thus,attraction between induced dipole and the nearby charges (resin charge) and increase the binding fo the ion. For example R-SO3

- resin shows greater Affinity for polarizable cations (e.g., Ag+, Tl+) compared to alkali metals (Li+,CS+)

SCN- SO42->

Ag+ Li+>

Affinity of

Affinity of

III. Influence of Stationary Phase Charge

The functional group of the ion exchanger. A sulfated stronger cation exchangerexhibit higher retention compared to a carboxylated weak cation exchanger.Similarly, a quaternary ammonium cation will be a strong cation exchanger thanSecondary or tertiary amine.

SO3-

SO3-

SO3-

SO3-

a) Strong cation exchanger

COO-

COO-

COO-

COO-

c) Weak cation exchanger

+N(CH3)3

+N(CH3)3

+N(CH3)3

+N(CH3)3

Strong anion exchanger

+NH(CH3)2

+NH(CH3)2

+NH(CH3)2

+NH(CH3)2

Weak anion exchanger

(B) Ion-exhange capacity of ion exchangerIncreasing the ion-exchange capacity increases the retention, but selectivitycoefficient remains constants

Cation ExchangeCu2+ 2H+

2H+ + 2OH- 2H2O

Application of IEC in the preparation of deionized waterWe need two ion-exchange columns (cation exchange RSO3

- and anion exchangeR-N+(CH3)3) to remove cations and anions, respectively. Suppose we needremove Cu(NO3)3 from the faucet water.

Cation exchanger will bind Cu2+

and H+ ionsare knocked off

2H+

Anion Exchange2NO3

- - 2OH-

Anion exchangerwill bind NO3

-

And OH- ionsare knocked off

2OH-

Applications of IEC (Contd)

(b) Convert one counterion of the salt to another

(CH3-CH2-CH2)4---N+I- (CH3-CH2-CH2)4---N+-OH-

Tetrapropyl ammonium iodide Tetrapropyl ammonium hydroxide

I- is a UV absorbing counterion OH- is a non-UV absorbing counterion

(c) Preconcentration of trace components

--For example we can pass large volume of fresh lake water through a cation exchange resin in the H+ form (using HNO3) to concentrate metal ions onto theResin.--Chelex 100, S-DVB resin containing iminodiacetate groups binds transition metals (e.g., Fe3+, Ni2+)

Resin --------N

CH2-COOH

CH2-COOH

: Fe2+

Fe3+1 pptr

Resin -

Fe3+ 1ppm

Metals can be eluted using HNO3

from the column in small volumefollowed by column regeneration in H+ form

ION CHROMATOGRAPHY--abbreviated as IC is a high pressure version of ion-exchange chromatography, has become the method of choice for anion analysis

Recent IC applications in rainwater, groundwater, surface water, wastewater, drinking water, fog samples, ice, snow, soil, sediments, sludge, plants, air, exhaust, aerosols, flue dust, fly ash, fuel oil and coal are reviewed with a majoremphasis on speciation of ions. IC shows great promise for the sequential determination of ionic species in a wide variety of water samples

A) Non-Suppressed ICaka. IECwith direct or indirectconductivity andindirect-UV detection

B) Suppressed IC withdirect conductivity Detection employs two columnsa) ion-exchange columns -- separate solute ionsb) suppressor column --- decreases the background conductivity of the eluent

Types of Ion Chromatography

Suppression

Suppression

Eluent(HCl) Inj

…....…....………………

Pump

An

alyt

ical

Co

lum

n

Su

pp

ress

or

Co

lum

n

ConductivityCell

Conductivitymeter

Recorder

Cation IC

Inj

…....…....………………

An

alyt

ical

Co

lum

n

Su

pp

ress

or

Co

lum

n

Pump EluentNaHCO3

Conductivitymeter

Conductivitycell

Recorder

Anion IC

Resin—SO3-H+ + Y+

Resin—SO3-Y+ + H+

Y+ = Na+, K+, Rb+, Cs+

Resin—N+ OH- + H+Cl-

Resin—N+ Cl- + H20

Resin—N+ OH- + Y+Cl-

Resin—N+ Cl- + Y+OH

Resin—N+HCO3- + X-

Resin—N+X- + HCO3-

Resin—SO3-H+ + Na+X-

Resin—SO3-Na+ + H+X-

Resin—SO3-H+ + Na+HCO3-

Resin—SO3-Na+ + H2CO3

Counterions associated with activeeluent species are replaced in the

M.P by either H+ or OH- to form H20 andH2CO3 Key to suppression

X- = F-, Cl-, NO2-, PO4

3-

Supppression

Comparison of Non-suppressed vs. Suppressed Conductivity Detection

Problems with Suppressor Column--Need to regenerate the suppressor column periodically (typically after every8-10 hrs) to convert the suppressor column back to acid/base form-- Increase dead volume (extra column band broadening) and decreases theOverall efficiency for analysis

Eluent and suppressor solution flows in opposite direction on either side of theIon-exchange membraneFor analysis of cations - membrane is a cation exchangerFor analysis of anaion - membrance is an anion exchanger

M.P used in non-suppressed IC(direct conductivity)

M.P used in non-suppressed IC(Indirect conductivity)Sodium Hydroxide (anion exchange)Nitric Acid (cation exchange)

List of Mobile Phases Used in Non-Suppressed IC

-OOC

-OOC

Phthalate

-OOC OH

p-hydroxybenzoate

COO-

Benzoate

Why non-suppressed IC is less sensitive than suppressed IC?We are measuring the change in conductance of the eluted solute. Thisis contrast to IC where eluent conductivity is suppressed to zero beforedetection

M.P used in suppressed IC

The two chromatogram shown to the leftshows the application of IC with a suppressorcolumn. In each of these cases analytes aredetected in ppb (mg/L) are getting popularthese days.

Chromatogram shows separation of doublycharged species (Mg2+------.Ba2+) but no resolution of singly charged species (Li+--Cs+)all coeluting as single band.

Simultaneous separation of both singly and doubly charged species is difficult inIC because differences in selectivity and interaction with S.P for 2 classes of cations is wider

TYPICAL APPLICATIONS OF IC WITH SUPPRESSED CONDUCTIVITY DETECTION

a) 28 mM NaHCO3/23 mM Na2CO3

b) 25 mM phenylenediamine dihydrochoride/25 mM HCl (Taken from Dionex, Inc, Sunnyvale, CA.)

Non-Suppressed IC with Indirect Photometric Detection

Principle: UV absorbing anions/cations (depending on mode of IEC) are added tothe M.P. So detector has a high UV-absorbance in the baseline

What happens when we inject a non-UV absorbinganions (e.g., Cl-)?

Baseline noise

A

Cl-A-OOC

-OOC

-OOC

-OOC

+N(CH3)3

+N(CH

3)3+N(CH3)3

+N(CH3)3

+N(CH3)3

+N(CH3

)3+N(CH3)3

+N(CH3)3

Cl-

-OOC

-OOC

-OOC

-OOC

-OOC

-OOC

+N(CH3)3

+N(CH3

)3+N(CH3)3

+N(CH3)3

2Cl-

Cl- elutesoff the columnOriginal condition

is restored