Chromatographic

Process
Mobile phase

B+A

Ion Exchange
Chromatography

Stationary
Phase

A
B

Dr. Shulamit Levin

Medtechnica

Distribution:
K = C s/C m

A
Elution through the Column

Ion Exchange Theory

Cation Exchange vs Anion Exchange
Cation Exchange

Anion Exchange

ION EXCHANGE
INSIDE A PORE IN THE STATIONARY PHASE
SAMPLE IONS IN

+++ ++1. INJECTION

Cation exchange columns have a negative charge to attract cations.

Anion exchange columns have a positive charge to attract anions

Chromatogram

- -- --

COUNTER IONS OUT

+
+ - -+ + -+
2. ADSORPTION:
DISPLACEMENT OF
COUNTER IONS

-- - -

MOBILE PHASE
ADDITIVES

-- - + - -+ -- + - - + +3. ELUTION

Dr. Shulamit Levin, Analytical Consulting, Medtechnica

ION EXCHANGER

Analysis of Ions - Ion Chromatography

ANION
ANION EXCHANGE
EXCHANGE

ION (Mono, Di, Tri - Valent)

K XY
B+Y-

+ X-

FUNCTIONAL

B+X- + Y-

ANION

CATION
CATION EXCHANGE
EXCHANGE

KM NH
A-M+

NH+

Organic
Acids
Phenols

A-NH+ + M+

Ion Exchange Theory

Strong vs. Weak Exchange Materials

Cation

NR3+
WEAK

STRONG

COO-

Organic
Amines

Inorganic
Metals
NH4+

Ion Exchange - Bonded Functionalities

Anion exchanger

SO3-

Inorganic
FCl-

Ions can be characterized as:

organic or inorganic, anion or cation, mono or polyvalent.

IMMOBILIZED ON THE STATIONARY PHASE

Cation exchanger

CATION

COO-

Anion

Na+

Carboxylic Acid

NH3+

STRONG

SO3- Na+
Sulfonic Acid

R
N+-R ClH
Primary, Secondary
or Tertiary Amine

R
N+-R ClR
Quaternary Amine

WEAK
Typical chemical functionalities used for commercial exchangers.

Strong Exchangers stay ionized as pH varies between 2 and 12.

Weak exchangers can lose ionization as a functionof pH.

Dr. Shulamit Levin, Analytical Consulting, Medtechnica

Conductivity and PDA Detectors in Series

1.60

Functional groups
-

Direct Conductivity after

Detection:

Suppression

CATION EXCHANGERS

ANION EXCHANGERS

1.20
4

5
7

0.80
S

0.40
0.00
3

0.05

Detection:

0.04
AU

1. Fluoride

1 ppm

2. Chloride

2 ppm

3. Nitrite

4 ppm

4. Bromide

4 ppm

5. Nitrate

4 ppm

6. Phosphate

6 ppm

7. Sulfate

4 ppm

UV (PDA) at 214 nm

TYPE
Sulfonic acid
Carboxylic acid
Phosphonic acid

0.03

Column:

Waters IC-Pak Anion HR

Eluent:

1.2 mM Sodium Carbonate/

phosphinic acid

1.2 mM Sodium Bicarbonate

0.02
0.01

Flow rate:

1.0 mL/min

Injection vol.:

50 L

Arsonic

0.00
0.00

4.00

8.00

12.00

Phenolic

16.00

20.00

Selenonic

24.00

Minutes

FUNCTIONAL
GROUP
- +
-SO 3 H
- +
-COO H
- +
PO 3 H
- +
HPO 2 H
- +
-O H

TYPE
Quaternary amine
Quaternary amine
Tertiary amine
Secondary amine
Primary amine

Hydrous Oxide
=M-O-H
=M-O-H

=M-O
+ H+
=M + OH

+
-NH 2(CH 3) 2 OH
+
-NH 3 OH

- +
-HAsO 3 H
- +
-SeO 3 H

Packing Supports
Resin

Polymer-based ion-exchangers

+
-N(CH 3) 2 (EtOH)
+
-NH(CH 3) 2 OH

Ion Exchange Theory

Columns Matrices
Silica-Based

FUNCTIONAL
GROUP
+
-N(CH 3) 3 OH

Silica-Based

Capacity
Swelling
Mass Transfer
Size Separation
Reverse Phase
Efficiency
pH Range
Equilibration
Literature

Both resin and silica based ion exchangers have advantages

and disadvantages which are summarized here.

Dr. Shulamit Levin, Analytical Consulting, Medtechnica

ION EXCHANGE

ION EXCHANGE

CATIONS
CATIONS

ANIONS
ANIONS

RETENTION & ELUTION STRENGTH

RETENTION & ELUTION STRENGTH

-

F , OH >

OAc

> H PO
>
2
4

HSO > CN > Br >

3

HCO

> Cl

>

NO

MONO-VALENT

NO > I
3

Li + > H + > Na

>

++
Be++ > Mn

Properties of Mobile phases

Compatibility with the detection mode Suppressed or Non-suppressed.
Nature of the competing ion
Concentration of the competing ion
Mobile phase's pH

4 + > K + > Rb
DI-VALENT

++
Mg

++

Zn

++

> Co

TRI-VALENT

Al

A--- > A -- > A-

+ > NH

+++

>

+++

> Ce

+++

+ > Cs + > Ag

++
++
++
> M > Ca
> Sr
transition
metals

Transition
metals

Ion capacity
The number of functional groups per unit
weight of the stationary phase.
A typical ion-exchange capacity in IC is 10100 mequiv/g.

Buffering capacity of the mobile phase

Ability to complex the ionic sample components
Organic modifiers

Dr. Shulamit Levin, Analytical Consulting, Medtechnica

ELUTION ORDER IN ANION EXCHANGE

ELUTION ORDER IN CATION EXCHANGE

DENSITY OF CHARGE

DENSITY OF CHARGE

1
R
E
S
P
O
N
S
E

OH-

F-

OAc-

1
Cl0

NO3

NO2

VOID
1

NO3-

SO42-

SO32-

TIME (MIN.)

R
E
S
P
O
N
S
E

HAc

k1
k2

H+ +

Ac-

+
HB
HB+

HH++++BB

pKa ~ 7-8
At pH < 7-8 the main species is BH+

Na+

Li+

Ag+

Zn++

Al+++

Ka =

(H+) (Ac)
(HAc)

pH and pKa

A-

WEAK
WEAK BASES
BASES

K+

The Equilibrium Constant

pKa~ 4-5
At pH >4-5 the main species is

TIME (MIN.)

WEAK
WEAK ACIDS
ACIDS
HH++ ++ AA- -

VOID

IONIZATION and RETENTION

HA
HA

(H+)

= Ka

(HAc)
(Ac)

pH = pKa - log

(HAc)
(Ac-)

A general understanding of ionization constants, pH, and pKa

are useful in understanding ion exchange and buffer
phenomena.

Dr. Shulamit Levin, Analytical Consulting, Medtechnica

Amino Acids Analysis In Plasma

ELUTION ORDER IN ION EXCHANGE

Ion Exchange with Ninhydrin detection

CATION EXCHANGE

ANION EXCHANGE
STRONGER ACID

STRONGER BASE

Gradient of pH and Ionic Strength

1
R
E
S
P
O
N
S
E

VOID

TIME (MIN.)

1
3

R
E
S
P
O
N
S
E

VOID

TIME (MIN.)

Dr. Shulamit Levin, Analytical Consulting, Medtechnica