Bioseparation Science

Chromatography

Clémence Coetsier
clemence.coetsier@univ-tlse3.fr

www.lgc.cnrs.fr
 LIQUID CHROMATOGRAPHY

I- Introduction – General
I-1 Definitions V- Different types of PLC
I-2 Implementation V-1 Partition Chromatography
I-3 Classification V-2 Adsorption Chromatography (Normal and
I-4 GC vs. LC Comparison Reverse)
II- Fundamental Quantities V-3 Affinity Chromatography
II-1 Introduction V-4 Ion Exchange Chromatography
II-2 Retention Quantities V-5 Steric Exclusion or Gel Permeation
II-3 Selectivity Chromatography
II-4 Effectiveness V-6 Choosing a Method
II-5 Resolution
II-6 Pressure Drop and Flow Resistance Factor
II-7 Performance Index – Separation Impedance
II-8 Reduced Quantities
III- Trade Kinetics
III-1 Mechanisms of peak dispersal
III-2 Knox's equation
III-3 Experimental Application
IV- Separation Optimization
IV-1 Introduction
IV-2 Resolution
IV-3 Analysis Time and Pressure Drop
Laboratoire de Génie
Cours Chromatographie Chimique – LGC, Toulouse
Analytique 2
 LIQUID CHROMATOGRAPHY

I - INTRODUCTION - GENERALITES

I-1 Definitions
A general term that includes all separation methods based on the distribution of a
solute between two phases, one being mobile (gas or liquid), the other stationary
(liquid or solid)

Two antagonistic effects allow for separation:

Entrainment by the mobile phase and retention effect by the stationary phase due to
selective distribution

Dynamic phenomenon: equilibrium state between the mobile and stationary phases

CS -solute
Distribution coefficient: K -Nature of phases M and S
CM - T°

1906 – Tswett, Russian botanist

Separating the pigments from a spinach leaf
(petroleum ether on a calcium carbonate column)
Laboratoire de Génie Chimique – LGC, Toulouse 3
I-2 Implementation Column Chromatography:

Mobile phase A+B

Injection
Injection of a small
Elution Mixing volume (10 to 200 μL)
(constant flow)

Column
Stationary
Separation
Phase

A Chromatogram
Detection
Collector
Volume
Time
Eluate
Difference in distribution between phases
Difference in elution velocity
Different elution times or volumes

Laboratoire de Génie Chimique – LGC, Toulouse 4

I-2 Implementation

Column Chromatography:

Planar chromatography:
On paper or in thin film (glass or plastic plate)
Capillary displacement

Planar chromatography:
Fixed travel time: measurement of ≠ distance traveled

Column Chromatography:
Constant Distance Traveled: Time ≠ Measurement (Analysis)

Laboratoire de Génie Chimique – LGC, Toulouse 5

I-2 Implementation

Development modes:

Elution Development: Most Used Mode

Constant-Composition Elution: Isocratic Elution

Variable Composition Elution: Gradient Elution

Separation of complex mixtures:
gradual modification of the Cond° Op. over time
Decrease the retention of the most retained molecules
Without interfering with the segregation of the least retained species

More complex apparatus (gradient pump)

Regeneration stage to return to the initial state

Laboratoire de Génie Chimique – LGC, Toulouse 6

 I-3 Classification
-Gas chromatography (GC)
1- Depending on the nature of the mobile phase:
-Liquid chromatography (LC)
--Supercritical Chromatography (SCC)
2- LC: Depending on the nature of the ph. Stationary or separating effect
Separating effect
Type of Chromatography
Stationnary Phase Nature of the phenomena
involved in the separation

Liquid fixed on a support Partition Solubility

Polar Solid Adsorbent Adsorption (ph. Normal)
Polarity
Nonpolar solid adsorbent Adsorption (ph. Reverse)
Grafted inert support Specific reaction
Affinity
(ligand) Solute / Ligand

Ion Exchange Resins Ion exchange Electrostatic charge

Steric Exclusion
Porous Solid Size (shape)
or Gel Permeation
Laboratoire de Génie Chimique – LGC, Toulouse 7
 I-4 Comparison GC – LC

● Limitations of GC: -Low-volatile substances (often the case for MW > 300g/mol)
- Substances sensitive to T° elevation (biological compounds)
- Ionized substances (because they are not very volatile)

● LC : No limitation due to volatility and thermal stability

● LC Often more effective in the case of difficult separations because:

1- GC only solute/pH interactions. Stationary while in

LC interactions between solute / ph S. - Solute / ph. M – ph. M/ph S.

solute GC solute

Stationary phase Stationary phase Mobile phase

LC

2- different stationary phases available in LC (Ion Exchange, Exclusion, etc.)

3- Lower LC temperature: greater molecular interactions

Laboratoire de Génie Chimique – LGC, Toulouse 8
 II – FUNDAMENTAL QUANTITIES
II-1 Introduction

Ideal Peaks: Low injected amounts: symmetrical peaks (Gaussian shape)

1   x² 
y exp 
2  2 
Rt : retention time:
x=0 y= 0,399
0,4

2 Inflection points:
0,3
x = ± 1 y = 0,242

0,242
s Width at 2 pts of inflection = 2 s

0,2
H
d s : standard deviation s ² variance
Width at half-height: 2.35s = d
0,1 H
2 Width at base: 4 s = w

0
w
Area between -2s and +2s = 0.954
-4 -3 -2 -1 0 1 2 3 4

Laboratoire de Génie Chimique – LGC, Toulouse 9

 II-1 Introduction

LC Separation:
A good separation involves:
Retention of constituents (affinity with ph. S sufficient) Vr > Vm.ph
Sufficiently separated peaks:
Separation of Solute Bands (Selectivity) > Peak Spreading (Efficiency)
Quick Separation

Improving separation: 2 possible steps

b- Keep the peaks away by using a pH. S more Selective

c- Decrease the width of the peaks (kinetics): Efficiency

Laboratoire de Génie Chimique – LGC, Toulouse 10

 II-2 Retention Quantities

1- Retention Time and Volume:

Rt : retention time = Time to peak from injection

VR : volume de rétention VR = Q Rt Q: m. ph flow rate

VR : volume of m. ph. necessary to elute the compound (to the nearest peak spread)

VR et Rt : characteristics of each compound

VR = Q Rt = Rt u S ec  Rt u  (dc2 /4) ec

ec: Column porosity 0.75 porous silica

0.35 – 0.4 exchange resins
Vc : Column Volume
Vi  V p Sc : Column Section
Vm
ec   dc : diameter of the column
VC VC Vm : Vacuum volume in the column

Laboratoire de Génie Chimique – LGC, Toulouse 11

 II-2 Retention Quantities

1- Retention Time and Volume:

Species not retained: t0 Zero retention time Vm vol of m. ph.
 Determination of t0 by injection of an unretained compound
Vi interstitiel vol.
Vm = Vi + Vp
Vp vol. in pores
L L : Column Length
t0 : Compound not retained t0  u : m. ph. Velocity
u

Retained species: VR = V m + K V s Vs vol. of S. ph.

Remark: Cs

Relation valid only for a linear elution

K independent of the C° of solute
Cm

Laboratoire de Génie Chimique – LGC, Toulouse 12

 II-2 Retention Quantities

2- Capacity Factor:

Vr and Rt: depend on the geometry of the column

Allows you to get rid of the geometrical parameters of the column
Q té soluté  S C s Vs V
k '  té  K s
Q soluté  M  Cm Vm Vm
𝑉 −𝑉 𝑅 −𝑡
𝑘′ = =
VR = V m + K V s 𝑉 𝑡

VR/Vm = 1 + k’
𝑅 = 𝑡 1 + 𝑘′

Low values of k' indicate poorly retained compounds, eluted shortly after the volume of Mobile phase contained
in the column, which corresponds to k'=0. High values indicate strongly retained compounds, eluted after a fairly
long analysis time: each increment of 1 unit corresponds to the elution of a column volume Vm to elute the
compound.
Trade-off: retention high enough to allow separation but not so high as to limit separation time

L L : length of column 𝐿
t0 : Compound not retained t0  u : m. ph. velocity
𝑅 =
𝑢
1 + 𝑘′
u
Laboratoire de Génie Chimique – LGC, Toulouse 13
II-3 Selectivity
characterizes the distance between 2 vertices of 2 consecutive peaks

𝑅 −𝑡 𝑘 𝐾
𝛼= ⇒ 𝛼= = (most retained compound 2)
𝑅 −𝑡 𝑘 𝐾

II-4 Effectiveness

Peak Spread = f (Efficiency)

Measurement by determining the number of theoretical plates N

Plateau Theory:

Statistical model to describe the functioning of a column (see distillation)

Instead of considering the continuous actual displacement of the Mobile phase, we consider
the progressive displacement of the Mobile phase

Progression of m. ph. by successive transfers (equilibrium between each transfer)

Column = successive zones at equilibrium = Theoretical plateaus

Calculation of the C° profile from step to step

Laboratoire de Génie Chimique – LGC, Toulouse 14
 II-4 Effectiveness
A real column is then said to have N theoretical plateaus if, under the given conditions, it
has the same efficiency as a fictitious column with N theoretical plateaus
Elution Peak = Gaussian Curve
𝑅
Standard deviation s or variance s² is link to the number of plateaus using: 𝜎 =
𝑁

𝑅 𝑅
Theoretical Relation of the Gaussian Curve 𝑁 = 16 = 5,54
𝜔 𝛿

w : Width from peak to base w = 4 s

d : Width to mid-height d = 2,35 s = (5,54)1/2 s (More accurate)
N: Accounts for the widening of the peak (% at column length)

To compare the performances of ≠ column

L
Height Equivalent to a Theoretical Plate: HEPT  L : length of column
N
Column Comparison (Column Length Independent)

Efficient column: high N spread and low HEPT

Laboratoire de Génie Chimique – LGC, Toulouse 15

 II-4 Effectiveness
Implications for Elution Curve Theory

Deduction of solute concentration at Rt (max curve)

qi N
Cmax 
VR 2

qi : injected quantity = C0V0

Dilution Factor:

C0 VR 2 Dilution is all the more important because:

f   Retention is high
Cmax V0 N The injected volume is small
The column is less efficient (low N)

Laboratoire de Génie Chimique – LGC, Toulouse 16

II-5 Resolution

𝑅 −𝑅 (most retained compound 2)

𝑅 =2
𝜔 +𝜔

High Rs  good separation

𝑅 −𝑅

𝑅 −𝑅

Rs = 1

w1 w2
2 2
w1 w2
2 2

Rs < 1 Overlap Virtually complete separation

Rs < 0.8 Insufficient separation 2% overlap

Laboratoire de Génie Chimique – LGC, Toulouse 17

 II-5 Resolution
Cas 1 : Same width at basew1 = w2
Relation of Purnell
1    1  k  '
Rs    2
  N 1/ 2
(most retained compound 2)
4    1  k 2' 
2

3 main parameters:

Selectivity Capacity Factor Efficiency

Improved separation by adjusting at least one of these 3 parameters

Cas 2 : Same number of trays N1 = N2 = N

1    1  k '  1/ 2 k '1  k 2 '

Rs     N  avec k' 
2    1  1  k '  2

Laboratoire de Génie Chimique – LGC, Toulouse 18

II-5 Resolution

Fixed Rs: Separation = f (Ratio of Peak Areas)

The separation is all the worse as this ratio increases

Influence of the R-term on the separation of

two peaks of equal intensity

Laboratoire de Génie Chimique – LGC, Toulouse 19

II-6 Pressure Drop and Flow Resistance Factor

Pressure drop in a column

 Lu P : Pressure Drop (Pa)
Darcy's Law P  : viscosity (Pa.s)
K0 K0 : Column permeability constante (m²)

d p2  : Flow Resistance Factor

K  0
dp : Particle Diameter (m²)

 depends on:
Particle shape
Particle size distribution   500 : Spherical porous particles
Texture S. ph.   1000 : Irregular porous particles
Filling quality

d p2 P d p2 t0
Experimental determination of  :  
K 0
 L2

Check Fill Quality and Compare Columns

Laboratoire de Génie Chimique – LGC, Toulouse 20

 II-8 Reduced Quantities

t R2 L
Variance s² of the elution peak Determination of N et HEPT s 2 HEPT 
N N
Identification of the global influence of all parameters

But there is no clarification on the impact of each of the parameters on distribution of the peak

Peak spread depends on:

Diameter and nature of pH. S
Nature and Velocity of pH M
Nature of the solute
Homogeneity of the filling

Need to use reduced quantities to determine the influence

of each parameter on HEPT

Column Comparison (≠ ph. S; ≠ ; ≠ C°)

Laboratoire de Génie Chimique – LGC, Toulouse 21

 II-8 Reduced Quantities

L
Reduced length: l
dp
Represents the number of particle slices contained in the column

For columns with the same reduced length, the solute will encounter the same number of
particles on average.
This leads to the same Resolution if these columns are implemented at the same reduced
speed (see below).

Example : l = 20 000 L = 10 cm dp = 5 µm
L = 100 cm dp = 50 µm

HEPT l
Reduced height:
h 
dp N

Represents the number of particle slices per theoretical plateau

Comparison of the efficiency of columns filled with particles of ≠ sizes

Laboratoire de Génie Chimique – LGC, Toulouse 22

 II-8 Reduced Quantities

u dp l d p2
Vitesse réduite : v  Dm : coef. diffusion of the solute in the M . ph.
Dm t0 Dm

𝑙𝑖𝑛𝑒𝑎𝑟 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝜙M

v=→
v diffusion of the solute over a distance equal to the particle diameter

INTRO - GENERALITES GRANDEURS FONDAMENTALES CINETIQUE OPTIMISATION Laboratoire TYPES

DIFFERENTS de GénieDE
Chimique
LC – LGC, Toulouse 23
 III – EXCHANGE KINETICS

III-1 Mechanisms of peak dispersal

Peak Spread N (Number of theoretical plateaus)

Narrow Peak N high  HEPT weak

Linear elution development case:
3 origins of the spread of a peak: (elution development)
-Dispersion of molecules by longitudinal diffusion
- Existence of multiple paths (flow anisotropy)
-- Resistance to material transfer
Independent Processes: variance totale   variance

HEPT   HEPTi  HEPTdiff HEPT flux  HEPTtransfert de matière

i

h   hi  hdiff h flux  htransfert de matière

i

Van Deemter Model (GasC)  Knox Model (LC)

This model has the advantage of being simple and allows you to get rid of the particle size.
It allows easy optimization of the conditions of an analysis and is in good agreement with many experimental results.
With the help of this model, we can explain the different terms of the previous equation.

Laboratoire de Génie Chimique – LGC, Toulouse 24

 III-1 Mechanisms of peak dispersal

B
Longitudinal diffusion: hdiff 
v
This term reflects the influence of the dispersion of solute molecules by longitudinal diffusion
(along the axis of the column).

Einstein's Diffusion Theory 

B  2  1 k'  : Tortuosity Factor
(Influence of Filling)
B2 k’ : Capacity Factor
Flow anisotropy:

-Existence of several possible pathways for ph. M within the ph. S.

- Different speeds within the same section

h flux  A v1/ 3
A depends on:
- Regularity of filling
- particle size distribution

Well filled column 0.5 < A < 1

Incorrect filling or too large a particle size A > 3

Laboratoire de Génie Chimique – LGC, Toulouse 25

 III-1 Mechanisms of peak dispersal

Resistance to material transfer:

Relative Motion of the Two Phases Distribution equilibrium is not achieved

2 concentration profiles whose phase shift increases with velocity.of M. ph hence a

widening of the peak

Limitation of the kinetics of Adsorption-Desorption (or TdM) by solute diffusion (mainly

in the ph. S)
t : Average dwell time in theph. S (dp , DS)

During t : Longitudinal transfer in the ph. M Band Dispersion

The phase shift depends on the path traveled in the elutant phase during the average residence
time of the ph stat.

avec C = f (Dm, Ds)

htransfert de matière  C v
0,01 < C < 2

Laboratoire de Génie Chimique – LGC, Toulouse 26

 III-2 Equation of Knox

Finally, the reduced plateau height varies with the reduced speed according to the equation:

B A=1
h  Av
1/ 3
 C v 1
B=2
v C = 0,1

Log h
A v1/3
B/v
0
Cv

-1
-1 0 1 2 3
h mini = 2,4 pour v = 2,7 Log v

For v = 1 to 10  h Varies little but for v = 10 : Faster separation

For values of v where Av1/3 and Cv are close (e.g. 10 < v < 100), it is possible to group
them in a single term: h = cte vn with 0.33 < n < 1
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 III-3 Experimental Application

Influence of the nature of the ph. S

Knox's model: based on the independence of particle diameter for the same pH S

Numerous experimental verifications

Alumine Sphérisorb AY
dp = 6 – 7,5 – 10 µm

Silice Sphérosil XOA 600

dp = 6,5 – 7,5 – 9 – 12 µm

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III-3 Experimental Application

Influence of the nature of the ph. S

Overall for Completed Goods Columns:0,01 < C < 0,15

However, for polymerized grafted phases or EI resins: C close to 1

Influence of filling quality

Incorrectly filled columns: A  2

Information on the properties of ph. S

h = f (v)
Indications on the quality of the filling

Laboratoire de Génie Chimique – LGC, Toulouse 29

 III-3 Experimental Application

Influence of filling quality

A=1 C = 0,1
vmini = 2,7 hmini = 2,4

A = 0,5 C = 0,1
vmini = 3,4 hmini = 1,5

A=4 C = 0,1
vmini = 1,3 hmini = 6

A = 0,5 C = 0,4
vmini = 2 hmini = 2,4

A=4 C = 0,4
vmini = 1,1 hmini = 6,4

Filling:  ,  et  : hmini with the quality of the filling

si hmini > 5 Incorrect Filling

Laboratoire de Génie Chimique – LGC, Toulouse 30

 III-3 Experimental Application

Influence of filling quality

A=1 C = 0,1
vmini = 2,7 hmini = 2,4

A = 0,5 C = 0,1
vmini = 3,4 hmini = 1,5

A=4 C = 0,1
vmini = 1,3 hmini = 6

A = 0,5 C = 0,4
vmini = 2 hmini = 2,4

A=4 C = 0,4
vmini = 1,1 hmini = 6,4

Transfer Resistance:

 et  : Column Filled in negligible loss of efficiency close to vmini

mais l’ ° of h with v is faster for C larger
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III-3 Experimental Application

Influence of filling quality

A=1 C = 0,1
vmini = 2,7 hmini = 2,4

A = 0,5 C = 0,1
vmini = 3,4 hmini = 1,5

A=4 C = 0,1
vmini = 1,3 hmini = 6

A = 0,5 C = 0,4
vmini = 2 hmini = 2,4

A=4 C = 0,4
vmini = 1,1 hmini = 6,4

 : Poor filling and slow transfer

hmini > 5 et vmini weak

Laboratoire de Génie Chimique – LGC, Toulouse 32

III-3 Experimental Application

Advantages of reduced quantities Characterization of the efficiency of a column

Column Comparison

h = f (v)

Characterization of the behavior of a substrate and/or the quality of the filling

Based on two determinations of h : at v1  3 and v2  100

Si h1 > 5 : Incorrect filling

Si h2 < 10 : Good filling and fast transfer
Si h2/h1 > 4-5 : Slow transfer

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 IV – OPTIMIZATION OF A SEPARATION

IV-1 Introduction

Preliminary Testing Unsatisfactory separation: - or too low a resolution

- or the duration is too long

Optimization of experimental conditions

Ideal : Rs high and time for analyse short while having a P acceptable

Example : Rs low  ° L with the same speed ° Time & P (% L)

 ° dp  ° efficiency but° P (% 1/dp2)

Temps élevé ° v (deviation % hmini )   ° Efficiency (N) et ° P

Optimization: a compromise between several conflicting requirements:Rs – Tps – P

1    1  k 2' 
Rs      N 1/ 2

4    1  k 2' 
2 (most retained compound 2)

3 main parameters: Selectivity Capacity Factor Efficiency

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 IV-2 Resolution

BUT

Selectivity -  Rs with   ' 

 k2 
Mobile and   1
' 
pour k’ >5-10
Stationary Phases  1  k2 
Temperature
° Time of Analysis
Capacity Factor– k’ Rs with k’

Efficiency N Rs with N

N  with L for the same speed  ° time and P (% L)

HEPT rather Rs with HEPT

Knox Equation  h mini  v
v Dm uopt  with Dm
 Velocity uopt : uopt 
uopt  with  dp
dp
° dp  ° P (% 1/dp2)
 HEPT = h dp  hmini dp
HEPT  With dp
(in the vicinity of hmini)
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 V – DIFFERENT TYPES OF LC

V-1 Partition Chromatography Liquid-Liquid Chromatography

Stationary phase : liquid fixed on a support

Separation according to the differential distribution of solutes between the two liquid phases

V-2 Adsorption chromatography Liquid-Solid Chromatography

Separation according to the differential distribution of solutes between ph. S (solid) and ph. Mobile (L)

Adsorbants : - Low Capacity (Alumina, Talc, Sodium Carbonate)

(solid particles) - High capacity (silica gel)

-High Electrical Polarity (Silica, Alumina)

-- Low polarity (activated carbon)

Solvants : Elutant power = f (solvent polarity)

Polar Solvent Important eluting power Rapid elution

ph. S : Polar / Solute: Polar
Non-polar solvent Low eluting power Slow Elution
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 Chromatographie Liquide
V-2 Chromatographie d’adsorption

Gel de silice : SiO2 x / 2 OH x n H 2O  p

Groupe silanol : propriétés adsorbantes

En chromatographie : différents types de sites mis en jeu

Silanol Silanol Pont Silanol libre Gel de silice

libre lié Siloxane hydraté fortement hydraté

Gpts siloxane  Si  O  Si 

Rque : si recouvrement total par H2O : chromatographie de partage

INTRO - GENERALITES GRANDEURS FONDAMENTALES CINETIQUE OPTIMISATION

Laboratoire de Génie Chimique – LGC, Toulouse
DIFFERENTS TYPES DE LC 37
 V-2 Adsorption Chromatography

Normal Phase:

Stationary phase : Polar Solid

(often silica grafted with organic polar patterns)

Mobile phase : Nonpolar solvent (often a mixture)

Separation based on hydrophobic interactions between the molecules to be separated and the ph. S.

+ The solute is polar and + it is retained by the ph. S.

+ The solute is nonpolar and + it is driven by ph. M.

Solute: Small Polar Molecules

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 Chromatographie Liquide
V-2 Chromatographie d’adsorption

Phase Normale :

Greffage de la silice : Réaction de silanisation

Réaction d’une silice avec un silane mono (chlorosilane),
di ou trifonctionnalisé chloro ou alkoxysilane

 Formation de liaisons  Si  O  Si 
Principale phases greffées polaires :

Aminopropyle :  CH 3   NH 2

Alkylnitrile :  CH 2 n  C  N

Glycéropropyle :

 CH 2 3  O  CH 2  CH (OH )  CH 2 (OH )

INTRO - GENERALITES GRANDEURS FONDAMENTALES CINETIQUE OPTIMISATION

Laboratoire de Génie Chimique – LGC, Toulouse
DIFFERENTS TYPES DE LC 39
 V-2 Adsorption Chromatography

Reverse Phase:

Stationary phase : Nonpolar Solid

(often silica grafted with non-polar organic patterns)

Mobile phase : Polar solvent (often a water-methanol or water-acetonitrile mixture)

Separation based on hydrophobic interactions between the molecules to be separated and the ph. S.

+ The solute is nonpolar and + it is retained by the pH. S.

+ The solute is polar and + it is driven by pH. M.

solute: nonpolar small molecules (lipids, A.A., peptides and proteins)

Remarks:
Analysis: Reverse-Phase High Pressure Liquid Chromatography (RP-HPLC)
Represents a large number of separations

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 Chromatographie Liquide
V-2 Chromatographie d’adsorption

Phase Inverse :

Greffage de la silice :

Greffons apolaires de tailles différentes (de 2 à 18 Carbones)

Suivant le nb de C  ph. S. de polarités ≠

Greffons les plus courants : C8 (octyle) et C18 (octadécyle)

INTRO - GENERALITES GRANDEURS FONDAMENTALES CINETIQUE OPTIMISATION

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DIFFERENTS TYPES DE LC 41
V-2 Adsorption Chromatography

Elutant Power

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V-2 Adsorption Chromatography

Solvent strength

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V-3 Affinity Chromatography

Stationary phase : inert macromolecular support onto which an effector is grafted

that has a biological affinity to a solute
(e.g. ph.S.: carboxymethylcellulose, Sephadex, polyacrylamide gel)

3 types of affinities: Enzyme – Substrate

Ligand – Receptor
Antigen – Antibody

3 steps:
1- Binding: only the molecule with an affinity is retained by the effector
2- Purification: elution of contaminant molecules
3- Elution: detachment of the molecule of interest(° pH, FI, compétition with a free ligand)

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 V-4 Ion Exchange Chromatography

Separation of ionized or ionizable species

Separation Mechanism: Electrostatic Interactions

Stationary phase : Ion exchanger

Solid with ionizable functions RX

R: Fixed
X: movable and interchangeable (counter-ion)

Force association of electrostatic attractions

Solanki & Saini, 2015

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