CHROMATOGRAPHY IN BIOSEPARATION

• Quantitative Analysis (Numerical Problems) and Analytical Problems

(Case Studies)

• Self-study - General principles of (a) Gel Filtration, (b) Ion Exchange

Chromatography, (c) Reverse Phase Chromatography, (d) HPLC,
(e) Affinity Chromatography

For self-study refer to any of the following books or any other reading
material on chromatography available with you.

Physical Biochemistry: Principles and Applications. 2nd Edition by David

Sheehan. © 2009 John Wiley & Sons.

Wilson and Walker’s Principles and Techniques of Biochemistry and

Molecular Biology. 8th Edition. Edited by Andreas Hofman and Samuel Clokie.
© 2018 Cambridge University Press.

Handout-I Bioseparation Engineering (BT 404) 1

 Essentials of Chromatography
Basic Working Principle in Liquid Chromatography

• A separation process achieved by distributing components of

a mixture between a stationary phase and a mobile phase
• The components retarded in the stationary phase are
retained longer in the system than those present selectively
in the mobile phase
• Solutes are eluted as local concentrations in the mobile
phase in the order of their increasing distribution
coefficients with respect to the stationary phase
• Primarily used in bioseparation for product purification,
especially recovery of small amounts of high value
components such as biotherapeutics
Handout-I Bioseparation Engineering (BT 404) 2
Function of Column - Separation
B B C A
A C
Column (Distribution System)

Peaks Not Separated

B C A

Peaks Separated
Peaks Very Broad – Not Good Enough

Handout-I Bioseparation Engineering (BT 404) 3

Function of Column – Minimize Dispersion
B B C A
A C
Column (Distribution System)

Peaks Separated and Broad

B C A

Peaks Separated and Relatively Sharp !

Handout-I Bioseparation Engineering (BT 404) 4

Typical Chromatogram is Gaussian

Retention volume (Vr)

Peak width at
Signal

Void volume (V0) 0.607 h = 2

Peak height (h)
Peak width at
base = 4

Volume

Handout-I Bioseparation Engineering (BT 404) 5

Partition Coefficient and Solute Distribution
Stationary Mobile Stationary Mobile

100 100
Plate 1

Initial number
Horizontal
of solute
Plate 2 and
molecules =
Longitudinal
200
Diffusion of
Molecules
Partition across
Coefficient Plate 3 theoretical
(K = 1.0) plates in the
column
No. of
theoretical Plate 4
plates in the
column = 5 Stage I

Plate 5

Handout-I Bioseparation Engineering (BT 404) 6

Partition Coefficient and Solute Distribution
Stationary Mobile Stationary Mobile

100 100 50 50

Horizontal
50 50 and
Longitudinal
Diffusion of
Molecules
across
theoretical
plates in the
column

Stage I Stage II

Handout-I Bioseparation Engineering (BT 404) 7

Partition Coefficient and Solute Distribution
Stationary Mobile Stationary Mobile

50 50 25 25

Horizontal
50 50 50 50 and
Longitudinal
Diffusion of
Molecules
25 25 across
theoretical
plates in the
column

Stage II Stage III

Handout-I Bioseparation Engineering (BT 404) 8

Partition Coefficient and Solute Distribution
Stationary Mobile Stationary Mobile

25 25 12.5 12.5

Horizontal
50 50 37.5 37.5 and
Longitudinal
Diffusion of
Molecules
37.5 37.5 across
25 25
theoretical
plates in the
column

12.5 12.5

Stage III Stage IV

Handout-I Bioseparation Engineering (BT 404) 9

Partition Coefficient and Solute Distribution
Stationary Mobile Stationary Mobile

K = 1.0

No. of Molecules
12.5 12.5 6.25 6.25

37.5 37.5 25 25

37.5 37.5 37.5 37.5

1 2 3 4 5
Plate Number
12.5 12.5 25 25

Stage Stage
IV V
6.25 6.25

Handout-I Bioseparation Engineering (BT 404) 10

Physical Cause of Peak Broadening
A. Eddy Diffusion

Initial Band Width

Broad Band

Stationary
Phase
Particle

Narrow Broad
Channel Channel

Handout-I Bioseparation Engineering (BT 404) 11

Physical Cause of Peak Broadening
B. Longitudinal Diffusion

Mobile Initial Mobile Mobile

Phase Band Phase Phase
Width

Broad
Band Width

Handout-I Bioseparation Engineering (BT 404) 12

Physical Cause of Peak Broadening
C. Mobile Phase Mass Transfer
Initial Band Width

Slower-moving Mobile Phase

Near Stationary Phase Particle

Stationary Broad
Phase Band Width
Particle

Faster-moving Mobile Phase

In the Middle of Channel

Handout-I Bioseparation Engineering (BT 404) 13

Size Exclusion Chromatography (Gel Filtration)

Source: Gel Filtration Principles and Methods. 18-1022-18. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 14

Size Exclusion Chromatography (Gel Filtration)

Source: Gel Filtration Principles and Methods. 18-1022-18. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 15

Affinity Chromatography
1. Affinity medium equilibrated in binding
buffer

2. Sample applied under conditions that favor

specific binding of the target molecule(s) to
ligand. Unbound material washed through the
column

3. Target protein elution performed by using a

competitive ligand, or by changing the pH,
ionic strength or polarity.

4. Affinity medium re-equilibrated with binding

buffer

Source: Affinity Chromatography Principles and Methods. 18-1022-29. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 16

Affinity Chromatography

Source: Affinity Chromatography Principles and Methods. 18-1022-29. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 17

Ion Exchange Chromatography: Protein Titration Curve

Source: Ion Exchange Chromatography & Chromatofocusing Principles and Methods.

11-0004-21. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 18

Anion Exchange Chromatography

Source: Ion Exchange Chromatography & Chromatofocusing Principles and Methods.

11-0004-21. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 19

Anion Exchange Chromatography

Source: Ion Exchange Chromatography & Chromatofocusing Principles and Methods.

11-0004-21. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 20

Anion Exchange Chromatography

Source: Ion Exchange Chromatography & Chromatofocusing Principles and Methods.

11-0004-21. Amersham Biosciences

Handout-I Bioseparation Engineering (BT 404) 21