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Separation Techniques
Theory of Column Chromatography
Definitions
Elution Chromatography
Partition Chromatography Theory
Vaq
KD
Countercurrent Fractionation
Partition coefficient
Capacity
Selectivity
Vorg
www.chem.unsw.edu.au/UGNotes/Guilhaus/
CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -2
Definitions
Chromatography (from Greek chromatos
meaning colour) - named by Tswett who
separated plant pigments in a column.
Michael Tswett
(1872-1919)
Russian Botanist
Elution Chromatography
1
2
4
5
Chromatographic Equilibria
Adsorption: solute
adsorbed on surface of
stationary phase
Chromatographic Equilibria
Partition: solute dissolved
in liquid phase bonded to
surface of solid support
Chromatographic Equilibria
Chromatographic Equilibria
Molecular Exclusion:
large molecules are
excluded and are therfore
not retained.
M. Guilhaus UNSW 1999 - All rights reserved.
Chromatographic Equilibria
Affinity: based on
molecular recognition
between covalently bound
molecule having specific
donor-receptor attraction.
Other molecules are not
retained.
mobile phase
stationary phase
v=
L
tR
u=
L
tM
A is not retained
B is retained by the
column
solute velocity
mobile phase
velocity
10
CM VM
1
1
=u
=u
V
C V
CM VM + CS VS
1+ S S
1+ Kp S
VM
CM VM
11
k' R =
1
1+ Kp
VS
VM
KVS
VM
v =u
1
L
L
=
t R t M 1 + k' R
k' R =
tR - tM
tM
12
13
14
COLUMN
stationary phase
mobile phase
15
COLUMN
stationary phase
mobile phase
peak width C u
CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -2
16
COLUMN
stationary phase
mobile phase
peak width A
CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -2
17
y = H = A + B/u + Cu
y = Cu
y=A
y = B/u
u
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19
The number of
theoretical plates
is estimated from
the width of the
peak
M. Guilhaus UNSW 1999 - All rights reserved.
t
N = 16 R
W
20
A quantitative
measure of column
ability to separate
solutes with similar
mobilities
M. Guilhaus UNSW 1999 - All rights reserved.
R=
2Dt R
WA + WB
21