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Chromatographic

separations
Chapter 26

The stuff you do before
you analyze a complex
sample
It is all about Reducing
Interferences

Chromatography basics
Mobile and Stationary
phase
Retention - Migration
Bands or zones
Equilibrium!
Column vs. planar
Liquid vs. gas vs. SF
High vs. low resolution
Partition
Adsorption
Ion exchange
Size exclusion
Chromatography
A. Column Chromatograpgy, B. Planar Chromatograpgy
Column Chromatography
Chromatogram
Dilution &
Peak broadening!
Chromatography: Peak separations
Chromatography: Peak Resolution
Poor resolution
More separation
Less band spread
Chromatography:
Distribution Constant (recommended by IUPAC)
(old term: partition coefficient)

M
S
c
c
c
K =
stationary
mobile
A mobile A stationary
K ~ constant linear chromatography

>>>K >>> Retention in the stationary phase Retention times

How to manipulate K?
C
S
= n
S
/V
S
, C
M
= n
M
/V
M
Chromatography Retention Times
t
M
= retention time of mobile phase (dead time)
t
R
= retention time of analyte (solute)
t
S
= time spent in stationary phase (adjusted retention time)
L = length of the column
Chromatography: Velocities
Linear rate of solute migration!
M
R
t
L
t
L
v
=
=

Velocity = distance/time length of column/ retention times



Velocity of solute:



Velocity of mobile phase:
Chromatography
Velocity/Retention time and Kc
S S M M
M M
V c V c
V c
v
v
v
+
=
=
=

solute of moles total


phase mobile in solute of moles
phase mobile in time of fraction
Chromatography
Velocity Relationships
M S
M
S
M M S S
S S M M
M M
V V K
v
c
c
K
V c V c
v
V c V c
V c
v
/ 1
1
Constant on Distributi
/ 1
1
+
=
=
+
=
+
=

Chromatography
Retention Factor : are we there yet?
M
M R
A
A M R
A
M S A A
M S
t
t t
k
k t
L
t
L
k
v
V V K k
V V K
v

=
+
=
+
=
=
+
=
1
1
1
1
Factor) (Retention /
/ 1
1

Adjusted retention time


Relative retention time:
RRT = t
R
/t
Rs

t
Rs
= retention time of internal standard
Chromatography
Selectivity Factor: can you separate from your neighbor
M A R
M B R
M
M B R
B
M
M A R
A
A
B
A
B
t t
t t
t
t t
k and
t
t t
k
k
k
K
K

=
=
=
) (
) (
) ( ) (
o
o
o
B retained more than A o >1
Distribution
Constant
Retention factor
Retention time
Chromatography
Column Efficiency - Theoretical Plates
Plate and Rate Theories
L
H
H
L
N
N
H
2
plates of number
height plate
o
=
=
=
=
o standard deviation o
2
/L variance per unit length.
L = length of column packing
Chromatography
Relation between column distance and
retention times
R
R
R
t L
t L
t
L
/
in time deviation standard
time retention
distance in deviation standard
(distance) length column
o
t
t o
t
o
=
=
=
=
=
=
Chromatography
Relation between column distance and
retention times
2
2 2
16
4
4
R
R
R
R
t
L W
L
H
t
L W
W
t
L
t L
= =
=
=
=
=
o
o
t
t
o
t o
~96%
2t
Tangent at
Inflection point
Chromatography
Determining the Number
of Theoretical Plates
2
2 / 1
2
54 . 5
16
pates of number
|
|
.
|

\
|
=
|
.
|

\
|
=
=
W
t
N
W
t
N
N
R
R
W
1/2
Summary of Plate Theory
Successfully accounts for the peak shapes
and rate of movement
Does not account for the mechanism
causing peak broadening
No indication of other parameters effects
No indication for adjusting experimental
parameters



Rate Theory
Zone broadening is related to Mass Transfer
processes




Column Efficiency
Kinetic variables
Zone Broadening
Flow Rate of Mobile Phase
Liquid chromatography
Gas chromatography
Note the differences in flowrate and plates height scales
Why GC normalluy has high H, but also high overall efficiency?
Zone Broadening
Kinetic Processes
) ( /
M S
C C B A H + + + =
Van - Deemter
Equation
and are constants that
depend on quality of the
packing.

B is coefficient of
longitudinal diffusion.

Cs and Cm are
coefficients of mass
transfer in stationary and
mobile phase,
respectively.
Zone Broadening
Kinetic Processes
) ( /
M S
C C B A H + + + =
Van - Deemter Equation
Zone Broadening
Multiple Pathways
Eddy Diffusion: band broadening
process results from different path
lengths passed by solutes.

1. Directly proportional to the
diameters of packing
2. Offset by ordinary diffusion
3. Lower mobile-phase velocity,
smaller eddy diffusion


Stagnant pools of mobile phase
retained in stationary phase.
Chromatography
Resolution
B A
A R B R
s
B A
s
B A
s
W W
t t
R
W W
Z
R
W W
Z
R
+

=
+
A
=
+
A
=
] ) ( ) [( 2
2
2 / 2 /
Chromatographic
Separations with a twist
Chromatographic Definitions
Chromatographic
Relationships
Quantitative Analysis
Peak areas
Peak height
Calibration and standards
Internal Standard method

Summary
Relate to column chromatography
Retention times
Velocities of mobile and component
Height equivalent of theoretical plates
Peak or zone broadening
Resolution