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
=
=
µ
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
+
× =
× =
× =
µ
µ
µ
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
+
× =
=
+
× =
+
× =
µ
µ
µ
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
µ
µ
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
µ µ ) ( /
M S
C C B A H + + + =
Van - Deemter
Equation
λ and γ 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
µ µ ) ( /
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

Sign up to vote on this title
UsefulNot useful

Master Your Semester with Scribd & The New York Times

Special offer for students: Only $4.99/month.

Master Your Semester with a Special Offer from Scribd & The New York Times

Cancel anytime.