Chromatographic

separations
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

Dilution &
Peak broadening!

Chromatogram

Chromatography:
separations

Peak

Chromatography:
Resolution

Poor resolution

More separation

Less band spread

Peak

Chromatography:
Distribution Constant
(recommended by IUPAC)
(old term: partition coefficient)

A mobile A stationary

cS
Kc
cM
CS = nS/VS,

stationary
mobile

CM = nM/VM

K ~ constant linear chromatography

>>>K >>> Retention in the stationary phase Retentio

How to manipulate K?

Chromatography
Retention Times

tM = retention time of mobile phase (dead

time)
tR = retention time of analyte (solute)
tS = time spent in stationary phase (adjusted

Chromatography:

Velocities
Linear rate of solute migration!

Velocity = distance/time length of column/ retenti

L
tR

Velocity of mobile phase:

L
tM

Velocity of solute:

Chromatography

Velocity/Retention time and Kc

v fraction of time in mobile phase

moles of solute in mobile phase
v
total moles of solute
cMVM
v
cMVM cSVS

Chromatography

Velocity Relationships
cM VM
v
cM VM cSVS
1
v
1 cSVS / cM VM
cS
K
cM

Distribution Constant

1
v
1 K VS / VM

Chromatography

Retention Factor : are we there yet?

1
v
1 K VS / VM
k A K AVS / VM

(Retention Factor)

1
v
1 k A
L
L
1

tR tM 1 k A
tR tM
kA
tM

Adjusted retention time

Relative retention time:

RRT = tR/tRs
tRs = retention time of internal
standard

Chromatography

Selectivity Factor: can you separate from

your neighbor
B retained more than A >1

KB

KA

Distribution
Constant

kB

kA

Retention
factor

(t R ) A tM
(tR ) B tM
kA
and k B
tM
tM
(tR ) B tM

(t R ) A tM

Retention
time

Chromatography

Column Efficiency - Theoretical

Plates
Plate and Rate Theories
H plate height
N number of plates
L
N
H
2
H
L

L = length of column pack

standard deviation 2/L variance per unit length.

Chromatography

Relation between column

distance and retention times
L column length (distance)
standard deviation in distance
t R retention time

standard deviation in time

L tR

L / tR

Chromatography

Relation between column

distance and retention times

Tangent at
Inflection
point

~96%
2

L tR
L

tR
W 4
WL

4 tR
2 W2 L
H

L
16 t R2

Chromatography

Determining the Number

of Theoretical Plates
N number of pates

N 16
W1/2

tR

t
N 5.54 R
W1/ 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

Why GC normalluy has high H, but also high overall efficien

Zone Broadening
Kinetic Processes

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,
Mrespectively.

H A B / (CS C )

Zone Broadening
Kinetic Processes

Van - Deemter Equation

H A B / (CS CM )

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
Stagnant pools of mobile pha
diffusion
retained in stationary phase.

Chromatography
Resolution

Z
Rs
WA / 2 WB / 2
2Z
Rs
WA WB
2[(t R ) B (t R ) A ]
Rs
WA WB

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