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CHROMATOGRAPHY

Separates components in
mixture
Based on:
- polarity
- boiling point
- ionic strength
- size

Partition
Chromatography

Movie

Used in GC & LC
Molecules will partition into
the stationary phase based
upon affinity for stationary
phase & eventually partition
into mobile phase again
Thin layer is coated onto
inside of GC column or on
small particles on LC
column

Chromatography

Mobile phase: phase which sample is

dissolved in may be gas, liquid, or
supercritical fluid
Stationary phase: phase which mobile phase
is forced through
Mobile and stationary phases are chosen so
the analyte will distribute itself between the
two phases

Adsorption
Chromatography

Very similar to
partition
chromatography
Adsorption just on
surface, partition into
thin layer
Not used as widely as
partition used mainly
in TLC & very small
particles in LC

Movie

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Ion Exchange
Chromatography

Movie

Separation of either
cations or anions
Separtion based on
relative strength of
ionic bond
Anion exchange has
cations on surface
Used in LC exclusively

Gel Electrophoresis

Separation based on
size and charge
Smaller molecules will
migrate further, less
tangled
Movie

Molecular Exclusion
Chromatography

Separation based on
size
Small molecules get
trapped in pores & take
longer to get out

Movie

Affinity
Chromatography

Very selective
Specific binding site is
used to concentrate
analyte on column
Used a lot in biological
applications

Movie

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Typical Gas Chromatogram

Typical Liquid
Chromatogram

Introduction to Chromatography Theory

General Relationships
1. Distribution constant
a. Craig counter current experiment
2. Retention time
3. Relationship between distribution constant
and retention time
4. Capacity factor k
5. Selectivity factor a

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Introduction to Chromatography Theory

Peak Broadening
1. Shapes
2. Column efficiency
a. plate height
b. number of plates
3. Kinetic factors Van Deemter equation

Craig counter current

movie

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2. Retention time t
Time it takes for analyte
r

to reach detector after

sample injection
Tm = retention time for
material to come through
column which is not
retained also called dead
time or void volume

tm rate of migration is the

same as the average rate of
motion of the mobile phase
molecules u = L/tm

v = u x moles of analyte in mobile phase

total moles of analyte

v=ux

CmVm

CmVm + CsVs

v=ux

1
1 + CsVs/CmVm

1
1 + KVs/Vm

4. Capacity factor k

Describes migration rates of analytes in column

For a species A
k = KAVs

3. Distribution constant &

retention time

v = u x 1/(1 + k)

kA = (tr- tm)/tm
For separations involving few components ideal capacity
factors are between 1 - 5

5. Selectivity factor a

Ability to distinguish
between 2 species, A &
B

What is k
for this
peak?

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Purpose of
Chromatography

Peak Broadening

Achieve separation

Elution movie

Peak Broadening

Is peak broadening a good or bad thing?

BAD Why?

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Column Efficiency

Plate Height

Plate height (H)

# theoretical plates
(N)
N = L/H
Efficiency of a
column goes up as
N increases and H
decreases
Typical 250 10,000
plates

3. Kinetic Factors: The Van

Deemter Equation

What variable do you think are important in

determining the efficiency of a separation?

Reality: column efficiency is

affected by kinetic factors

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In your notebook predict what the effect of

increasing linear velocity (flow rate) will have
on column efficiency (H)

Van Deemter Equation

Van Deemter Equation

How can band broadening be reduced?

(and thus column efficiency be enhanced)

H = A + B/u + Cu

A = Eddy diffusion: Due to

different paths molecules
can take as they go through
particles
B/u = longitudnal diffusion Band diffuses in and
against direction of mobile phase movement
Cu often broken into 2 terms Csu + Cmu
Mass transfer coefficient: Time it takes for analyte to
diffuse into stationary phase

1.
2.
3.
4.

Decrease particle diameter

Decrease column width
Lowering temperature in GC (reduces diffusion
coefficient)
Minimize thickness of liquid stationary phase

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Resolution
is called General
ThisElution
Problem
Rs = 2((tr)B (tr)A)
wA + wB