19/03/2012

Chromatography
What you should be able to do after
these
lectures
:To explain the effect of
chromatography
chromatograph
To
explain different kinds of
y
chromatograph

yTo characterise the performance of

To illustrate a GC (and an HPLC)

Analysis of a mixture of organic

compounds
1st step is often to produce an extract!

Like dissolves like

Lipids, alkanes, aromatics,
non polar solvent
(e.g., pentane,
hexane)
Sugars, alcohols,
carboxylic acids,
proteins
polar solvents (e.g.,
water, alcohols)

19/03/2012

How does chromatography

work the general
principles
(e.g., 60
%) at 2
Onephases
extraction:
partitioning
between

equilibrium

Multiple extraction (successive)

2

1st extraction 60 %

2nd extraction 60 % (overall 84 %)

3

3rd extraction 60 % (overall 94 %)

stationary phase; mobile

phase is a continuous
mobile phase

Definitio
ns
Chromatography is a process in
which we separate compounds from
one another by passing them all
through a column that retains some
compounds longer than other.
Stationary phase stays in the
column and is either a solid or a
liquid coating
Mobile phase is either liquid or gas
(eluent)
Gas chromatography (GC, mobile phase
is a gas)
Liquid chromatography (LC or HPLC,
mobile phase is a liquid)

start

19/03/2012

Chromatography

Chromatography is a process in which we separate

compounds from one another by passing them all
through a column that retains some compounds longer
than other.

? Which Species
Separation: Liquid/Gas
Chromatography
Chromatographic

Mobile Phase )Gas/liquid(

signal

separation system

Stationary Phase
Sample injection:
Species in

time

(gas/liquid
)
Mixture
of
matrix

Compound Separation:
equilibria
Repeated repartition

(resolved signal
Detection:

Chromtogram )time

19/03/2012

Peak broadening

Peak Broadening

signal

Ideal, no peak broadening

time

signal

Peak Broadening

time

Laminar flow profile

19/03/2012

Peak broadening

Peak broadening or spread comes from

Eddie Diffusion
Multiple flow paths (smaller particle in stationary
phase narrow the peaks, but absent in a hollow tubular
column)

How can we
describe a
chromatogram
?

Retention time (s)

19/03/2012

Gaussian peaks
Gaussian distribution curve: normal distribution

68.2%
95.5%
99.7%

How can we
describe a
chromatogram
?

Retention time (s)

!Gaussian shaped peaks

19/03/2012

Theoretical plates and

HETP
Traditional: distillation was the most
powerful tool to separate
compound A from B

Distillation columns were divided into section in

which AB in vapour are in equilibrium with AB in
liquid (one plate!)
The more plates the more equilibriums (multiple
extractions) the better the separation.
HETP as Criterion for chromatographic
performance

How does chromatography

work the general
principles
en

One extraction: partitioning

betwe
2 phases (e.g., 60 %) at
equilibrium

Multiple extraction
(successive)
1st extraction 60 %
2nd extraction 60 % (overall 84 %)
3rd extraction 60 % (overall 94 %)

stationary phase; mobile

phase is a
continuous mobile phase

start

1
2
3

19/03/2012

Theoretical plates and

HETP

separation

The more plates the more equilibriums (multiple

.
extractions) the better the
Separation columns are divided into theoretical
plates
Calculating HETP for a given system and a given
compound:
N = 5.55 * ( tr / w1/2)2 ,

(or N=16* ( tr /

w)2 )
N=number of plates, tr retention time, w1/2 width at
half height.
HETP = L / N
HETP: Height Equivalent of a Theoretical Plate, L:
length of column
(usually HETP is given in mm)

Peak resolution

Determination of HETP for:

-

comparison of different systems

- Resolution
between neighbouring peaks is equal
Optimisation of separation conditions
tothe peak separation (tr) divided by the average
peak
width (wav) , Resolution = tr/ wav
The better between
the resolution,
separation
peaksthe more
complete is the

Shape of peaks like Gaussian curves !!

19/03/2012

What information can

we get from a
chromatogram ?
Qualitative
information from
the retention time
Comparing
with
standards

Quantitative
information from
the signal
generated
Needs a comparison
with standards
(calibration)

Types of
chromatography 1

Adsorption chromatography: solute is adsorbed on the

surface of solids
(GC, LC) not often used !

Partition chromatography: thin layer of liquid on the solid

surface.
Solute equilibrate between stationary liquid and mobile phase.
(continuous multiple extraction) often used for separating
organics!

19/03/2012

Brief Summary
Chromatography is used to
separate compounds
Chromatography combined with
detectors can give qualitative and
quantitative information
The goodness of separation can be
measured and calculated by HETP.
If number of plates increase the
separation
gets better, that is the case when
HETP decreases
Length of column increases