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CHROMATOGRAPHY
Basic Principles
WHAT IS CHROMATOGRAPHY?
Separation Detection:
Identification/
Quantification
Chromatography Detector +
LEMONS and
ORANGES
CHROMATOGRAPHY COUPLED TO MS?
Chromatography:
Separation
Detection:
Identification/
Quantification
Chromatography MS +
Salt
Drug
Plasma
Proteins
NaCl
MWT: 371 MWT: 58.5
MWT: e.g.
10000
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INVENTION OF CHROMATOGRAPHY BY M.
TSWETT
Ether
CaCO
3
Chlorophyll
Chromatography
Colors
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COMPARING CHROMATOGRAPHY TO THE
FLOW OF A RIVER...
Base
Water flow
Light leaf
Heavy stone
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MOBILE PHASE / STATIONARY PHASE
A site in which a moving
phase (mobile phase) and
a non-moving phase
(stationary phase) make
contact via an interface
that is set up.
The affinity with the
mobile phase and
stationary phase varies
with the solute.
Separation occurs due to
differences in the speed of
motion.
Strong
Weak
Mobile
phase
Stationary
phase
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LIQUID CHROMATOGRAPHY
Chromatography in which the mobile
phase is a liquid.
The liquid used as the mobile phase is called the
eluent.
The stationary phase is usually a solid
or a liquid.
In general, it is possible to analyze any
substance that can be stably dissolved
in the mobile phase.
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INTERACTION BETWEEN SOLUTES,
STATIONARY PHASE, AND MOBILE PHASE
Differences in the interactions between the solutes
and stationary and mobile phases enable separation.
Solute
Stationary
phase
Mobile phase
Degree of adsorption,
solubility, ionicity, etc.
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COLUMN CHROMATOGRAPHY AND PLANAR
CHROMATOGRAPHY
Separation column
Packing material
Column Chromatography
Paper or a
substrate coated
with particles
Paper Chromatography
Thin Layer Chromatography (TLC)
Column chromatography:
Stationary phase is held in a narrow tube
Mobile phase is forced under pressure through
Stationary phase.

Planar chromatography:
Stationary phase is supported on a flat plate or in the
small spaces of a paper.
Mobile phase moves through the stationary phase by
capillary action or under the influence of gravity.

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SEPARATION PROCESS AND CHROMATOGRAM
FOR COLUMN CHROMATOGRAPHY
O
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p
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t

c
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Time
Chromatogram
Detector
CHROMATOGRAM
A detector that responds to solute concentration
is placed at the end of the column.

Detector signal is plotted as function of time, a
series of peaks is obtained.

The plot, called a chromatogram, is useful for
both qualitative and quantitative analysis.
The positions of peaks on the time axis may serve
to identify the components of the sample.
The areas under the peaks provide a quantitative
measure of the amount of each component.

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CHROMATOGRAM
t
R
t
0
I
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n
s
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t
y

o
f

d
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t
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Time
Peak
t
R
: Retention time
h

A

t
0
: Non-retention time
A : Peak area
h : Peak height
k =

0

Retention
Factor
APPLICATIONS OF CHROMATOGRAPHY

Chromatography has grown to be
the premiere method for separating
closely related chemical species. In
addition, it can be employed for
qualitative identification and
quantitative determination of
separated species.
QUALITATIVE ANALYSIS

A chromatogram provides only a single piece of
qualitative information about each species in a
sample, namely, its retention time or its position on
the stationary phase.

It is a widely used tool for recognizing the presence or
absence of components of mixtures containing a
limited number of possible species whose identities
are known.

Positive spectroscopic identification would be
impossible without a preliminary chromatographic
separation on a complex sample.
QUANTITATIVE ANALYSIS

Chromatography can provide useful quantitative
information about the separated species.

Quantitative column chromatography is based upon a
comparison of either the height or the area of the analyte
peak with that of one or more standards.

For planar chromatography, the area covered by the
separated species serves as the analytical parameter.

If conditions are properly controlled, these parameters
vary linearly with concentration.
GAS CHROMATOGRAPHY (GC)
LIQUID CHROMATOGRAPHY (LC)
GC
Excellent Resolving power
Analysed compounds should be
Volatile
Thermally Stable.
Suitable for quantitative and qualitative analysis.

LC
Suitable for non-volatile and polar compounds (e.g.
pharmaceuticals)
High Performance Liquid Chromatography (HPLC)
Suitable for quantitative and qualitative analysis.
TLC (Thin Layer Chromatography) used mainly for
qualitative analysis.