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Normal-phase chromatography is really not that normal. That is to say that it is used much less frequently than reversed-phase chromatography. – PowerPoint PPT presentation

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Normal-phase chromatography
Normal-phase chromatography is really not that
normal. That is to say that it is used much
less frequently than reversed-phase
chromatography. The main reason for this is
simply that many separations can be accomplished
using either reversed-phase or normal phase
chromatography, but reversed-phase is easier,
and hence more common.
Separation in Normal-Phase Chromatography
-- samples with many different functional
groups -- samples with molecules containing
double bonds -- samples containing isomers --
samples that are very hydrophobic -- samples that
are very hydrophillic
Normal-phase chromatography is also called
adsorption chromatography or liquid-solid
chromatography. Retention of analytes occurs
by an adsorption process rather than by a
partition process (as in reversed-phase
Silica is the most common of the non-bonded
phases and can provide very high selectivity for
many applications, but water adsorption by the
silica can make reproducible retention times
Comparison of different types of silica columns.
(a) acidic type A silica (b) less acidic type B
1, toluene 2, benzanilide 3, phenol 4, benzyl
alcohol I, impurity
Other Stationary Phases
Alumina, has unique selectivity, but it is little
used because it has problems such as low
theoretical plate number (N), variable retention
times, and low sample recovery. Cyano columns
are the best for general analysis because they
are the most stable and are more convienent to
use than silica columns. Diol and amino
columns can offer different selectivities, but
are less stable than cyano columns.
Parameters for Commonly Used Normal-Phase
Separation in Normal and Reversed-Phase
In normal-phase chromatography, the stationary
phase is polar and it interacts with the polar
parts of the molecule, therefore it would not be
an effective method for separating molecules such
as butanal, hexanal and octanal.
In reversed-phase chromatography, the stationary
phase is non-polar and therefore it interacts
with the non-polar portion of molecules.
Reversed-phase chromatography would be a good
choice for separating butanal, hexanal and
The adsorption of analyte molecules decreases in
the following order carboxylic acids
amides amines alcohols ketones
aldehydes esters nitro compounds ethers
sulphides organic halogen compounds
aromatics olefins saturated hydrocarbons
Separation of Isomers
The adsorption of an analyte is based on the type
of functional group present and also steric
factors which makes is similar to chiral or
affinity chromatography, the difference being
that the adsorption sites on silica are not very
Separation of Very Hydrophobic Molecules
Hydrophobic analytes are more soluble in these
solvents than they would be in the aqueous
mobile phases used in reversed phase
Very hydrophobic molecules are strongly retained
in reversed phase chromatography often resulting
in poor separations. These molecules can be
analyzed using normal phase chromatography.
Separation of Very Hydrophilic Molecules
Just as there is non-aqueous reversed-phase
chromatography there are also aqueous mobile
phases used in normal phase chromatography. In
this case, very hydrophyllic samples that are
not retained in reversed phase conditions can be
chromatographed. Carbohydrates are often
separated on an amino column with mobile phases
consisting of 60-80 acetonitrile/water.
Normal phase separation of carbohydrates using
an amino column and 75 acetonitrile-water as
the mobile phase. 1 fructose 2 glucose
3 sucrose 4 maltose
Gradient Elution
Polar solvents can interact strongly with the
surface of a silica. This strong interaction
makes changing solvents difficult because it
takes a long time for the column and solvent to
come to equilibrium (typically from 45 min to 1
Solvent Demixing - Example 100 Hexane --gt 100
Isopropanol As the gradient changes from 100
hexane with the addition of isopropanol, all
the added isopropanol is adsorbed to the column
surface and 100 hexane continues to elute from
the column. After a while, the column becomes
saturated with isopropanol, and a sudden jump in
isopropanol concentration is seen in the mobile
phase. This rapid change in mobile phase solvent
strength will elute sample components with low k
values and poor separation.
Stationary Phase Water Content
What else is silica used for?
Even fairly non-polar solvents will adsorb some
water from the air. The dissolved water will be
adsorbed on the surface of the column during the
chromatographic run, changing the mobile phase
polarity which can have a drastic effect on
analyte retention times
The k value (relative retention time) for phenyl
propanol when there is no water in the mobile
phase is about 18 and at 100 water saturation of
the mobile phase (0.15 water), the k value is
about 4.
Improving reproducibility of retention times add
from 0.1 to 0.5 methanol or propanol to the
mobile phase equilibrate the mobile phase with an
intermediate concentration of water
Temperature Effects
Changes in operating temperature have little
effect on selectivity.
Some changes in selectivity with temperature can
be observed with the used of localizing solvents
such as acetonitrile.
Changes in overall retention time can vary with
temperature so controlling column temperature
may be needed to achieve reproducible retention
Summary of Advantages and Disadvantages of
Normal-Phase Chromatography
ADVANTAGES -the sample can be dissolved in a
non-polar solvent -it can be used for analytes
that may decompose in water -it is good for
separating isomers and very hydrophopic
or hydrophillic analytes -it can use higher flow
rates due the use of low viscosity solvents.
DISADVANTAGES -higher costs for purchase and
disposal of solvents, -difficulty in
controlling solvent strength, -lower boiling
point solvents are subject to evaporation
and bubble formation, -retention may be
variable - gradient elution can be difficult.