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 chromatography).
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 difficult.
Comparison of different types of silica columns.
(a) acidic type A silica;
(b) less acidic type B silica.
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 Solvents.
Solvent
Hexane
Chloroform
Methylene chloride
Ethyl ether
Methyl t-butyl ether
Ethyl acetate
Acetonitrile
Tetrahydrofuran
1- or 2-Propanol
Methanol
Strength(o)
0.00
0.26
0.30
0.38
0.48
0.48
0.52
0.53
0.60
0.70
Localization
no
no
no
yes
yes
yes
yes
yes
yes
yes
Basic?
UV-cutoff
Not relevant 201
Not relevant 247
Not relevant 234
yes
219
yes
225
no
256
no
192
yes
230
Proton donor 214
Proton donor 210
Separation in Normal and Reversed-Phase Chromatography
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 octanal.
O
BUTANAL
O
HEXANAL
O
OCTANAL
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 specific.
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 chromatography.
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 hour).
Solvent Demixing - Example: 100% Hexane --> 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 times.
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.