The Theory of HPLC
Normal Phase (Absorption) Chromatography
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Aims and Objectives
Aims and Objectives
Aims
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To give an overview of the mechanism of Normal Phase Chromatography
(NPHPLC) and explain the basis of the retention mechanism
To highlight typical NPHPLC Applications
To explain retention order in NPHPLC and demonstrate the influence of mobile
phase composition on retention
To explain how the mobile phase composition and constituents might be
manipulated to optimise chromatographic separations in NPHPLC
To illustrate the principles which are used to select appropriate stationary phases
and column geometry in NPHPLC
Objectives
At the end of this Section you should be able to:
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To outline the advantages and limitations of NPLHPLC compared to RPHPLC
To outline the issues with water in NPHPLC mobile phases and give strategies to
practically overcome problems
To explain the best way to get started with NPHPLC and to optimise the chances
of a successful separation
Content
Mechanism of Normal Phase Chromatography
Applications of Normal Phase Chromatography
Retention and Selectivity in Normal Phase Chromatography
Separation of Isomers using Normal Phase Chromatography
Mechanism of Isomer Recognition in Normal phase HPLC
Stationary Phases for Normal Phase HPLC
Typical Mobile Phases HPLC
Controlling Retention
Mobile Phase Optimisation
Problems with Water in the Mobile Phase
Getting Started with Normal Phase HPLC
Glossary
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Mechanism of Normal Phase Chromatography
Normal phase chromatography was the first Liquid Chromatographic technique,
chronologically. As we have seen, Tswett used this mode to separate plant pigments
using a calcium carbonate stationary phase with a petroleum ether mobile phase.
By definition, normal-phase HPLC utilises a stationary phase that is more polar than the
mobile phase.
Typical stationary phases include bare silica as well as cyano, diol, and amino bonded
phases. Typical mobile phase constituents include organic solvents such as hexane and
ethyl acetate.
The retention mechanism in normal phase HPLC is based on polar adsorption of either
the solvent molecules or the analyte onto the polar stationary phase surface. If the solvent
molecules are ‘localising’ they will be adsorbed onto the stationary phase surface.
If the analyte molecule contains highly polar functional groups, it may also be capable of
‘localising’ onto the stationary phase surface – essentially displacing the solvent molecule
and gaining retention. Mass action will then ‘displace’ the analyte from the stationary
phase surface back into the mobile phase, where it will be transported down, and
eventually elute from, the column.
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Initially the acetonitrile solvent molecules
(a ‘localising’ solvent), are adsorbed to
polar retention sites on the silica surface
(silanol groups). The analyte molecule
will compete for retention sites with the
solvent molecule.
The nature and
concentration of localising solvent in the
mobile phase will have a large effect on
normal phase retention characteristics.
In normal phase chromatography – less polar (hydrophobic) compounds elute first, whilst
more polar (hydrophilic) compounds elute later.
As can be seen in the example – the hydrocarbon portion of the analyte is only weakly
attracted to the stationary phase, whereas the polar hydroxyl functional group is strongly
attracted. The polar phenol molecule localises onto the stationary phase and displaces the
acetonitrile molecules.
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In normal phase chromatography – less polar (hydrophobic) compounds elute first, whilst
more polar (hydrophilic) compounds elute later.
Vitamin molecules generally show poor water solubility and may be analysed using
normal phase chromatography.
The order of elution is least polar first, followed by increasingly polar (less hydrophobic)
analytes.
This separation uses a bonded phase column. Hexane is a non-localising (non-polar,
weak) solvent and Ethanol is the localising (strong) solvent used to displace the analyte
from the silica surface. As the vitamin molecules are relatively non-polar, only a very
small amount of strong solvent is required for elution.
Applications of Normal Phase Chromatography
In normal phase chromatography, polar (hydrophilic) analytes are retained longer than
less polar (hydrophobic) analytes.
Normal phase chromatography has been used for the separation of both neutral and
ionisable compounds, although neutral sample separations predominate the literature.
Reverse phase chromatography is usually attempted first, if the required retention or
selectivity is not obtained using the strategies outlined, then normal phase
chromatography is used as a second choice.
Some samples are only sparingly soluble, or insoluble in aqueous media. This renders
them unsuitable for reverse phase HPLC. Whilst it is possible to introduce samples into
reverse phase HPLC systems using 100% organic solvent diluents, peak shapes are often
very poor. Normal phase is often a good alternative as samples are much more soluble in
the organic solvent systems used.
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Table 1. Advantages and Disadvantages of Normal Phase Chromatography
Normal Phase Advantages
Normal Phase Disadvantages
 Separation selectivity can be greatly
 Most amenable to low and mid polarity
influenced by altering the mobile phase
samples – ionic samples are best
constituents and ratio of solvents
analysed by reverse phase (although
addition of triethylamine to the mobile
 Organic compounds are highly soluble
phase assists with analysis of bases in
in the solvent systems used – a big
normal phase)
advantage
for
preparative
chromatography
 Controlling solvent strength can be
unpredictable
 Solvent viscosity is lower – therefore
 Solvents are more prone to air bubble
higher flow rates can be used to achieve
formation – giving rise to instrument
improved sample throughput
problems and noisy baselines
 Care must be taken to exclude mobile
phase water with non-bonded stationary
phases
 Gradient elution is often not feasible
due to solvent de-mixing
 Solvents used have a much higher
cost of disposal and environmental
impact
Due to the localising behaviour on the stationary phase, normal phase systems are
excellent at discriminating between compounds whose spatial geometry differs. Hence,
normal phase systems are popular for the separation of chiral enantiomers as well as
positional isomers.
If large amounts of analyte need to be recovered from solution using preparative
chromatography normal phase systems are usually employed due to the ease of solvent
removal. Normal phase solvent fractions are more easily evaporated to dryness than the
highly aqueous systems encountered with reverse phase chromatography.
In the next example a 21.2 mm i.d. preparative HPLC column is used to separate
phospholipid analytes using normal phase chromatography with a fairly complex mobile
phase system.
Normal phase preparative separation of Soy Phospholipids
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Normal phase has several advantages for this separation:
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High volatility solvents for easy fraction (analyte) recovery
Low viscosity phases for high sample throughput at increased flow rate
Enhanced selectivity via the adsorption mechanism and better selectivity control using
normal phase localising solvents
Lack of analyte chromophore means alternative detection mechanism – the volatile
solvent systems are highly compatible with evaporative light scattering detectors
Retention and Selectivity Stationary Phases
The retention order in normal-phase HPLC is generally the opposite of reversed phase
HPLC. The stationary phase is very selective for the number, type, and orientation of
polar functional groups. A general elution order is shown opposite. Adding more polar
functional groups to a molecule increases the retention. As a general rule it is the most
polar functional group that determines retention.
General Retention Trends in Normal Phase HPLC
The retention factor data shows tremendous selectivity for polar functionalities in normalphase mode. The only difference in the molecules on the left is the polar functionality, yet
their k values range from 0.6 to 5.5.
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Separation of Isomers using Normal Phase Chromatography
The example on the right illustrates the selectivity for structural isomers on bare silica.
The compounds differ only in the location of the polar constituents, one in the meta
position and one in the para position. The selectivity for structural isomers is primarily
restricted to the bare silica columns.
Separation of Positional Isomers of Baythroid using Normal Phase CHromatography
Mechanism of Isomer Recognition in Normal phase HPLC
The selectivity movie indicates the primary reason for the ability to discriminate
between such closely related compounds – localisation onto the silica surface.
Depending upon the geometry of the analyte molecule, and the relative strengths
of the dipoles or hydrogen bonding capability, the analytes will bind more or less
well to the stationary phase surface – resulting in excellent selectivity.
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Mechanism of Positional Isomer Separation in Normal Phase HPLC
Stationary Phases for Normal Phase HPLC
Bonded stationary phases offer several advantages over bare silica in normal
phase HPLC applications. They equilibrate more rapidly than silica columns;
therefore gradient elution is possible. Strong (localising) solvents tend to bind very
strongly to bare silica and equilibration can take 20 column volumes or more.
In gradient elution the strong solvent being introduced tends to be irreversibly
adsorbed to the silica surface. Once the surface is saturated, a sudden increase in
the modifier (strong solvent) eluting from the column is seen, and some
compounds may elute with low retention and inadequate separation. This problem
is much less apparent when using bonded phase columns and as such gradient
elution is possible.
Bonded phase columns for Normal phase chromatography are available in a wide
variety of polarities for better selectivity. These columns also have a higher sample
capacity than bare silica columns.
The mobile phase water content does not have to be strictly controlled as it does
with silica columns (more on this later).
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Advantages of Bonded Phases:
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Mobile phase trace water does not have to be controlled
Gradient elution possible
Column equilibrates rapidly
Wide variety of polarities, selectivity
High capacity
Peaks don’t tail as with bare silica
Silica first choice for preparative separations as bonded phases have higher
cost, lower stability and lower loadability
Silica
Amino
Diol
Cyano
Typical Bonded Phase Stationary Phases used in Reverse Phase HPLC
As a result of all these advantages, it is recommended that method development
in normal-phase mode is initially carried out using bonded phase columns. In
particular, with cyano columns, which have intermediate polarity and good stability.
Diol columns are the most polar and silica like. They however, like amino columns
are not as stable.
Cyano: Most popular phase to begin normal phase method development. Dipolar
compounds such as chloro, nitro and nitrile substituents are more strongly retained on
cyano columns relative to amino or diol columns.
Diol: The most polar of the bonded phases. Basic compounds such as amines, ethers,
esters and ketones are preferentially retained on amino and diol columns relative to cyano
columns.
Silica: The use of silica phases is less convenient for analytical applications due to
problems with adsorption of trace water and solvent de-mixing affecting reproducibility and
the ability to use solvent gradients. Silica is the phase of choice for many isomer
separations and for large-scale preparative chromatography applications.
Amino: Basic compounds such as amines, ethers, esters and ketones are preferentially
retained on amino and diol columns relative to cyano columns. Amino columns should not
be used with adehydes and ketones as they can form Schiff bases. Amino columns have
been useful for the separation of vitamins A and D in normal phase mode.
If the selectivity obtained is not appropriate on the bonded-phase columns, then switch to
bare silica. Bare silica is recommended as the starting point for the separation of
structural isomers.
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Separation of herbicides using different normal phase stationary phases
Differences in stationary phase selectivity are demonstrated in this application of
preparative and analytical scale chromatography of herbicide analytes from a sample of
natural oats. Separation a) is carried out on a cyano column – due to the large number of
sample components the separation is impossible in a single analysis. The area of the
chromatogram indicated by the arrow is diverted via column switching onto a diol column
using the same mobile phase – this is separation b), which showed improved separation
characteristics and the herbicide is seen but is heavily interfered. The collected fraction
from this chromatogram was re-analysed using a silica column – this is separation c) and
again showed a markedly different selectivity to the two previous stationary phases.
Potential for poor peak shape in Normal Phase HPLC when using bare silica stationary
phases
Care should be taken when working with silica stationary phases. Surface acidity (related
to the silanol conformation, metal ion content etc.) can cause poor peak shape with some
polar compounds – Zorbax Rx-Sil has lower surface acidity and improved peak shape.
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Typical Mobile Phases HPLC
Common normal-phase solvents along with their elution strengths can be seen behind the
solvent strength button opposite. The data shown is for solvents on bare silica columns
and all strengths are relative to n-pentane.
Weak solvents such as fluoroalkanes and n-hexane have negative or low elution
strengths. The stronger solvents can be divided into three groups: non-localising, basic
localising, and non-basic localising, referring to the solvents ability to compete with
various analyte types. Remember that localising refers to the ability to interact with the
stationary phase surface through dipole or hydrogen bonding interactions.
Solvent Selectivity Triangle for Normal Phase Solvents
When developing a normal-phase method, select a weak solvent, such as hexane or
1,1,2-trifluoro-1,2,2-trichloroethane, and one of the stronger solvents such as methylene
chloride or ethyl acetate and vary the concentration from strongest to weakest mobile
phase composition.
This example illustrates the nature
of the normal-phase mechanism.
The three sample components are
xylene (0.24), toluene (0.28), and
benzene (0.20). The methylene
chloride is stronger than all three
sample components with elution
strength of 0.32. Therefore, the
sample components are not strong
enough to displace the methylene
chloride from the active sites. All
sample components elute as one
peak in the dead volume. When
the mobile phase is changed to
npentane, the samples are found to
be stronger than the mobile phase
and retention takes place.
Separation of Benzologs by Normal Phase Chromatography illustrating the elution
strengths of various normal phase solvents
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Take note that analyte retention will not vary in a linear fashion with changes in mobile
phase composition as with reversed-phase HPLC. If the solvent combination does not
provide the desired selectivity, switch to one of the other categories (e.g. basic to nonbasic) for strong solvent and test combinations.
It may be necessary to use all three strong solvent types (non-localizing, basic localizing,
and non-basic localizing) in combination with the weak solvent to achieve the desired
selectivity.
Hexane is good for low UV adsorption, but is not miscible with all strong solvents and care
should be taken in this respect. Add methylene chloride to the mobile phase to ensure
miscibility.
Controlling Retention
Use the slider to see how the chromatogram changes at different mobile phase strengths.
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1-nitronaftalene
dimetoxynaftalene
1,7-
 You should particularly notice
the shape of the plots for the
retention of peaks 1 and 2 and
notice how large retention
changes occur at lower %B
concentrations and changes at
higher concentrations have little
effect on retention -this is a
general observation about all
normal phase separations
 You should also notice how
the
separation
selectivity
changes as the modifier
concentration is increased – this
is also a facet of normal phase
chromatography.
It is more
useful to introduce a different
type of strong solvent than to
vary the modifier concentration
over a wide range
Optimising Mobile Phase Strength in Normal Phase
HPLC
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Mobile Phase Optimisation
As we have seen, different solvents may be employed to change the selectivity in normal
phase chromatography. There are many charts and graphs to help you in this regard,
however the solvent optimisation process is much more empirical than with reverse phase
HPLC, usually involving a good deal of trial and error.
Perhaps all the components in your sample elute within the correct k range when you use,
for example, 92% n-pentane with 8% methyl acetate. Some of your chromatographic
peaks, however, are not well separated. You can refer to a chart or graph found in a text
or paper and find solvent combinations of equivalent elution strength. The chart of
isoeluotropic mobile phase combinations indicates that in this case you may try 62% npentane with 38% methylchloride to achieve similar overall analysis time, but with altered
selectivity.
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Nomographic Relationships between mobile phase systems for Normal Phase HPLC
Where
Table 2. List of compounds
Compound
Name
MTBE
Methyl tertiarybutyl ether
EtOAc
Ethyl Acetate
MC
Methylene Chloride
PrOH
1-propanol
Nomomgraphs of the type shown opposite are also available in the literature and may be
used to ensure isoeluotropic behaviour, whilst changing solvents to adjust selectivity. You
can use the slider to investigate isoeluotropic compositions for various solvents
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Problems with Water in the Mobile Phase
Bare silica columns exhibit a number of problems not associated with bonded phase
columns including peak tailing, irreproducible retention times, and long equilibration times.
The problems are caused by silanol groups, which have varying ‘strengths’ on the
stationary phase surface and trace water in solvents.
Due to ambient humidity, trace water will be taken up by the mobile phase. This dissolved
water is then taken up by the column, which can lead to chromatographic variability.
The trace water level is not easily controlled leading to different water concentrations at
different times. Water can be picked up from glass surfaces and the air. The trace water
will adsorb to the strongest of the silanol groups, leading to reduced (and variable)
retention of analyte components.
Table 3. Effect on Retention Factor of some typical analytes for wet and dry solvents in
Normal Phase HPLC
Compound type
Dry solvent
50% H2O st. Solvent
Aromatics
0.05 – 0.25
-0.2 – 0.25
Halides
0.0 – 0.3
-0.2 – 0.1
Mercaptans
0.0
-0.2
Ethers
0.1
0.0
Nitros, esters, nitrites, carbonyls
0.2 – 0.3
0.1
Alcohols
0.3
0.2
Phenols
0.3
0.2
Amides
0.2 – 0.6
0.0 – 0.4
Acids
0.4
0.3
Amides
0.4 – 0.6
0.3 – 0.5
Researchers have also published data indicating the approximate elution
strength (εo) necessary to separate a given class of compounds. An
example appears above. The values can be used in conjunction with the
solvent strength values given earlier.
Snyder and Kirkland have proposed that the mobile phase is equilibrated with an
intermediate (‘50% saturation’) amount of water. A portion of mobile phase is saturated
with water, then this portion is blended with an equal volume of dry (over molecular sieve)
solvent which has not been treated with water. This can often result in much improved
retention time reproducibility and column equilibration times can be shortened from many
hundreds of column volumes to only a few.
In some cases, the effect of varying mobile phase water concentrations on sample
retention may be minimised by adding 0.1 to 0.5% methanol or propanol to the mobile
phase.
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Getting Started with Normal Phase HPLC
Some suggested starting conditions for normal phase HPLC are shown. The mobile
phase compositions are recommendations only and, as has been discussed, the
optimisation of the solvent system will be very application dependant.
Switching between basic and non-basic localising solvents is recommended to investigate
selectivity in the early stages of method optimisation.
The temperature does not have a marked effect on selectivity in normal phase
chromatography. However, it does alter retention characteristics, and as such, it is
important that temperature is controlled.
A list of critical issues in normal phase chromatography is shown. Note that the addition of
a sacrificial base such as Triethyamine (TEA) or acid such as acetic acid can markedly
improve peak shape in normal phase HPLC. This is analogous to the situation found in
reversed phase HPLC.
Table 4. Some suggested starting conditions and critical issues for Normal Phase HPLC
Bonded Silica
Silica
CN
SIL
Column
Packed in normal phase solvents
ZORBIX Rx-Sil-basic solutes
Mobile phase
Hexane with 1- or 2- propanal
Methylen chloride with 0.05% 0.5% methanol
Temperature
Ambient – 60oC
Ambient – 60oC
Table 5. Critical Issues in Normal Phase HPLC
Poor peak shape
 Injection solvent stronger that mobile
phase
 Basic samples give better peak shape
using high purity silica
 Basic samples may require 20mM TEA
in the mobile phase
 Basic samples may require 20mM acetic
acid in the mobile phase
 Strongly retained polar materials can
build on the column
Reproducibility
 Silica requires addition of water or
methanol to maintain reproducibility
 Use columns packed in normal-phase
solvents
It is important to match the solvent strength of the sample diluent with the mobile phase in
order to avoid poor peak shape – if necessary a weaker diluent strength is acceptable –
otherwise use a sample concentration as high as possible in strong solvent and inject
under 10μL.
It is often necessary to flush both bonded and non-bonded phase with 100% strong
solvent to remove adsorbed sample components – this will restore column performance.
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Glossary
Localising – refers to the ability of an analyte or solvent to interact, via polar functional
groups, and be adsorbed onto the stationary phase surface. A basic representation of this
process might be:
Mass action – a concentration effect in which a species in vast excess of another is able
to displace the species which is dilute.
Preparative chromatography - a mode of chromatography in which the large columns
(21 – 50 mm i.d. are common), are overloaded using large volumes or masses of analyte.
The mobile phase eluting for the column around the retention time of the peak of interest
is collect, with the intention of drying down the solvent to recover the purified analyte for
further characterisation or use a standard material or use in a further reaction etc.
Snyder and Kirkland - L.R. Snyder and J.J. Kirkland, Introduction to Modern Liquid
Chromatography, 2nd. Ed., Wiley-Interscience, New York, 1979, pp. 374-383
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