    
     
HPLC
Separation Mechanisms
Retention
Mechanisms
In general,
three primary characteristics of
chemicals can be used to create HPLC
separations. They are:
-
Polarity
-
Electrical Charge
-
Molecular Size
First,
we’ll discuss an overview of Polarity
and the two Retention Mechanisms which
employ this characteristic; Normal Phase
and Reversed-Phase chromatography.
Polarity
All
chemicals have a unique behavioral
characteristic related to their
molecular structure. They can be
described as being “ Polar ” or “
Non-Polar ”, with a range of polarities
between the most polar and most
non-polar.
Depending
on the structure of the molecule and
its’ electron charge distribution,
molecules will be “Very Polar” and some
will be “Very Non-Polar”. Others will
have different degrees of polarity
between these two, so there is a range
of polarity. For any individual compound
molecule, it will have a “certain
polarity” within that range. Water is a
good example of a very Polar liquid, and
paraffin based oil is a good example of
a very Non-Polar liquid.
We use this
“polarity” characteristic of chemicals
to build chromatographic “ Retention
Mechanisms ” that are used to create
many HPLC separations. Because of
polarity, there can be either
“Attraction”, “No Attraction”, or
“Repulsion” between two chemical
species.
A
simple rule describes this behavior
for polarity-based retention
mechanisms: “Like Attracts
Like, and Opposites are Not
Attracted When We Use Polarity”
Polars will
attract Polars (like), and repel Non-Polars
(opposites). Non-Polars will attract
Non-Polars (like), and repel Polars
(opposites). Oil, which is non-polar,
does not mix with water, which is polar
(opposites repel!) (This is just the
reverse of magnetism, where opposite
poles of the magnet attract, and like
poles repel).
To make a
chromatographic separation system, we
create a “Competition” for the different
sample compounds by making the mobile
phase and the stationary phase have
different polarities. Since the
compounds in the sample will have
different polarities, compounds which
have the same polarity as the stationary
phase (column packing material) will
slow down (because they are attracted to
the particles). Any compounds that have
the same polarity as the moving mobile
phase (attracted), will move along at a
faster speed. We have created a
separation by changing the relative
attractions (and, therefore the speeds)
of each compound.
The
chromatographer carefully chooses the
polarity of the mobile phase and
stationary phase to develop the
competition needed for the HPLC
separation of the sample compounds.
As shown
above, water is one of the most polar
mobile phase solvents while hexane is
one of the most non-polar solvents.
Miscible solvents can be blended in
different proportions to create the
exact separation performance required.
Here, the
stationary phase/packing material
polarity range is shown. Particles of
pure silica which are not bonded, are
the most polar. Column manufacturers can
change the polarity by chemically
bonding a modifier to the silica
particle surface. For example, by
bonding C18 (or “OctaDecylSilane,
ODS”) to the surface, the particle
can be made to be very non-polar (very “Hydrophobic”).
Today in HPLC, a bonded C18 –
silica particle is the most popular
type. Looking at the sample polarity
below, the different compounds in the
sample will typically have different
polarities with which the
chromatographer will be able to separate
with the competition developed between
the mobile phase and stationary phase
conditions. Now, we can discuss the
retention mechanisms based on Polarity.
Remember, “Like Attracts Like”
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Thinking
back to the Tswett experiment with plant
extracts, the conditions he used were
basically to have a very POLAR
Stationary Phase (in the column) and a
more non-POLAR Mobile Phase to create
the competition for separation. When we
use these same conditions today, this
mode of chromatography is called, “NORMAL
PHASE”.
The Polar
sample compounds will be attracted to
the Polar Stationary Phase and slow
down. The Non-Polar compounds in the
sample will be attracted to the
Non-Polar Mobile Phase and move faster
to create the separation. The mobile
phase will be 100% organic solvent. No
water is present. Remember that in
Normal Phase chromatography, the
Stationary Phase is Polar, therefore, a
Polar compound(s) (Yellow) will be
retained, and the Non-Polar(s) (Blue),
will elute early. |
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If we
“reverse” the conditions used in Normal
Phase chromatography, now the Stationary
Phase is Non-Polar (hydrophobic), and
the Mobile Phase is Polar. This is
called
REVERSED-PHASE chromatography.
Here, the
sample compounds, which are Non-Polar,
will be attracted to the Non-Polar
Stationary Phase and slow down, while
the Polar compounds in the sample will
be attracted to the Polar Mobile Phase
and move faster. Remember, that in
Reversed-Phase chromatography, the
Stationary Phase is Non-Polar, therefore
the Non Polar compound(s) (Blue) will be
retained, and the Polar(s) (Yellow) will
elute early.
Today,
approximately 75% of all HPLC methods
utilize Reversed-Phase
chromatographic conditions because
they tend to provide reproducible
results. Most methods will utilize a
blend of water (aqueous) with a miscible
organic solvent to insure the proper
interaction of the sample compounds with
the non-polar, hydrophobic particle
surface. As mentioned earlier, the most
popular type of reversed-phase column is
a “C18” stationary phase,
also referred to as an “ODS” column.
an
interactive chart which compares
different types of reversed-phase
packing materials.
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Summary Table
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Retention Mechanism
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Particle (Stationary
Phase) |
Mobile Phase
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Normal
Phase |
POLAR |
Non-POLAR |
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Reversed-Phase |
Non-POLAR |
POLAR |
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HPLC done
in the HILIC mode features the use a
very polar stationary phase column to
retain polar compounds. This is similar
to Normal Phase chromatography with one
major difference in the mobile phase
conditions. In Normal Phase, the mobile
phase is 100% organic solvent. No water
is present. In HILIC, water is always
present in the mobile phase. We are
retaining polar compounds because of
their attraction to the polar stationary
phase particle surface. Water, which is
a very polar solvent, is used as the
elution solvent for the polar compounds.
A HILIC separation can be achieved using
either an isocratic or gradient mode.
When using a gradient mode, the initial
mobile phase will have a low
concentration of water, approximately 5%
water (95% acetonitrile), to promote
retention of the polar compounds. The
concentration of water (elution solvent)
is then increased during the gradient,
to elute the polar compounds from the
column. |
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This mode
of chromatography is typically used for
proteins and other large biomolecules. A
moderately
hydrophobic, reversed-phase packing
material (stationary phase) is used but
with very different mobile phase
conditions, because these samples can be
adversely affected when exposed to
organic solvents that are typically used
in reversed-phase methods. Here, the
sample is loaded onto the column in
very-high-salt concentration, aqueous
mobile phase. This causes the compounds
to “salt out” and be retained on the
packing material. For elution, a
gradient is run from the initial high
salt, to a low salt concentration. This
allows the compounds to go back into the
mobile phase and be eluted. No organics
are used so that the proteins do not
denature. |
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In this
mode of HPLC, the stationary phase
particle has a charge (positive or
negative). Ionized compounds with the
opposite charge will be attracted to the
particle surface (ion–exchange) and be
retained (slowed down). The compounds
can be eluted by one or two approaches.
First, adjust the pH of the mobile phase
to create the un-ionized form of the
compound (no charge), which shuts off
the ion-exchange mechanism and allows
the compound to elute from the column.
The second approach is to add counter
ions to the mobile phase which are more
highly attracted to the charged
particles in the stationary phase, thus
replacing the compound from the particle
surface and allowing it to elute from
the column. |
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Size Exclusion Chromatography is an
HPLC technique where the different sizes
of the compounds are used to create the
separation. There is no chemical
attraction involved. The stationary
phase particles have openings, called
pores, which are of a controlled
size. Simplistically, if the size of
compound A in solution, (which is
related to its’
molecular weight (MW))
is too large to fit into the particle’s
pore, it can just pass by the particle,
being carried down the length of the
column by the mobile phase. However, if
compound B is small enough (lower
molecular weight) to fit inside the
pore, then it will spend some time in a
pore before it eventually comes back
out. As this process continues down the
full length of the column, compound A
will be able to elute from the column
first, while compound B will be delayed
in time, (more retained), thus creating
a separation based on compound size.
A
simple phrase to summarize Size
Exclusion Chromatography is:
“Big Ones Come Out First”
This
technique is very useful in
characterizing the molecular weight
distribution of polymers, or the
relative amounts of larger molecular
weight components to the smaller
components in the polymer sample. From
the elution pattern on the chromatogram,
you can determine the molecular weight
distribution for the polymer. This
distribution determines the physical
properties performance of the polymer,
which is important for quality control.
Molecules,
such as proteins, DNA and peptides, can
also be separated by size. This is often
called
Gel Filtration Chromatography. |
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