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Originally, this was the gravity‐driven, large column chromatography as
you have performed in your organic labs. These columns are long (5 –
50 dm), wide bore (1 – 5 cm) and are packed with large (150 – 200 μm)
silica particles. This large particle size is necessary to leave enough
room between the particles so that the force of gravity can pull the
solvent quickly enough through the column. Still, elution is very slow,
but the column capacity is very large, and this kind of chromatography
is very useful for synthetic work.
Analytical liquid chromatography is based on high performance liquid
chromatography (HPLC). This technique replaces the gravity driven
elution with a system of solvents and pumps. The much higher driving
force (100’s of atm) requires shorter columns (5 – 25 cm) to prevent a
large pressure drop. The columns are narrower (3– 5 mm), and the
packing materials are also much smaller (3 – 5 μm). Current HPLC
columns have an i.d. of 1 – 4.6 mm, while in the 1980s this was 3 – 7.5
mm.
HPLC is much faster, and has much higher resolution compared to
gravity LC, and has entirely replaced it in analytical applications.
However, due to the much volume of smaller columns, HPLC capacity is
lower, and is not so useful for synthetic applications.
The ultra performance liquid chromatography (UPLC) that we have at
UOIT is a refinement of the HPLC idea. The Waters company has been
able to reliably manufacture uniform packing particles of 1.7 – 1.8 μm
size. Such particles pack the column much more tightly, requiring
higher pressures (~1000 atm) and shorter columns (3 – 15 cm). Clearly
the column capacity will be smaller, but analysis times are three to 13
times faster compared to HPLC. Here is the pitch for Waters’ website:
“Because of its revolutionary technology, the ACQUITY UPLC
System can squeeze more out of a minute than any other system. You've
got five-hundred twenty-four-thousand three-hundred-sixty
opportunities a year for peak performance.
Cheesy, but you get the idea.
The mobile and stationary phases
In the original gravity‐driven LC an organic (non‐polar) mobile phase
was used, and the solid column packing was silica (polar). This
arrangement of stationary and mobile phase polarities is called normal
phase LC.
Majority of LC separations today are performed on reverse phase
systems (non‐polar stationary phase, polar mobile phase). Reverse
phase separations have several advantages. The principal advantage is
the ability to use water or aqueous solutions as the mobile phase.
Water is cheap, environmentally benign and is the solvent of choice for
many biological molecules (amino acids etc.) The cost aspect is further
accentuated by the fact the solvents for LC must be ultra pure (to avoid
non‐reversible interactions with the column which could ruin it).
Purifying water is very simple compared to the purification of organic
solvents.
Types of LC
Compared to GC, LC provides a rich collection of stationary phase
compositions. We will look at partition, adsorption, ion, size exclusion,
affinity and chiral separations.
Partition LC is the most common type of LC.
In partition LC, the differential solubility of a chemical between the
mobile and stationary phases is the key to the separation. The
stationary phase was historically a liquid adsorbed on the packing. In
modern columns the liquid is bonded to the silica packing. The
permanent bonding of the liquid makes for a more durable stationary
phase. Furthermore this configuration is more flexible, in that the
stationary phase can be used with a wider range of solvents without
worrying about solvation/mixing. (Note that the ‘liquidness’ of a
chemical immobilized on a surface is an interesting question to think
about).
The bonding of the liquid to the silica is
accomplished via surface silanol groups. (Note that
this chemistry is the same for the FSWC columns
used in GC).
The silanol groups are reactive towards chlorinated species like
Cl‐Si(CH3)2‐R :
This chemistry allows placing various R‐ groups on the silica surface.
These groups are chemicals that would be liquids under normal
circumstances. e.g. if R‐ is CH3(CH2)4‐, then the stationary phase will be
pentane‐like.
Note all surface silanol groups are functionalized, due to the steric bulk
of the ‐Si(CH3)2‐ groups. This creates a problem since some reactive
silanol groups remain exposed to the mobile phase, which can lead to
adsorption and hence tailing. So, after functionalization, a further
treatment with Cl‐Si(CH3)3 is performed to cap remaining silanols. This
chemical is less bulky, and thus more likely to have access to remaining
silanols.
More polar R‐ groups for normal phase stationary phase include (in
increasing polarity):
‐(CH2)2CN – cyano functional group
‐(CH2)3OCH2CH(OH)CH2CH2OH – diol functional group
‐(CH2)3NH2 – amino functional group
‐(CH2)3N(CH3)2 – dimethylamino functional group
Remember, the separation will depend on the partitioning reaction,
Amp Ý Asp
The equilibrium constant K will depend on the interactions between the
analyte and the two phases.
A subset of partition chromatography is chiral chromatography. Here
the stationary phase interacts differently with the two enantiomeric
components of a racemic mixture. This allows separation of such a
mixture – which of immense use in the pharmaceuticals field.
Adsorption chromatography is no longer used very much, since liquid
bonded columns are readily available. The polar stationary phase is
either silica (SiO2) or alumina (AlO2).
Ion exchange chromatography
The stationary phase packing begins with
polystyrene:
The styrene functional group functionalized
with a strong or weak acidic or basic group. e.g. the strong acid –SO3H.
The acid can give up a H+, and leave behind a negative surface site,
which can then interact with a cation in the mobile phase. For a singly
charged cation,
–SO3H(sp) + M+(mp) Ý –SO3M(sp) + H+(mp).
sp and mp refer to the stationary and mobile phases, respectively. This
chemistry will be governed by
[H ]mp [ SO 3M]sp
K
[M ]mp [ SO 3H]sp
ex
The mobile phase is buffered, so [H+]mp remains constant. Furthermore,
the number of functionalized sites on the stationary phase surface is
typically far grater the amount of ion present in the mobile phase that
can be changed – so [–SO3H]sp also remains constant. (This is similar to
pseudo first rate reactions). Therefore the equilibrium constant for the
exchange reduces to
[ SO 3M]sp
K ex
[M ]mp
Which, mathematically is identical to partition chromatography.
Mechanically speaking, polystyrene
is not a very good materially – it’s
basically like a wet noodle. During
polymerization, a cross linker is
added, which leads to spherical
beads.
Unfortunately these spheres are
porous – which means that the mobile phase can diffuse into these
pores and be removed from the general flow. (Remember, we saw this
when we were looking at the theoretical treatment of column
efficiency). Note that silica spheres do not have holes, and this does not
occur.
To overcome this problem, newer packing materials begin as polymer
beads or silica particles, and then have a coating of the exchange resin
(polymer) applied to their surface.
Polymer cores are more pH resistant (i.e. can be used over a broader
pH range). Silica cores, on the other hand are more efficient.
Acids, like the strong acid –SO3H or the weak acid –COOH are used to
make cation exchange columns. Strong bases like –N(CH3)3OH, or weak
bases like –NH3OH are used to make anion exchange columns.
Size exclusion chromatography relies on the problem discussed above.
The stationary phase is purposely made with a porous polymer or silica
beads (10 μm diameter). The pores are carefully designed to cover a
range of sizes.
Large molecules cannot enter the pores, and elute with the mobile
phase. Very small molecules can readily enter all the pores, and are
delayed. Intermediate sized molecules are delayed less than small
molecules, but more than large ones – hence different size molecules
can be separated.
Note that the basis of separation – unlike the other chromatographies
we have studied – is not chemical, but is physical.
This type of separations are very useful for biological molecules,
homologs and oligomers.
Affinity chromatography is also somewhat different from other types,
in that the interaction with the stationary phase highly specific for one
or few chemicals. This is useful in purifying a chemical that is found in a
complex mixture.
The packing surface is coated with a chemical the reversibly binds a
specific chemical – enzymes, antibodies etc. As the mixture moves
through the column all other chemicals, these chemicals are retained
on the stationary phase. Note that they are not delayed, but are
permanently retained – until the mobile phase is changed in such a way
as to un‐bind the chemical (usually accomplished with a change in pH).
Now the desired chemical elutes, and can be recovered in a purified
form.
Ideal detectors of LC share many of the properties of an ideal GC
detector. They need not be useable at high temperatures, but
otherwise, the same ideal characteristics hold.
In general, GC detectors tend to be developed specifically for GC (e.g.
one does not see flame ionization detectors outside of a GC). LC
detectors, on the other hand tend to be general detectors that have
been modified for use in LC. The modifications are to allow the detector
work on small volumes, on flowing samples.
Another concern is extra‐column band broadening. This can occur if the
diameter of the pipes used outside of the column are wider than the
column itself. To prevent this, pipes used for the detector must be of
the same diameter as the column.
Detectors are distinguished by whether they work on bulk or solute
properties. An example of the former is thermal conductivity, which
changes in the presence of an analyte. An example of the latter is UV‐
vis absorption.
UV‐Vis, IR and fluorescence detectors for LC are
similar to full sized instruments, but small. Here
is an example of a UV detector:
A good question would be to figure out why this
arrangement would be useful for an LC detector.
A bulk property detector for LC is based on the refractive index. These
detectors are not affected by the flow rate, but are highly sensitive to
temperature fluctuations. They must be used with a thermostat.
EVAPORATIVE LIGHT SCATTERING detectors are based on the familiar
atomizer. As the liquid exits the column, it is nebulized. Small droplet
sizes makes it easy to evaporate the solvent. As the solvent evaporates,
analyte molecules form small particles which can scatter laser light,
giving rise to a signal.
A specific detector for ion chromatography is a conductivity meter. It is
a very simple detector, with a cell containing two electrodes. Such
detectors can suffer from high backgrounds, as the mobile phase itself
if often charged. An ion suppressor column before the detector can
reduce the background signal and improve sensitivity.