LECTURER: In this session we'll consider the additional steps in isocratic
reverse phase HPLC method development, which must be considered
when separating ionizable compounds,
including acids and bases.
We'll then outline the strategy for developing a method based on
gradient elution.
Neutral solids will be retained to some extent
on nonpolar reverse phase columns,
ionised solutes will strongly favour the polar mobile phase
and will not be significantly retained.
When it comes to acidic or basic solutes,
the charge there depends on the pH of the mobile phase.
The observe attention factor would depend on the average
extent of ionisation of the solute species.
For an acid the fraction in the neutral form, for example, HA
is given by the acid association constant
which to avoid confusion, will be given the symbol alpha.
Acid-base equilibrium constants are normally given the symbol K,
if the extent of ionisation is very low, a weak acid than alpha
would be close to unity and the observe retention
will almost be as high as a retention
factor for the purely neutral form HA.
We can control the acid base equilibrium by changing the pH of
the solution, this in turn will change the observe retention factor,
more ionisation means less retention on the reverse phase column.
Retention changes dramatically at a pH within one unit
of the solute pKa, this example shows at low pH, benzoic acid,
a weak acid, is nearly fully protonated and its retention
is unaffected by pH in the range of pH 1 to 4.
The pKa benzoic acid is 4.2, this parameter you will recall
equals a negative of the logarithm or the acid equilibrium constant
which here we are calling alpha HA,
the lower the pKa, the stronger the acid.
As the pH of the mobile phase approaches the pKa,
there is a decrease in the fraction of the acid
that's in the neutral form HA
and there's a decrease in user retention,
a pH well above pKa, ionised benzoic dominates is not retained.
A similar rationale can be used to explain
the behaviour for weak base, with the base equilibrium constant alpha B,
a weak base such as 4-Methylaniline pKa 5,
is weakly retained at low pH but is more retained
as the pH approaches the pKa.
As the base equilibrium shifts towards the neutral form
but in base separations are greater than pH of 8,
to maintain the protonated form, requires special stationary phases
to avoid dissolving the silica support material.
pKa values, are measured in purely aqueous solutions,
organic solvents modify pKa value slightly,
raising the values for weak acids, lowering the value for weak bases,
however, aqueous phase pKa values are a useful
guide method development when choosing the pH of the
mobile phase for separation weak acids and bases.
Controlling the pH of the mobile phase is achieved by
appropriate selection of a buffer, a solution of a weak acid or a base
and a corresponding salt, buffers maintain the pH at a desired value
producing a source and a sync of protons.
This table illustrates the buffering pH range of common HPLC buffers,
in addition to pH rang, buffers are selected according to the effect
they have on the rest of the system.
For example, their compatibility with the chosen method of detection
and their solubility in the solvent mixture
which comprises the mobile phase.
Buffer solubility increases in the order THF, acetonitrile and methanol,
the presence of the buffer has little effect
on the retention of the neutral form of the acid or basic analyte.
In optimising the separation of ionizable components
by isocratic reverse phase HPLC,
method development generally starts with a pH of 2.5 to 3.
For UV detection phosphate buffers are common,
due to their low UV cut off approximately 200 nm,
so lettuce spray ionisation MS detectors
are more volatile buffers required such as ammonium formate.
Changing the buffer pH is a powerful means
of controlling the relative attention but reproducibility
can deteriorate if operating within 1 pH unit of a component pKa.
Let's return to the example of our A component mixture of organic solute
separated on a C18 column, the optimum isocratic condition
were determined to be 35,40% of acetonitrile.
However, not all components were eluted with K in the range
of 0.5 to 20, and when the mobile phase strength was low enough
to separate the list of our components one to three,
the most well retained component eight, was very slow to elute
extending the runtime to over two hours.
Gradient elution was suggested in this case,
we saw previously how gradient elution improves the separation,
we saw in all components with the reduced runtime,
the approach taken was a segmented gradient,
using three different solvent compositions
of increasing element strength, between each composition,
the percentage of B was increased linearly over program timescale.
So how do we decide when to use isocratic or gradient elution?
And how do we develop a method for the latter?
The recommended approach is to run a wide linear gradient
e.g 10 to 90% B also called a scouting gradient
and to determine the difference delta t,
in the retention time between the first
and last peak in the chromatogram.
By dividing delta t by the gradient time tG over which
the solving composition is changed and numerical values obtained,
which can guide method selection.
Use a gradient method if the value is greater than 0.4,
use an isocratic method if the value is less than 0.25,
if the value is in between 0.25 and 0.4
either method can be used.
The complexity of the sample would dictate
which approach is more successful.
Developing a gradient elution method involves the following steps,
having first run a wide gradient 10 to 90% B,
and establish the gradient elution's appropriate
one could decide from the following chromatogram
which portions of this wide gradient are not necessary.
From the retention time for the first and last peak,
determine the corresponding solvent compositions
and run second shallower gradient covering a narrow range
of percentage B, over the same gradient time.
This will improve the peak separation and reduce the retention
time of each component, if the separation is acceptable,
running a steeper gradient over shorter time,
can be trailed to further reduce the runtime.
Let's look at an example at this methodology.
When they attain to 90% gradient, it forms the elution method
for this A component sample mixture,
starting with a delta t of 21.5 minutes divided by 40 minute runtime,
gives the value of 0.54.
Therefore, gradient elution is recommended,
reducing the range of solvent compositions
starting with a composition at the time peak one eluted in 1A,
30% of B and finishing with a composition the time eight eluted,
at 2% B, improved the separation and reduce the runtime.
To see if runtime could be further reduced,
while still resolving all components, a steeper gradient was trialled
in the final one, this time using a 20 minute gradient.
Peak six and seven were no longer fully resolved,
so these methods the conditions of 1B,
best met the goals of the analysis.
The order of elution can change during the solvent gradient
as indicated by non parallel lines in a plot of
low k versus composition.
If these lines cross the order evolution will change,
after gradient elution 10 to 20 volumes of mobile phase
with the starting composition should be run through the column
to equilibrate the stationary phase
and prepare for the start of the next gradient.
One further point to note, we're looking to transfer gradient elution
methods between different instruments or harbour configurations,
is that columns contained dwell volumes between the solvent
mixing point and the start of the column.
Dissociated dwell time is required for the gradient to reach the column,
dwell time is the dwell volume divided by the flow rate,
different dwell times in different instruments
mean the gradient separation methods are not directly transferable.
High pressure pumps generally have a smaller
dwell volume than low pressure pumps.
You should measure the dwell time and report it
with the gradient method to facilitate transfer
to other instruments, it can be measured
by injecting two solvents directly into the detector
bypassing the column.
One solvent B, is chosen to give a stronger detector response,
e.g UV absorption.
And one solvent A is chosen to give no response.
With a gradient of not to 100% B, the dwell time
is indicated by the rise in the detective response
as a mixture exits the injunction system
and directly enters the detector.