Mobile Phase Optimization Strategies for Reversed

The Essential CHROMacademy Guide
Mobile Phase Optimization Strategies for
Reversed Phase HPLC
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Speakers
Kevin Schug
Tony Taylor
HPLC Dept. Dean
CHROMacademy
Technical Director
CHROMacademy
Moderator
Dave Walsh
Editor In Chief
LCGC Magazine
Mobile Phase Optimization Strategies
for Reversed Phase HPLC
Aims & Objectives
1. Review of Reversed Phase
Retention Mechanisms
2. Methanol or Acetonitrile - which
one is best?
3. Eluotropic Strength - quick ways
to reach the ideal!
4. Changing Solvent - useful tools
and approaches
5. Mobile phase pH - understand
the effects
6. Optimising pH vs. retention
7. Which buffer - what strength?
8. Strategies for when all else fails
Reversed Phase Retention Mechanisms
1. Mobile Phase - mixture
of organic and
aqueous solvents
2. Stationary phase hydrophobic moiety
chemically bonded to
silica
3. Mobile phase MORE POLAR than Stationary Phase
4. Analyte ‘partitions’ between the two phases depending upon its
chemistry (hydrophobicity)
5. Increasing the % organic in the mobile phase increases the
‘elution power’ of the mobile phase
6. Retention and Selectivity are altered by changing the chemistry
of the stationary phase mobile phase and the temperature
Controlling Retention and Selectivity
Retention and Selectivity are altered by changing:
Stationary Phase - chain length and chemistry, inclusion of polar
moieties, exposure of silanol surface, polar end capping reagents
Mobile Phase - organic solvent type, % organic, pH, buffers and
other additives
Temperature - especially with ionisable analytes
Solvents for Reversed Phase HPLC (I)
1. Acetonitrile has lower viscosity - reduces back pressure and
often results in slightly better peak shape
2. Acetonitrile has lower UV cut-off - advantage for UV detection
3. Methanol is less expensive and less toxic
4. Methanol is more polar - reducing the risks of solid buffer
precipitation
Solvents for Reversed Phase HPLC (II)
1. Acetonitrile forms binary
mixtures with water
2. Methanol is ‘protic’ and can
undergo polar polar / ionic
interactions with solutes
3. Usually results in better
selectivity for more polar
compounds - at the expense of
longer run times and increased
peak asymmetry
4. Acetonitrile shows good wetting
properties when low % B
gradients are required - better
early peak retention time
reproducibility
MeOH
MeCN
Effect of Organic Modifier on Retention
Neutral HPLC Test Compound Mixtures - Selectivity also changes
Selecting Optimum Eluotropic Strength (1)
1. Carry out separation at
high %B (80%)
2. This saves time vs.
starting at low pH
3. Reduce by 5-10% B in
steps to assess
retention
4. Look out for changes in
selectivity
5. Really only works for
neutral compounds
6. Ionisable species need
to employ pH control
MobilePhase_01.flv
Scouting Gradient Methods
If Dtg < 0.25 tG then isocratic
analysis is possible !
Isocratic composition:
tr(avg) = ti + tf /2
tr(avg) = 12.8 + 21.2 / 2
tr(avg) = 17.0 mins.
At 17.0 mins. eluent
composition = 80% B
(Note: account for dwell
volume!)
That’s the ‘BEST ESTIMATE’
isocratic composition for this
separation
We will discuss the pH of the mobile phase and choice of buffer shortly
Simulation to Optimise %B
1. Use modelling for predicting separation
2. This DryLab® model was achieved using 2 injections at 30% B
and 50% B
Mixture of 6 Neutral Compounds
MobilePhase_02.flv
Altering Selectivity
1. What if separation
isn’t achieved with
the ‘OPTIMUM’
isocratic
composition from
the scouting
gradient?
2. Adjust %B either
side of optimum by
5-10% B
3. Use an alternative
solvent to adjust
selectivity
Iso-Eluotropic Mobile Phases
1. Iso-eluotropic - same elution power
2. Iso-eluotropic solvents elute
analytes in the same time frame
with different selectivity
3. Can use an iso-eluogram
(nomogram) to find
iso-eluotropic solvent compositions
MobilePhase_03.flv
Mobile phase pH effects
1. pH reflects the hydrogen
ion (or hydroxonium ion)
concentration in solution
2. Adding acid (proton
donor), increases the
hydrogen ion
concentration (lowers pH)
of the solution
3. Adding base (proton
acceptor), lowers the
hydrogen ion
concentration (increases
pH) of the solution
MobilePhase_04.flv
Extent of Analyte Ionisation
1. Extent of analyte ionisation
changes with mobile phase pH
2. Le Chateliers principle applies
to the equilibrium when
adding acidic or basic species
to the mobile phase
3. Ionised form is more polar less well retained under
reversed phase conditions
4. Non-ionised (ion-suppressed
form) is less polar - retained
longer under reversed phase
conditions
pKa = 50% Ionised (pH 4.6)
MobilePhase_05.flv
MobilePhase_06.flv
Retention Control using Mobile Phase pH
Optimising Separations using pH
What pH? Nicotine pKa? Robustness?
Simulation to Optimise pH
1. Use modelling for predicting separation
2. This DryLab® model was achieved using 5 runs injections at:
pH 2.9 / 3.0 / 3.5 / 5.0 / 6.5
Mixture of Acidic and Neutral Analytes
MobilePhase_07.flv
Buffers for Reversed Phase HPLC (I)
1. A ‘buffer’ - an aqueous
solution of a mixture of a
weak acid and its conjugate
base or a weak base and its
conjugate acid. pH of a
buffered solution changes
very little when a small
amount of strong acid or base
is added to it.
2. Where on the HPLC system do
we expect pH to change?
3. Buffer capacity is critical work within 1pH unit of the
buffer pKa!! 20% of maximum
buffer capacity is critical!
Buffers for Reversed Phase HPLC (II)
4. 25-50mM is a good
starting point for buffer
solution concentration
5. Chose appropriate
conjugate acid or base!
6. Always buffer the
aqueous component of
the mobile phase
separately
7. pH of the mobile phase
and pKa of analyte will
change in the presence
of organic solvents consistency is the key!
Buffers for Reversed Phase HPLC (III)
8. Increase buffer
concentration or capacity
if peak shape is poor note this may affect
selectivity!
9. Consider UV-Cutoff
10. TEA and TFA degrade and
their UV cut-off increases
11. Citrate buffers corrode
stainless steel
12. Solubility of salts
NH4 < K< Na
Effects of Buffer Concentration on Retention
1. Buffers change the polarity
and ionic strength of the
mobile phase
2. Doesn’t usually effect
analyte retention except
where secondary effects are
taking place with ionisable
analytes (i.e. Silanol
interactions)
3. Beware that some species
used as buffers (i.e. TFA) are
excellent ion-pairing agents
and can drastically alter
analyte retention times at
the wrong concentration!
MobilePhase_08.flv
Working at Low pH with Basic Analytes!
Amphetamine – peak 7
Amphetamine – peak 7
pH 2.5
pH 7.0
Using a sacrificial base
1. Triethylamine (TEA) and
Dioctylamine (DOA) can
be used to ‘end cap’ the
column on the fly
2. Improves peak shape
3. Can be used in
conjunction with other
buffers at lower
concentrations (0.1%)
Using Ion Pair Reagents
1. How do we control retention
with amphoteric
moleculeswhich cannot be
rendered neutral using pH
control?
2. Some modern reversed
phase HPLC column
chemistries are suitable
3. More usual solution is to use
ion pairing reagents
4. Ionic / Hydrophobic reagents
(e.g. Sodium Dodecyl
Sulphonate) are used to
‘pair’ with the analyte
ionised moiety in solution
MobilePhase_09.flv
Using Ion Pair Reagents
1. Actual mechanism is a
mixture of ion-pairing and
ion-exchange
2. Ion Pair Concentration is
important and has an
optimum concentration
3. More on Ion Pairing
Chromatography in a
future Essential Guide!
When all else fails!!
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