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HPLC method development requires optimizing a wide variety of mobile phase and column
parameters. The pH of the mobile phase is just one. In a reversed-phase HPLC separation of
acids and bases, pH plays a crucial role in determining retention and selectivity, and in
controlling the reproducibility and ruggedness of a method. Understanding how mobile phase pH
affects your analytes and HPLC column can help you quickly determine the best mobile phase
conditions for a separation, reducing the amount of time it takes to develop a rugged method.
The pH range most often used for reversed-phase HPLC is 1 - 8 and can be divided into low pH
(1 - 4) and intermediate pH (4 - 8) ranges. Each range has a number of advantages. Low pH has
the advantage of creating an environment in which peak tailing is minimized and method
ruggedness is, maximized. For this reason, operating at low pH is recommended.
The best initial mobile phase pH will be 3.0 or lower for I the majority of basic samples,
including these antihistamines, since they are fully protonated at this pH. The plot of the
antihistamines data shows good, consistent retention and selectivity for these basic compounds
over the pH range 3 - 5. Figure 2 shows the excellent separation obtained at pH 3 (see Why Use
a B offered Mobile Phase). At this low pH excellent peak symmetry is obtained. At pH 3.5, 4, or
5 the separation will be the same, with retention and selectivity unchanged.
The pH 3 mobile phase has an added benefit for the separa- tion of these antihistamines. It is at
least one pH unit away from the pKa values of the analytes (see Understanding pKa). This will
often be the most rugged and reproducible mobile phase pH choice.
Separations at Intermediate pH -
Retention and Selectivity
The same type of pH vs. retention plot constructed for the antihistamines
FIGURE 3 can be established for any group of basic and 'dic analytes. This plot
Separation of 'nd'cates the sensitivity of a separation to pH changes and serves as the
Benzodiazepines at primary guide to selecting the most rugged mobile phase pH for a
pH7 separation. For optimum ruggedness, select a pH for the mobile phase
where retention and selectivity do not change if the pH changes slightly.
For most basic compounds this will be at low pH. The goal in
determining the impact of pH on your analytes is to balance sample
retention and selectivity, so that the best method ruggedness is obtained.
Peak Shape
Column Stability and Durability
Reproducibility
The pH of the mobile phase also affects the column. It can help determine peak symmetry,
column lifetime, and method reproducibility.
Peak Shape
Figure 4 shows a separation of highly basic amphetamines at pH 3 and pH 7. The USP tailing
factor is used to measure peak tailing. Some methods recommend that this value be less than 2.0.
At pH 7, one compound, methamphetamine, has a tailing factor of 2.35. At pH 3 the tailing
factor for this compound is 1.28, similar to other compounds in the sample. At pH 3 all silica
surface silanols are protonated, eliminating strong interactions with protonated basic compounds
that cause tailing. Any mobile phase pH below ~4.0 will protonate these silanols, improving
peak symmetry and general method ruggedness and reproducibility. At low pH, analytes
experience a consistent silica surface from day to day and column to column.
In the intermediate pH range there are more possible interactions with underlying silanols. Figure
5 shows the type of interaction that occurs with an ionized silanol. These types of interactions are
substantially reduced by using a high-purity, fully-hydroxylated silica surface, extra dense ligand
bonding and double-endcapping to produce a column packing such as the Eclipse XDB-C8. This
packing neutralizes the maximum number of silanols and benefits applications in the
intermediate pH range.
Figure 6 shows the dramatic difference that double endcapping makes. Here several very basic
tricyclic antidepressants are separated at neutral pH on an Eclipse XDB-C8 column and a column
containing single endcapped packing. The double endcapped column packing exhibits better
peak shape (indicated by improved tailing factors) and shorter retention times for these
compounds. These results indicate that silanol interactions have been significantly minimized.
Excellent peak shape and high efficiency result for very basic compounds.
Understanding pKa
The pKa value (acid dissociation [ionization] constant) for a compound is the pH at which
equal concentrations of the acidic and basic forms of the molecule are present in aqueous
solutions.
For basic compounds this is [BH+;]= [B] and for acidic compounds this is [HA] = [A-;].
Analytes may sometimes appear as broad or tailing peaks when the mobile phase pH is at,
or near, their pKa values. A more rugged mobile phase pH will be at least 1 pH unit
different from the analyte pKa. This shifts the equilibrium so that 99% of the sample will
be in one form. The result is consistent chromatography.
FIGURE 5
Peak Tailing Interaction
At Low pH
FIGURE 6
At low pH the major mechanism of column degradation is different Double Endcapping
from that at high pH. At low pH, acid hydrolysis of the bonded phase More Effectively
from the silica surface results in changes in solute retention over time. Reduces Silanol
The rate of hydrolysis increases with decreasing pH. A 1% Interactions
trifluoroacetic acid mobile phase (pH 0.8) will reduce the lifetime of
most HPLC columns. The buffer strength and organic modifier have less
of an effect at low pH than at high pH. FIGURE 7
Column Stability
We compared the stability of three C8 columns at pH 3 and 60oC Testing at pH 3.0,
(Figure 7). The columns were purged with a mobile phase at this low pH 60o;C
and periodically tested with this same mobile phase to determine if the
column was changing. The test sample included the highly basic
compound amitriptyline. An increase in retention for amitriptyline
indicates a loss of bonded phase and an increase in exposed acidic
surface silanols. The ZORBAX® StableBonde® SB-C8, manufactured
with a special stabilized bonded phase for acidic mobile phases, shows
no change during the stability testing. The Eclipse XDB-CS column also
showed no change at this pH. But a third endcapped C8 column showed
a substantial increase in retention for amitriptyline over time. This
indicates the endcapping reagent was stripped off the column packing by
these low pH conditions.
Reproducibility