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Tel: 1-800-255-8324 Fax: 847-948-1078 Specialists in Chromatography

Selecting the proper buffer pH is
necessar y to reproducibly separate
ionizable compounds by reversed-phase
liquid chromatography. Selecting an
improper pH for ionizable analytes often
leads to asymmetric peaks that are broad,
tail, split, or shoulder. Sharp, symmetrical
peaks are necessar y in quantitative
analysis in order to achieve low detection
limits, low relative standard deviations
(RSD) between injections, and
reproducible retention times. We will
discuss when buffering is needed, how to
select buffer pH, and how to choose
the proper buffer system for that pH.
When is Buffering Needed?
I n reversed-phase chromatography,
mobile phase pH values are usually between
2 and 7.5. Buffers are needed when
the analyte is ionizable under reversed-
phase conditions or the sample solution is
outside this pH range. Analytes ionizable
under reversed-phase conditions often
have amine or acid functional groups with
pKas between 1 and 11. A correctly chosen
buffer pH will insure that the ionizable
functional group is in a single form,
whether ionic or neutral. I f the sample
solution is at a pH damaging to the
column, the buffer will quickly bring the
pH of the injected solution to a less
harmful pH.
Howto Choose Buffer pH
I t is necessar y to know the pKa of the
analyte before buffer pH can be chosen.
A buffer 2 pH units above or below pKa is
recommended for good peak shape. From
the Henderson-Hasselback equation,
pH = pKa + log([ A
-
] /[ HA] )
it can be determined that 99% of an
analyte is in a single form if the solution pH
is 2 units above or below the analytes pKa.
Good peak shape is possible only when an
analyte is in a single form.
Lets work through some examples for
choosing mobile phase pH. Suppose we
are analyzing benzoic acid, which is shown
in its neutral and ionic forms in Figure 1.
The pKa of benzoic acid is 4.2 therefore by
the Henderson-Hasselback equation 99%
of the benzoic acid molecules will be in the
protonated, neutral form at pH 2.2. 99%
will be in the unprotonated, anion form at
pH 6.2. The protonated, neutral form is
required for carboxylic acid retention on
reversed-phase columns therefore an acidic
buffer at about pH 2.2 will work. Table 1
lists common buffers and their buffering
ranges. From Table 1 both phosphate and
citrate buffers are effective at pH 2.2.
Using Buffer pH to Adjust Selectivity
As mentioned earlier, amine retention
decreases as the buffer pH is decreased, a
characteristic that can be used to change
selectivity. I f two compounds co-elute and
one contains an amine functional group,
changing buffer pH will help to separate
the compounds.
Because selectivity for ionizable
compounds is pH dependent, changing
buffer pH can help identify the functional
groups on unknown analytes. I f a peak
elutes faster when the pH is made more
acidic, then the analyte probably contains
an amine group. I f a peak elutes earlier or
at the void when the pH is made more
basic, the analyte may be a carboxylic acid
since the ionic form elutes faster than the
protonated neutral form.
Summary
A properly buffered mobile phase is ver y
important in a successful reversed-phase
HPLC method for optimal peak shape,
detection limit, and consistent retention
times. Selecting the proper buffer pH is
easy if you know the analytes functional
groups and pKa values.
Table 1
Common Buffers for Reversed-
Phase Chromatography
Figure 1
The neutral and anionic forms of
benzoic acid
I f the analyte contains only amine
functional groups, buffer selection is easier.
Most amines will be in the cation form at
pH values less than 9, so any buffer
effective at pH 7 or lower will work. You
may ask why use a buffer at all since water is
already about pH 7. Buffering increases a
methods ruggedness since amine retention
and peak shape are pH dependent. As pH
is lowered amine retention times shorten
and peak shape sharpens as the buffer
protonates the acidic silanols on the silica
surface. From Table 1 any buffer with a
pKa less than 7 is suitable, but we have
found potassium phosphate at pH 3 is the
best for amines.
I n both examples potassium phosphate
buffer at pH 3 works well and in general is
an excellent buffer for analytes that contain
acid and amine functional groups. We have
found the potassium salt works better than
the sodium salt for amines.
Selecting Buffers for Ion-Pair Reagents
A similar process is followed to select a
buffer for methods that use ion-pair
reagents. I on-pair reagents such as
tetrabutylammonium chloride and sodium
heptane sulfonate are ionic mobile phase
modifiers that pair with the ionic functional
groups of the analyte. A buffer that puts
the analyte in its ionic form is required in
ion-pair chromatography.
C OH
O
C O
O
2259
Buffer pKa Buffer Range
REVERSED-PHASE HPLC BUFFERS
Phosphate
pK1 2.1 1.1 - 3.1
pK2 7.2 6.2 - 8.2
pK3 12.3 11.3 - 13.3
Citrate
pK1 3.1 2.1 - 4.1
pK2 4.7 3.7 - 5.7
pK3 5.4 4.4 - 6.4
Formate 3.8 2.8 - 4.8
Acetate 4.8 3.8 - 5.8
Tris (hydroxymethyl) aminomethane
8.3 7.3 - 9.3
Ammonia 9.2 8.2 - 10.2
Borate 9.2 8.2 - 10.2
Diethylamine 10.5 9.5 - 11.5
Selecting Buffer pH in Reversed-Phase HPLC