Which Buffer Should I Select

Which Buffer Should I Select?
The suitability of a buffer system depends upon many factors,

the first being the pKa value of the buffer acid or base. Other
factors that should be taken into account include the solubility
and stability of the analytes in the electrolyte, the effect of
temperature and heat generation. A selection of commonly used
buffers are listed in Table 2.
"For a buffer to be effective its pH must be centred around
its pK a 1 buffers should be selected accordingly."
Table 2: Commonly used

buffers in CE.
Common Problems in Buffer Preparations

Dilution of stock pH adjusted buffer: A common laboratory practice in buffer preparation is to prepare
concentrated buffer solutions. These stock solutions are then diluted to obtain the required run buffer
concentrations. This experimental problem was examined by initially preparing a stock solution of 2 M
sodium borate adjusted to pH 9.4.2 This solution was then diluted with water to obtain a final
concentration of 500 mM sodium borate. The pH was re-examined and was found to be 9.33.
The same procedure was repeated on a 1 M solution of sodium di-hydrogen orthophosphate adjusted to
pH 2.50 with concentrated phosphoric acid (~15 M). This solution was diluted with water to obtain a
final concentration of 500 mM and the pH was 2.58.
"Good working practice would be the preparation of a buffer at the required pH rather than diluting a
stock solution."
Buffer description errors: Another common problem is the description of preparation procedures given
in the literature, where many research papers in CE include a simple but vague description of the buffer
composition. Consequently, an analyst attempting to repeat the work is regularly faced with an
inadequate description. Taking the term "borate" as an example, this description is extremely
ambiguous and has appeared consistently in the literature. For example, sodium borate solution (15
mM) was used in the separation represented in Figure 4(a) and sodium tetraborate (15 mM) in Figure
4(b) for resolution of the acidic test mix.2 Because 1 mole of tetraborate is equivalent to 4 moles of
borate then 15 mM tetraborate will be equivalent to 60 mM borate.
The migration times are very different as the tetraborate solution is four times more concentrated in
borate ions, thus generates a larger current and longer migration times.
"It is strongly suggested that buffer solution preparations are described in exquisite detail to ensure
consistent preparation."
Not specifying when to measure pH: Addition of organic solvents to buffers alters the number of
protons in solution and, therefore, invalidates the true measurement of pH. Consequently, it is advisable
to specify measuring the pH before the addition of a specific volume of solvent. A full method should
specify when to measure pH a good recent example specifies that the pH should be measured before
adding cyclodextrin to the buffer5 sulphated cyclodextrins are acidic and will lower the pH when
added. Heat is usually generated when acids and bases are mixed. Buffer solutions should be allowed to
reach room temperature (after mixing or removing from a fridge) before measuring pH as pH is
temperature dependent.
Incorrect use/care of pH meter: Manufacturers of modern pH meters have made operation so simple
that successful operation is often taken for granted, but it is not quite as easy as it looks. Electrodes
must be clean and properly filled. Calibration buffers must be fresh and span the pH range of interest.
Temperature is an important factor; not only does the pH of most buffered systems change with
temperature, the response of a pH electrode has a temperature dependence of its own. The
temperature setting on a pH meter is used to adjust the temperature of the electrode during the
measurement, not to correct the temperature where the buffer solution will be used. Specific volumes
of specific concentrations of acids and bases can be mixed to make pH values, for example, phosphoric
acid and triethanolamine, which avoids use of a pH meter.3
"The best way to use a pH meter is to avoid its use it is more reliable to mix measured volumes of
acids and bases together to generate a specific pH rather than rely on a pH meter."
Buffer not used within its buffering range: A buffer is only a buffer within 1 pH unit of its pKavalue;
working within 0.5 pH units of pKa is better for critical work. A buffer is needed not only to provide an
appropriate pH for the desired process but also to maintain that pH in the face of outside influences
such as temperature changes, reaction product formation or factors entering from the environment
(i.e., gases such as CO2).
However, it has been found during literature investigations that many methods cited6,7 do not use the
buffer within their adequate range, for example, phosphate pH5 is an electrolyte solution not a
buffer.6 As a result, in some instances, low capacity buffers used require frequent buffer replenishment
because they are quickly affected by "buffer depletion".
Inconsistent reagent grades or descriptions: The grade and source of reagents should be specified
because this affects the purity (and cost) of the reagents such as cyclodextrins and sodium dodecyl
sulphate. Variations in purity will lead to alterations in ionic strength, current, separation selectivity and,
hence, lack of method robustness.
Not specifying shelf-life and storage conditions of buffers: Shelf-life for buffers cannot be assumed.
They must be experimentally assessed and proven before writing into methods. For example, materials
such as pthalates and metal ions can leach from containers, microbial growth can occur in buffers
containing carbohydrate sources such as cyclodextrins or carbon dioxide can be absorbed from the
atmosphere.
Insufficiently described buffer filtration procedure: Generally all electrolytes (and sample) should be
filtered through at least a 0.45 m filter to remove particulates, which would appear as apparent noise
on the detector baseline. The buffer filtration procedures can result in removal, or addition, of
components to the buffer. This may be a result of adsorption onto the filter or extraction from the filter.
The filtration process should be assessed, validated and stated in the method. Failure to do this can
result in non-robustness. For example, it was observed that up to 15% of the sodium dodecyl sulphate
(SDS) was being retained using certain filter types when filtering small volumes.8
"The exact filtration process should be described in the method including the volumes filtered and the
filter type used."

Which Buffer Should I Select

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Which Buffer Should I Select

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Which Buffer Should I Select?

The suitability of a buffer system depends upon many factors,

Table 2: Commonly used

Common Problems in Buffer Preparations

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