Accurate preparation and correct selection of buffers is essential to obtain reproducible and consistent results in capillary
electrophoresis (CE). A number of factors should be considered in buffer optimization, including the pKa of the buffering
ion and the analyte as well as the molarity of the acid or base used in the adjusting procedure. Accurate recording of the
precise reagents used and the procedures performed is necessary to prepare buffers consistently.
"Poor reproducibility of results and poor quantitative precision will be attainable in CE assays without significant attention
being paid to the preparation of buffers used."
In this instalment of "CE Currents" buffer selection, buffer preparation and a selection of problems encountered when preparing
working buffers for capillary electrophoresis (CE) will be examined. Variation in the buffer preparation in CE has more pronounced
effects on the analyte separation than HPLC, therefore, analysts must take great care when describing buffer preparation and
following the preparation instructions.
Why Use a Buffer?The primary role of the buffer is to generate and maintain a set pH because this affects solute ionization and the level of
electrosmotic flow (EOF) generated in the capillary. The ionic strength of the buffer also influences the solute migration
times and the level of current and EOF generated in the capillary. The term electrolyte is often used in CE. This simply refers
to a solution of ions but does not indicate any buffering ability. Failure to use a buffered electrolyte can result in severe
operational difficulties because the method will be significantly less robust than a buffered electrolyte.
Electrolytic changes — termed "buffer depletion" — of the buffer can occur, especially in prolonged injection sequences. This
results in gradual changes of buffer pH in the separation buffer vials, which leads to changes in migration times and/or separation
selectivity. If the electrolyte used has a good buffering capacity then it can actively resist these pH changes.
How Do you Prepare a Buffer?
Let's look at a common buffer described in CE literature — "25 mM phosphate pH 7.0". How exactly is that prepared? From this
simple description it is impossible to tell the exact composition and ionic strength and is, therefore, impossible to reproduce.
Table 1: Variants on buffer preparation procedure.
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Table 1 shows how a buffer described in the literature as "25 mM phosphate pH 7.0" could have been prepared: the ionic strengths,
buffering capacity, EOF flow-rates and currents (the internal heating in the capillary) will be different for each preparation
variant.
"The exact procedure for electrolyte preparation should be defined in the method including the electrolyte salt form used
and, if used, an exact pH adjustment procedure. The pH adjustment procedure should include details of the nature and concentration
of the solution used to adjust the pH of the electrolyte."
Which Buffer Should I Select?
Table 2: Commonly used buffers in CE.
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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 pKa ± 1 — buffers should be selected accordingly."
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