INFRAESTRUCTURA URBANA

Based on the literature to date, the zwitterionic buffer MES/His has been the buffer of choice for every research group investigating conductivity detection since Zare first introduced an on-column conductivity detector for capillary electrophoresis in 1987.16 This still holds true for both capillary and microfluidic based separations (Table 2-5). While there has been discussion of the use of ampholytes as background electrolytes in conductivity detection due to their low conductivity at high concentrations, there has

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been no clear indication as to the reason for the superior low baseline noise of MES/His with this detection method.

2-N-morpholinoethanesulfonic acid (MES) is a substituted taurine with a pKa of 6.15 at 20 °C. Traditionally, this acid is paired with an equimolar amount of an amino acid, usually Histidine, which has a pKa of 6.00 at 20 °C (Figure 2-14). Additionally, Pospichal et al. found that the ionic mobilities of MES and His are -26.8 x 109 _m2 _V-1 _s-1 and +26.7 x 109 m2 V-1 s-1, respectively.17 It is interesting to note that this particular buffer combination results in a unique situation where the constituents have similar pKa values and essentially equal, but opposite, mobilities.

In a comparative study, the noise and separation efficiency of several different buffers were tested using a mixture of 10 ppm Na+_{, K}+_{, and Li}+_{. Buffer components were} dissolved in deionized water at concentrations that would give similar background conductivities to 20 mM MES/20 mM His buffer. Initially, the buffers were not pH adjusted, but were left at their native pH when combined in solution. While this meant that some buffers would not be at their optimal pH and hence, operating under conditions not suitable to give their maximal buffering capacity, it prevented any increased

conductance from additional chloride or sodium ions in solution.

Initially, another zwitterionic buffer, piperazine-N,N’-Bis(2-ethanesulfonic acid) or PIPES, was used in conjunction with Histidine to test the effect of the anionic species in the buffer system. PIPES is a symmetrical disulfonic acid with a pKa of 6.8. The absolute ionic mobility of this molecule has not been determined. In a comparison of these two buffer systems, it was found that the MES/His system was less noisy, had a lower DC current, and gave higher conductivity detection signal-to-noise values.

3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), which has a pKa of 10.4, was used with Arginine, which has a pKa of 12.5. The experiment was conducted at pH 10.4, well within the accepted buffering zone for CAPS. According to theory and given the experimental electrophoresis current, the shot noise with this system should be 600 nV. The expected noise with the additional contribution from the function generator brings this value up to 2.2 µV for this system. As can be seen from Table 2-6, the

experimental noise is an order of magnitude greater than that predicted (~21 µV) and the signal to noise ratio for the lithium ion is much smaller than for MES/His. Additionally, CAPS was paired with lysine to give two buffer components with similar pKa values; however, this proved to be even worse due to the increased current and noise obtained.

Table 2-6 summarizes the results from this extensive study, with a few

representative electropherograms shown in Figure 2-15. Based on the tabulated data, it can be seen that while the background conductivities may be similar in magnitude, the noise associated with each buffer is much greater than the theoretically predicted value. The combination of MES and His is the only buffer combination that is on the order of the predicted value. Several other zwitterionic buffers were tested in addition to

MES/His; however, it was found that the while these buffers had very low conductivities, the noise in these systems was still greater than that seen in MES/His, even while at a pH value within their suggested buffering range. The use of MES/His as the buffer for conductivity detection meets the necessity of using a low conductivity background electrolyte while still providing the current conduction medium necessary without an increase in measured noise.

In a series of studies further investigating the use of MES/His as a buffer, the two components were tested individually, in an attempt to elucidate whether one component has a more significant contribution to the decreased noise values than the other. To keep the ionic strength of the buffer systems similar to that of the 20 mM MES/20 mM His system, the concentration of each individual species was increased to give an overall ionic concentration of 40 mM. As shown in Table 2-7, when dissolved in deionized water, each solution was at a pH value that was significantly far away from the pKa value. It is known that buffer systems have the highest buffer capacity within 1 pH unit of their pKa value.18 It should be noted that although the solutions of MES and His were beyond this boundary, the noise in the system was very near the expected values, based on the electrophoretic currents observed. However, the separation of the standard simple inorganic ions (Na+, K+, and Li+) was extremely poor (Figure 2-16A and B). It was thought that the low buffer capacity at the native pH could be the cause for the poor peak shapes. Each solution was adjusted to the pKa value using a strong acid or base. It was found that the noise increased by an order of magnitude when each solution pH was adjusted. The increased noise was presumably due to the increased conductance from sodium and chloride (Table 2-7) ions in solutions. In a practical sense, all of these experiments illustrate the importance of the buffer composition to the noise, as well as the separation, associated with conductivity detection.