Buffer viscosity

Since all electrophoretic mobility values are proportional to the reciprocal viscosity of the buffer, as derived in Chapter 1, the experimental mobility values n must be normalized to the same buffer viscosity to eliminate all other influences on the experimental data besides the association equilibrium. Some commercial capillary zone electrophoresis (CZE) instruments allow the application of a constant pressure to the capillary. With such an instrument the viscosity of the buffer can be determined by injecting a neutral marker into the buffer and then calculating the viscosity from the time that the marker needs to travel through the capillary at a set pressure. During this experiment the high voltage is switched off. 

Organic solvents Affect EOF and buffer viscosity Complex changes in separation system... 

The concentration of 25 mM was shown to increase resolution as well as migration times. Acetonitrile was added as an organic modifier due to its capability to change the zeta potential as well as the buffer viscosity, which resulted in changes in the electroosmotic flow (EOF). Therefore, the distribution of solutes between the buffer and the micelle was strongly affected by the involvement of acetonitrile. This was then shown to improve the poor resolution between ezetimibe and simvastatin [57]. 

The extent of the flow is related (Eq. (3.3)) to the charge (zeta potential) on the capillary, the buffer viscosity and dielectric constant of the buffer ... 

The control of EOF is critical to the migration time precision of the separation. Among the factors affecting the EOF are buffer pH, buffer concentration, buffer viscosity, temperature, organic modifiers, cationic surfactants or protonated amines, polymer additives, field strength, and the nature of the capillary surface. 

In CE, an increase in temperature reduces buffer viscosity leading to higher mobilities and shorter analysis times. Unfortunately the buffer conductivity increases at the same time, resulting in higher currents and extended Joule heating. With increasing temperature the risk of poor reproducibility and/or the total collapse of migration due to bubble formation also increases. It is not, therefore, commonly used for CE optimization. 

Therefore, as the viscosity decreases the mobility increases and the migration time gets faster. The volume injected via hydrodynamic injection (pressure or vacuum) is also inversely related to the buffer viscosity through the Hagen-Poiseuille equation (Eq. 2). 

As we have seen from Eq. 1, the mobility is dependent upon the buffer viscosity, assuming that the buffer make-up is constant and its pH is stable. Therefore, from an instrumental point of view, the electric field and the capillary temperature are the two main parameters which must be stabilised to effect reproducible migration time. Where a buffer is selected for properties other than its buffering capacity, e.g. indirect detection techniques, it may be necessary to replace the buffer frequently in order to ensure reproducible migration times. 

Separations should initially be attempted with the capillary thermostatted at close to ambient temperature. The capillary temperature can be increased on most commercial CE units to as high as 60 C without substantially increasing current with most buffers. When this is done using the same applied voltage, decreased buffer viscosity leads to an increase in analyte electrophoretic mobility, thus, decreasing separation times. Also, it is important to note that, when sample introduction is hydrostatic (same pressure/vacuum and time), increased capillary temperature will lead to an increase in the injected sample volume as a result of decreased buffer viscosity. " Sensitivity may not necessarily be increased. However, Undesirable effects include concurrent changes in buffer pH, band broadening due to increased diffusion, and possible thermal denaturation of the sample. 

Increasing the buffer viscosity ( ) affects the apparent mobility through the electrophoretic mobility ((iep. see Equation 18.9) and electroosmotic mobility ((Ceof. see Equation 18.10) by increasing the resistance to movement... 

To a good approximation, there is an inverse relationship between the viscosity and the resulting current doubling the buffer viscosity will halve the current and ATRadiai- In nonaqueous solvents, electrical currents are generally lower than that for the same electrolyte dissolved in water, but predicting ATRadiai becomes complex as one needs to take into account variations in the mobilities of species, dissociation equilibria, dielectric constant, zeta potential, and thermal conductivity [18]. 

The HX rates are also dependent on temperature. An increase in temperature affects HX rates primarily hy altering the water ionization constant, K, and thus increasing the concentration of OH . Further, some evidence suggests that temperature may also affect the collisional rate constant, k, in Equation 1.2 hy altering buffer viscosity and thus the diffusional collisional rate constant [24, 25]. A more recent study, however, has indicated that the effect of bulk viscosity on HX is negligible [30]. Theoretical HX rates can be determined as a function of temperature by a modified form of the Arrhenius equation (Eq. 1.4) and reference HX rate constants determined experimentally at 20°C ... 

An increase in temperature will result in faster ion mobilities as buffer viscosity decreases with increasing temperature. Care should be exercised as the buffer pH, and therefore the overall separation, can also be affected. 

T emperature Decreases buffer viscosity Can alter buffer pH, thereby... 

---