Borate buffer, free-solution separations

The supporting electrolyte for all free-solution separations reported here was a borate buffer (pH 8.1 or 8.26, 0.10 M or 0.20 M), prepared from reagent-grade sodium borate decahydrate and boric acid (J.T Baker). 

Fig. 3 Effect of external pH on internal vesicle pH and MCP hydrolysis. In all experiments, a vesicle sample of 1 ml, was eluted from a Sephadex G-25 column (1 x 50 cm) with the appropriate solution. The column was previously saturated with sonicated vesicles of (DODA)B or lecithin to avoid vesicle disruption due to adsorption on the Sephadex. The vesicles eluted in the void volume were pooled and a 0.05 ml aliquot was added to 1.5 ml buffer. Borate buffer (5 mM) was used at pH 10.2 and NaOH for all other pHs. The samples were maintained at 30 °C and the hydrolysis of MCP was followed at 265 nm. A Effect of pH on the hydrolysis of free (o) and (DODA)B-entrapped MCP ( ). Chloroformic (DODA)B vesicles were prepared in 5 mM MCP chloride and 0.95 M erythritol. The vesicles were eluted from the column with erythritol 0.95 M and NaCl 5 mM. Erythritol 0.95 M was used in all buffers and the ionic strength was maintained at 5 mM with NaCl in order to avoid osmotic and ionic stress. Final concentrations of MCP and (DODA)B were 5 x 10 M and 7 x 10 M, respectively. B Effect of, pH on hydrolysis of free (o) and Lecithin entrapped MCP ( ). Lecithin vesicles were prepared by reverse phase evaporation (0.01 M, with 10% DCP) in 0.16 M KCl and 5 mM MCP, and the sample was eluted from Sephadex with KCl (0.165 M). All buffers contained KCl (0.16 M) and the kinetics were obtained with lecithin (5.6 X 10" M) and MCP (5 x 10" M). (a) Lecithin vesicles eluted from the column were incubated with 5 x 10" M Val for 15 min. After pH change the rate of MCP hydrolysis was the same as in aqueous phase. In a separate experiment, lecithin vesicles containing MCP only in the internal compartment were incubated with Val for 90 min and refiltered in Sephadex. No free MCP was found. An aliquot of the refiltered vesicles was added to NaOH and the rate constant was measured (a). C Kinetics of the effect of pH on the absorbance of intravesicular 2-HTAB. (DODA)B vesicles (5 mM) prepared with 5 mM 2-HTAB, 5 mM NaCl and 0.95 M erythritol were eluted from Sephadex G-25 with NaCl 5 mM and erythritol 0.95 M. Aliquots (0.05 ml) of (DODA)B vesicles were added to 1.5 ml of a solution containing erythritol (0.95 M), NaOH, pH 11.59 and the absorbance was measured at 340 nm. At the end of the reaction, HCl was added. A new addition of NaOH promotes a similar increase of absorbance [42]...

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