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Buffer capacity falls to 33% of the maximum value at pH = pK a ± 1, to 10% at pH = pK a ± 1.5 and to 1% at pH = pK a ± 2. For this reason the most useful range is approximately pK a ± 1. When choosing a buffer for use at a specific pH, it should have a pK a value as close as possible to that pH. [2]
The following table presents pK a values at 20 °C. Values change by about 0.01 per degree of temperature. [1] [3] Good's original 1966 paper had two older buffers (marked with italics) for comparison.
It consists of a mixture of 0.04 M boric acid, 0.04 M phosphoric acid and 0.04 M acetic acid that has been titrated to the desired pH with 0.2 M sodium hydroxide. Britton and Robinson also proposed a second formulation that gave an essentially linear pH response to added alkali from pH 2.5 to pH 9.2 (and buffers to pH 12).
Add 2.84 mM of HCl to shift the buffer to 7.3 mM HPO 4 2− and 4.6 mM H 2 PO 4 − for a final pH of 7.4 and a Cl − concentration of 142 mM. The pH of PBS is ~7.4. When making buffer solutions, it is good practice to always measure the pH directly using a pH meter. If necessary, pH can be adjusted using hydrochloric acid or sodium hydroxide.
The effect of buffer identity on the kinetics of the restriction enzyme EcoRV has been studied in various buffers, including Bis-Tris propane. [5] Bis-Tris propane wide buffering range is also useful for calibration of genetically encoded pH indicators expressed in the cytosol or mitochondria. [ 6 ]
In general, as temperature decreases from 25 °C to 5 °C the pH of a tris buffer will increase an average of 0.03 units per degree. As temperature rises from 25 °C to 37 °C, the pH of a tris buffer will decrease an average of 0.025 units per degree.
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