Practical Corrections for p(H,D) Measurements in Mixed H2O/D2O Biological Buffers

Published

May 1, 2017

Author(s)

Kenneth A. Rubinson

Abstract

Mixtures of light and heavy water are used in NMR, small-angle neutron scattering (SANS), growth media for producing deuterated biological molecules, and analytical methods such as Hydrogen-Deuterium Exchange (HDX) mass spectrometry. It is common to measure the pH of these solutions with a combination glass electrode filled with an aqueous (H₂O) potassium chloride solution. In the daily measurement with mixtures of H₂O with D₂O-call this measurement {pH,pD}-we generally do not control for all of the contributions to the differences measured in carefully controlled electrochemical experiments. For example, the calibration solutions contain relatively low concentrations of the calibrant buffer with no added salt. Meanwhile the tested solutions can contain widely varying levels of any number of different salts as well as both polar and nonpolar organics and polymers and proteins. In this note, the {pH,pD} behaviors of 50 mM solutions of five different buffers used in biological in vitro solutions were measured over the full range of H₂o:D₂O rations in the open atmosphere. After calibration, pH measurements were made with the buffer solutions alone and with 100 mM KCl added to model a significant ionic strength difference. The solution consisted of 1:1 volume mixtures of the acid and base forms of acetate, monobasic/dibasic phosphate, tris, HEPES, and glycine to span the common, full range for biological buffers. The pH values of the 1:1 mixtures means that the measurements were, in fact, of their pK_a values. Each of the buffers exhibited a unique pattern of behavior, and none of them exhibited a measured {Δ}pK_a = pK_a^D-pK_a^H as large as 0.4, which is a value that has been suggested to be added to a pH measured in H₂O to match the equivalent pD measured D₂O. The results do indicate that when a reasonable accuracy for pH measurement is 0.1 units, three general rules of thumb apply 1) Where {pH,pD} values are less than 8 for any D₂O content, no correction is needed for the {pH,pD} measurement when comparing it to pH^H; 2) For less than 50% D₂O, if the 8H; 3) When the D₂O content is greater than 50% and the {pH,pD}>8, any corrections required will depend on the specific conditions and the specific buffer. Outside of this region or for needed greater accuracy, any correction required will depend on the specific conditions and the identity of the buffer.

Citation

Analytical Methods

Volume

9

Issue

18

Pub Type

Journals

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Keywords

Buffer isotope effect, pH measurement, pH correction, SANS, NMR, HDX

Citation

, K. (2017), Practical Corrections for p(H,D) Measurements in Mixed H₂O/D₂O Biological Buffers, Analytical Methods, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=922733 (Accessed April 25, 2024)