Low frequency Δν̄=0–350 cm−1, Raman intensity data were obtained from liquid water between 3.5 and 89.3 °C using holographic grating double and triple monochromators. The spectra were Bose–Einstein (BE) corrected, I/(1+n), and the total integrated (absolute) contour intensities were treated by an elaboration of the Young–Westerdahl (YW) thermodynamic method, assuming conservation of hydrogen-bonded (HB) and nonhydrogen-bonded (NHB=bent and/or stretched, O–H O) nearest-neighbor O–O pairs. A ΔH°1 value of 2.6±0.1 kcal/mol O–H ⋅⋅⋅ O or 5.2±0.2 kcal/mol H2O (11 kJ/mol O–H ⋅⋅⋅ O, or 22 kJ/mol H2O) resulted for the HB→NHB process. This intermolecular value agrees quantitatively with Raman and infrared ΔH° values from the one- and two-phonon OH-stretching regions, and from molecular dynamics, depolarized light scattering, neutron scattering, and ultrasonic absorption, thus indicating a common process. A population involving partial covalency of, i.e., charge transfer into, the H ⋅⋅⋅ O units of linear and/or weakly bent hydrogen bonds, O–H ⋅⋅⋅ O; is transformed into a second high energy population involving bent, e.g., 150° or less, and/or stretched, e.g., 3.2 Å, but otherwise strongly cohesive O–H O interactions. All difference spectra from the fundamental OH-stretching contours cross at the X(Z,X+Z)Y isobestic frequency of 3425 cm−1. Also, total integrated Raman intensity decreases occurring below 3425 cm−1 with temperature rise were found to be proportional to the total integrated intensity increases above 3425 cm−1, indicating conservation among the HB and NHB OH-stretching classes. From the enthalpy of vaporization of water at 0 °C, and the ΔH°1 of 2.6 kcal/mol O–H ⋅⋅⋅ O, the additional enthalpy, ΔH°2, needed for the complete separation of the NHB O–O nearest neighbors is ∼3.2 kcal/mol O–H ⋅⋅⋅ O or ∼6.4 kcal/mol H2O (13 kJ/mol O–H ⋅⋅⋅ O or 27 kJ/mol H2O). The NHB O–O nearest neighbors are held by forces other than those involving H ⋅⋅⋅ O partial covalency, i.e., electrostatic (multipole), induction, and dispersion forces. The NHB O–O pairs do not appear to produce significant intermolecular Raman intensity because they lack H ⋅⋅⋅O bond polarizability, but the corresponding NHB OH oscillators do contribute weakened Raman intensity above 3425 cm−1. An ideal solution thermodynamic treatment employing ΔH°1 =2.6 kcal/mol O–H ⋅⋅⋅ O, the HB mole fraction, and the vapor heat capacity, yielded a very satisfactory specific heat value of 1.1 cal deg−1 g−1 H2O at 0 °C. The NHB mole fraction, fu, from the YW treatment is negligibly small, 0.06 or less, for t<−50 °C. However, fu increases to 0.16 at 0 °C, and fu≊1 at 1437 °C, where recent shock-wave Raman measurements indicate loss of all partially covalent, charge transfer hydrogen bonding.

Precise isosbestic points occur in the Raman OH-stretching spectra from liquid water between 3 and 85 °C if cell alignment is accomplished with Newton’s rings. Isosbestic frequencies measured for the orientations X(Y,X+Z)Y=6β2, X(ZX)Y=3β2, X(Y+Z,X+Z)Y=45α2+13β2, X(Z,X+Z)Y=45α2+7β2, and X(ZZ)Y=45α2+4β2 are 3524, 3522 (note β2 agreement), 3468, 3425, and 3403 cm−1, respectively. Isosbestic points from two different measurements calculated by the relations, X(ZZ)Y-(4/3)X(ZX)Y and X(Z,X+Z)Y-(7/6)X(Y,X+Z)Y agree exactly for 45α2, 3370 cm−1. (α and β2 correspond to the mean polarizability and square of the anisotropy.) The pure α2 isosbestic frequency, 3370 cm−1, coincides with the peak of the highest frequency hydrogen-bonded (HB) Gaussian OH-stretching component. The pure β2 isosbestic point, 3522–3524 cm−1, coincides with the peak of the nonhydrogen-bonded (NHB) Gaussian OH-stretching component, next above in frequency. The α2 and β2 isosbestic points are thus thought to provide an experimental distinction between, and a clear definition of, the HB and NHB OH-oscillator classes for water. Moreover, the various OH-stretching combinations of α2 and β2 simply provide different measures of the HB→NHB equilibrium—no special information concerning the temperature dependence of this equilibrium results from use of any one linear polarizability combination over any other, including pure α2 or pure β2. The present results agree with mercury-excited data [Walrafen, J. Chem. Phys. 47, 114 (1967)] for X(Y+Z,X+Z)Y and with the corrected α2 data of d’Arrigo et al. [J. Chem. Phys. 75, 4264 (1981)]. Furthermore, the new data are in accord with the spectroscopic mixture model, but the continuum model conflicts with the observation of exact points. The isosbestic frequencies are also found to be strongly nonlinear in the amount of α2 or β2 involved in the spectra.

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (i.e., a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of a single protein molecule by measuring volumes of 0.1 nm per read.

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Raman OD-stretching (first) overtone spectra, X(Z,X+Z)Y, X(ZZ)Y, and X(ZX)Y, from pure D2O between 22 and 98 °C yield an isosbestic frequency near 5130 cm-1. This value corresponds to the X(ZX)Y fundamental isosbestic observed near 2624 cm-1, but not to the X(ZZ)Y fundamental isosbestic near 2429 cm-1, because the fundamental 2375 cm-1 correlated deuteron OD stretch, ν1, and its overtone, 2ν1, are weak in the X(ZX)Y spectra, compared to the three higher-frequency fundamentals, ν2, ν3, and ν4, and their overtones. The 2375 and 2475 cm-1 fundamentals and their overtones probably engage in coupling with a ≈175 cm-1 LA phonon which is supported by H-bonded aggregates or patches, whereas the corresponding data for H2O provide clear and unequivocal evidence for this LA coupling. Deconvolution of the X(Z,X+Z)Y overtone spectra, 22−152 °C, plus van't Hoff treatment yields an enthalpy for H bond, O−D···O, rupture of 2.8 ± 0.2 kcal/mol O−D···O, in good agreement with present values of 2.6 ± 0.2 and 2.8 ± 0.2 kcal/mol O−D···O, obtained from the fundamental X(ZZ)Y and X(ZX)Y spectra. Raman overtone measurements are important because the spacing between the four-Gaussian components is large in the overtone OD- and OH-stretching contours compared to the fundamentals.