Complex χ(2) spectra of buried silica/isotopically diluted water (HOD-D2O) interfaces were measured using multiplex heterodyne-detected vibrational sum frequency generation spectroscopy to elucidate the hydrogen bond structure and up/down orientation of water at the silica/water interface at different pHs. The data show that vibrational coupling (inter- and/or intramolecular coupling) plays a significant role in determining the χ(2) spectral feature of silica/H2O interfaces and indicate that the doublet feature in the H2O spectra does not represent two distinct water structures (i.e., the ice- and liquid-like structures) at the silica/water interface. The observed pH dependence of the imaginary χ(2) spectra is explained by (1) H-up oriented water donating a hydrogen bond to the oxygen atom of silanolate, which is accompanied by H-up water oriented by the electric field created by the negative charge of silanolate, (2) H-up oriented water which donates a hydrogen bond to the neutral silanol oxygen, and (3) H-down oriented water which accepts hydrogen bonds from the neutral silanol and donates hydrogen bonds to bulk water molecules. The broad continuum of the OH stretch band of HOD-D2O and a long tail in the low frequency region represent a wide distribution of strong hydrogen bonds at the silica/water interface, particularly at the low pH.

Despite recent significant advances in interface-selective nonlinear spectroscopy, the topmost water structure at a charged silica surface is still not clearly understood. This is because, for charged interfaces, not only interfacial molecules at the topmost layer but also a large number of molecules in the electric double layer are probed even with second-order nonlinear spectroscopy. In the present study, we studied water structure at the negatively charged silica/aqueous interface at pH 12 using heterodyne-detected vibrational sum frequency generation spectroscopy, and demonstrated that the spectral component of the topmost water can be extracted by examining the ionic strength dependence of the Imχ spectrum. The obtained Imχ spectrum indicates that the dominant water species in the topmost layer is hydrogen-bonded to the negatively charged silanolate at the silica surface with one OH group. There also exists minor water species that weakly interacts with the oxygen atom of a siloxane bridge or the remaining silanol at the silica surface, using one OH group. The ionic strength dependence of the Imχ spectrum indicates that this water structure of the topmost layer is unchanged in a wide ionic strength range from 0.01 to 2 M.

Infrared spectra of mono- and dihydrated clusters of guanosine (Gs) formed by laser desorption combined with supersonic jet cooling have been measured by the technique of IR-UV double resonance spectroscopy. The results are compared with those of 9-methylguanine (9MG), in which the sugar group of Gs is replaced with a methyl group, to elucidate the importance of the sugar group in the hydration structures. It is shown that the UV spectrum observed for the monohydrated cluster of Gs is composed of multiple structural isomers of larger stabilities. One of the monohydrates is identified to possess a hydration structure involving the 5'-OH group of the sugar and the amino group of the guanine moiety. The IR spectrum of the dihydrated clusters reveals that the 2'-OH group is significantly influenced by the addition of the second water, which suggests the possibility of specific dihydrate structures for Gs.

Elucidation of the molecular mechanisms of protein adsorption is of essential importance for further development of biotechnology. Here, we use interface-selective nonlinear vibrational spectroscopy to investigate protein charge at the air/water interface by probing the orientation of interfacial water molecules. We measured the Im χ spectra of hemoglobin, myoglobin, serum albumin and lysozyme at the air/water interface in the CH and OH stretching regions using heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy, and we deduced the isoelectric point of the protein by monitoring the orientational flip-flop of water molecules at the interface. Strikingly, our measurements indicate that the isoelectric point of hemoglobin is significantly lowered (by about one pH unit) at the air/water interface compared to that in the bulk. This can be predominantly attributed to the modifications of the protein structure at the air/water interface. Our results also suggest that a similar mechanism accounts for the modification of myoglobin charge at the air/water interface. This effect has not been reported for other model proteins at interfaces probed by conventional VSFG techniques, and it emphasizes the importance of the structural modifications of proteins at the interface, which can drastically affect their charge profiles in a protein-specific manner. The direct experimental approach using HD-VSFG can unveil the changes of the isoelectric point of adsorbed proteins at various interfaces, which is of major relevance to many biological applications and sheds new light on the effect of interfaces on protein charge.

We have employed IR-UV double resonance spectroscopy to identify the tautomeric and isomeric structures of uric acid and its monohydrated clusters which are produced by the techniques of laser-desorption and supersonic-jet cooling. The IR spectrum obtained for bare uric acid exhibits four distinct NH stretching transitions assignable to those of the most stable triketo form. We have also observed the two most stable monohydrated clusters, each with uric acid in the triketo form and water bonded to either the N3H or N9H site. It is demonstrated that the R2PI spectra of these monohydrates can be separated by using the IR-purified spectroscopic method.