Water (H2O and D2O) molar absorptivity in the 1000-4000 cm(-1) range and quantitative infrared spectroscopy of aqueous solutions
Water (H2O and D2O) molar absorptivity in the 1000-4000 cm(-1) range and quantitative infrared spectroscopy of aqueous solutions
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DOI：
10.1006/abio.1997.2136
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发表时间：
1997-06-01
影响因子：
2.9
通讯作者：
Prendergast, FG
中科院分区：
文献类型：
--
作者：
Venyaminov, SY;Prendergast, FG
水（H2O 和 D2O）摩尔吸光率通过傅里叶变换红外透射光谱在 25 摄氏度下在 1000-4000 cm(-1) 范围内测量。这些测量使用了一系列光程长度为 1.2 至 120.5 μm 的组装电池。计算所有水吸光带的最佳光程长度（经水吸光度校正后的溶质红外光谱具有最高信噪比时水溶液的光程长度）。这里给出的结果表明，最佳路径长度不取决于溶质性质，并且与溶剂（水）摩尔吸收率成反比。在生物应用中主要关注的 1650 cm(-1) 光谱区域中水溶液红外光谱测量的最大信噪比可以在 3-4 μ m (H2O) 和 40-60 μ m (D2O) 的最佳池路径长度下获得。例如，信噪比计算为溶菌酶水溶液的酰胺 I (H2O) 和酰胺 I' (D2O) 带的细胞路径长度的函数。水波段的摩尔吸光率比相同光谱区域中生物大分子最强波段的摩尔吸光率弱几个数量级。水溶液中的高净吸水率仅仅是由于水的摩尔浓度非常高。提出了一种定量测量池路径长度的方法，该方法利用最强水带（拉伸振动）或不与溶质吸光度重叠的带的摩尔吸光率。对于已知浓度的样品，使用该技术可以高精度地确定从大约 0.01 μm 到大约 1.0 mm 范围内的路径长度。讨论了生物分子水溶液红外光谱中强水吸光度的正确校正所涉及的问题，包括细胞内的多次反射、pH、温度的影响和极性溶质对水光谱特性的扰动，以及可以获得最精确的吸光度校正的最佳光谱区域的选择。 (C) 1997 年学术出版社。
Water (H2O and D2O) molar absorptivity was measured by Fourier transform infrared transmission spectroscopy in the 1000-4000 cm(-1) range at 25 degrees C. A series of assembled cells with path lengths from 1.2 to 120.5 mu m was used for these measurements. The optimal path length (the path length of aqueous solution at which the IR spectrum of solute, corrected for water absorbance, has the highest signal-to-noise ratio) was calculated for all water absorbance bands. The results presented here show that the optimal path length does not depend on solute properties and is inversely proportional to the solvent (water) molar absorptivity. The maximal signal-to-noise ratio for measurements of IR spectra of aqueous solution in the 1650 cm(-1) spectral region, of primary interest in biological applications, can be obtained at an optimal cell path lengths of 3-4 mu m (H2O) and 40-60 mu m (D2O). As an example, the signal-to-noise ratio was calculated as a function of the cell path length for the amide I (H2O) and amide I' (D2O) bands of an aqueous lysozyme solution. The molar absorptivities of water bands are several orders of magnitude weaker than those of the strongest bands of biological macromolecules in the same spectral regions. High net water absorbance in aqueous solutions is due simply to the very high molar concentration of water. A method is proposed for the quantitative measuring of the path length of the cell which exploits the molar absorptivity of the strongest water bands (stretching vibrations) or of bands which do not overlap with solute absorbance. A path length in the range from similar to 0.01 mu m to similar to 1.0 mm can be determined with high precision using this technique for a samples of known concentration. Problems involved in the proper correction of strong water absorbance in IR spectra of aqueous solutions of biomolecules are discussed, including multiple reflections within the cell, the effects of pH, temperature, and perturbation of water spectral properties by polar solutes, as well as the selection of optimal spectral regions in which one may obtain the most precise absorbance corrections. (C) 1997 Academic Press.