Rapid Detection of Low Concentrations of Aqueous Species in the Presence of Spectral Overlap Using Planar Array Infrared Spectroscopy

It is well known that water is the Achilles’ heel of infrared spectroscopy. The rotational-vibrational manifold of gas-phase water spans several hundred wavenumbers in the fingerprint region and necessitates that instruments be continuously purged with dry gas. This problem is especially vexing when studying aqueous systems, since the absorptivity of condensedphase water is relatively high, necessitating a balance between path length and sensitivity when attempting to study dilute aqueous samples. A common approach to the removal of spectral contributions from water is to perform a spectral subtraction, using pure water as a reference. This is only possible if the path length of the sample cell is small enough to prevent saturation of the absorption signals from water itself. Typically, path lengths of several micrometers are employed to keep the absorbance intensity of water within the linear range. However, this short path length requires that high concentrations of the species of interest be employed to maximize the signal-to-noise ratio of the final spectrum. Many species of physiological relevance exist at concentrations well below 1% w/w in vitro. Of particular interest is the analysis of proteins in the aqueous humor of the human eye, which has a typical total protein concentration of approximately 0.1% w/w. Additionally, specific proteins of interest to the detection and diagnosis of eye diseases, such as vascular endothelial growth factor (VEGF), exist at much lower concentrations, on the order of 10 7 % w/w. Thus, in order to study these species under realistic conditions, care must be taken to remove any spectral interference. The common approach to avoiding interference from H2O is to use D2O solutions, but it is well known that this can cause changes in the structure of proteins. Several instrumental and data processing approaches have been devised to minimize water interference, including attenuated total reflection, Fourier self deconvolution, spectral subtraction, and optimization of transmission cell thickness. Recently, we have developed planar array infrared (PA-IR) spectroscopy as a method to acquire infrared spectra rapidly, from both static and dynamic chemical systems. Here, we use PA-IR as a means of acquiring spectra of dilute aqueous solutions that are free from the interference of both gaseous and liquid water.