Stopped-Flow FT-IR Spectroscopy of Aqueous Solutions Using Attenuated Total Reflectance

Abstract

An apparatus for the study of solution phase kinetics using FT-IR spectroscopy has been developed. The observation chamber consists of an integrated tangential mixer-flow cell and a ZnSe element permitting attenuated total reflectance (ATR) measurements. The short optical pathlength afforded by ATR allows mid-IR observation of chemical reactions in aqueous solution, including the spectral region near the water bending vibration (1640 cm−1). High hydraulic backpressures required to force solution rapidly through a thin layer flow cell are not necessary with the ATR flow cell because the optical pathlength and the flow cross-section have been decoupled, allowing for a relativity large flow chamber when compared with instruments incorporating a transmission flow cell. Overall system performance has been evaluated using the hydrolysis of methylchloroacetate as a test reaction. The feasibility of observing reactions with initial half-lives of approximately 250 ms is demonstrated. The system is very robust, with little risk of damaging the optics during routine maintenance.

Stopped Flow Apparatus for Time-Resolved Fourier Transform Infrared Difference Spectroscopy of Biological Macromolecules in 1H2O

“…An alternative solution for stopped ow FT-IR spectroscopy in 1 H 2 O has been presented by Dunn et al, 29 using attenuated total re ection measurem ents to achieve a low effective optical path length while m aintaining a large ow cross-section. However, attenuated total re ection suffers from the complications of a wavelength-dependent optical path length, anomalous dispersion in the sample refractive index, and deviations from Beer's law for strongly absorbing bands.…”

Stopped-Flow Ultra-Rapid-Scanning Fourier Transform Infrared Spectroscopy on the Millisecond Time Scale

“…The temporal resolution of contemporary Fourier transform infrared (FT-IR) spectrometers 1 along with the rapid mixing time offered by stopped-flow (SF) instrumentation allows these techniques to be used for kinetic and mechanistic investigations of rapid chemical reaction processes. Over the past few decades, several research groups [2][3][4][5][6][7][8][9][10][11][12] have utilized SF techniques with conventional FT-IR spectrometers to study chemical reaction processes on time scales of 100 ms or more. However, the majority of recent advances in SF/FT-IR spectrometry have been focused on engineering of the mixing apparatus while using commercially available rapid-scan FT-IR spectrometers.…”

“…These short path lengths usually require high pressures inside the cell to achieve useful flow rates, a problem that was successfully addressed by White et al 2 Work by George and co-workers [3][4][5] showed that cells that are able to withstand fairly high pressures can be used for the study of bioinorganic systems, such as the reaction of H 2 , O 2 , and CO with [NiFe]-hydrogenase and nitrite with cytochrome c 0 . To circumvent problems caused by high pressures in short-pathlength transmission cells, Dunn et al 6 developed an attenuated total reflection stopped-flow cell for the investigation of aqueous biological systems that achieved a time resolution of 20 ms. Although most SF mixing instruments allow for the study of aqueous systems, transmission cells with longer path lengths can be used to study non-aqueous systems.…”

A Millisecond Infrared Stopped-Flow Apparatus

“…A variety of spectroscopic methods in conjunction with the stopped-flow initiation technique have been used in the study of the kinetics of chemical and biological processes. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] For example, stopped-flow fluorescence and circular dichroism (CD) are extensively used in the study of protein folding kinetics. [1][2][3][4] Among the commonly used stopped-flow methods (e.g., fluorescence, ultraviolet-visible (UV-Vis) absorbance, far-UV CD, small-angle X-ray scattering, infrared, and Raman), stopped-flow infrared (IR) is particularly appealing because of the high sensitivity of infrared spectroscopy to protein conformations.…”

“…[1][2][3][4] Among the commonly used stopped-flow methods (e.g., fluorescence, ultraviolet-visible (UV-Vis) absorbance, far-UV CD, small-angle X-ray scattering, infrared, and Raman), stopped-flow infrared (IR) is particularly appealing because of the high sensitivity of infrared spectroscopy to protein conformations. However, the existing stopped-flow infrared (IR) methods [5][6][7][8][9][10][11] are based on the rapid-scan mode of a Fourier transform (FT) spectrometer; therefore, their timeresolution is limited by the scanning rate of the interferometer (or the mechanical movement of the mirror). Depending on the specific FT-IR spectrometer used, the time resolution of such stopped-flow FT-IR instruments may vary, but it is typically around 60 to 80 ms. 11 While the time resolution can be greatly improved by using the continuous-flow method, 16,17 i.e., to the microsecond time scale, such continuous-flow IR methods suffer from high sample consumption and are normally limited to an observation time window of only a few milliseconds.…”