Evanescent-wave cavity ring-down spectroscopy for enhanced detection of surface binding under flow injection analysis conditions.

The feasibility of liquid-phase evanescent-wave cavity ring-down spectroscopy (EW-CRDS) for surface-binding studies under flow-injection analysis (FIA) conditions is demonstrated. The EW-CRDS setup consists of an anti-reflection coated Dove prism inside a linear cavity (with standard or super-polishing of the total internal reflective (TIR) surface). A teflon spacer with an elliptical hole clamped on this surface acts as a 20 muL sized flow cell. The baseline noise of this system is of the order of 10(-4) absorbance units; the baseline remains stable over a prolonged time and the prism surface does not become contaminated during repeated injections of the reversibly adsorbing test dyes Crystal Violet (CV) and Direct Red 10 (DR10). At typical FIA or liquid chromatography (LC) flow rates, the system has sufficient specificity to discriminate between species with different surface affinities. For CV a much stronger decrease in ring-down time is observed than calculated based on its bulk concentration and the effective depth probed by the evanescent wave, indicating binding of this positively charged dye to the negatively charged prism surface. The amount of adsorption can be influenced by adjusting the flow rate or the eluent composition. At a flow rate of 0.5 mL/min, an enrichment factor of 60 was calculated for CV; for the poorly adsorbing dye DR10 it is 5. Super-polishing of the already polished TIR surface works counter-productively. The adsorbing dye Crystal Violet has a detection limit of 3 muM for the standard polished surface; less binding occurs on the super-polished surface and the detection limit is 5 muM. Possible applications of EW-CRDS for studying surface binding or the development of bio-assays are discussed.

Cavity ring-down spectroscopy for detection in liquid chromatography at UV wavelengths using standard cuvettes in a normal incidence geometry.

Liquid chromatography (LC) with cavity ring-down spectroscopy (CRDS) detection, using flow cuvettes (put under normal incidence inside the ring-down cavity), is demonstrated. Fresnel reflections are maintained within the capture range of a stable cavity of 4 cm length. This method circumvents the need for specific Brewster's angles and possible mirror degradation is avoided. The flow cuvettes are commercially available at low cost. At 355 nm (the frequency-tripled output of a Nd:YAG laser), the system surpasses the performance of conventional absorbance detectors; the baseline noise was 1.3 x 10(-5)AU and detection limits (injected concentrations) were between 40 and 80 nM for nitro-polyaromatic hydrocarbons with an extinction coefficient epsilon of 7.3-10.2 x 10(3)M(-1)cm(-1). The system was also tested at 273 nm, but in the deep UV the reflectivity of the currently best available mirrors (R>or=99.91%) is still too low to show a significant improvement as compared to conventional UV-vis detection.

Cavity ring-down spectroscopy for detection in liquid chromatography: extension to tunable sources and ultraviolet wavelengths.

In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid chromatography (LC) can be improved significantly by using cavity ring-down spectroscopy (CRDS). Thus far, CRDS experiments have been performed using visible laser light at fixed standard wavelengths, such as 532 nm. However, since by far most compounds of analytical interest absorb in the ultraviolet (UV), it is of utmost importance to develop UV-CRDS. In this study, as a first step towards the deep-UV region, LC separations with CRDS detection (using a previously described liquid-only cavity flow cell) at 457 and 355 nm are reported for standard mixtures of dyes and nitro-polyaromatic hydrocarbons (nitro-PAHs), respectively. For the measurements in the blue range a home-built optical parametric oscillator (OPO) system, tunable between 425 and 478 nm, was used, achieving a baseline noise of 2.7 x 10(-6) A.U. at 457 nm, improving upon the sensitivity of conventional absorbance detection (typically around 10(-4) A.U.). An enhancement of the sensitivity can be seen at 355 nm as well, but the improvement of the baseline noise (1.3 x 10(-5) A.U.) is much less pronounced. The sensitivity at 355 nm is limited by the quality of the UV-CRDS mirrors that are currently available: whereas the ring-down times as obtained at 457 nm are around 70-80 ns for the eluent, they are only 20-25 ns at 355 nm. Critical laser characteristics for LC-CRDS measurements, such as pulse length and mode structure, are given and prospects for going to shorter wavelengths are discussed.

Liquid-phase and evanescent-wave cavity ring-down spectroscopy in analytical chemistry.

Due to its simplicity, versatility, and straightforward interpretation into absolute concentrations, molecular absorbance detection is widely used in liquid-phase analytical chemistry. Because this method is inherently less sensitive than zero-background techniques such as fluorescence detection, alternative, more sensitive measurement principles are being explored. This review discusses one of these: cavity ring-down spectroscopy (CRDS). Advantages of this technique include its long measurement pathlength and its insensitivity to light-source-intensity fluctuations. CRDS is already a well-established technique in the gas phase, so we focus on two new modes: liquid-phase CRDS and evanescent-wave (EW)-CRDS. Applications of liquid-phase CRDS in analytical chemistry focus on improving the sensitivity of absorbance detection in liquid chromatography. Currently, EW-CRDS is still in early stages: It is used to study basic interactions between molecules and silica surfaces. However, in the future this method may be used to develop, for instance, biosensors with high specificity.

Miniaturized cavity ring-down detection in a liquid flow cell.

A novel method for applying cavity ring-down spectroscopy in the liquid phase, compatible with LC analyses, is presented. The core of the setup is a home-built cavity ring-down flow cell (cell volume 12 microL) that is constructed using a silicon rubber spacer, which is clamped leak-tight between two high-reflectivity mirrors. The mirrors are in direct contact with the liquid flow, which provides for a small path length and short ring-down times. Inside the cavity there are no windows, reflection losses, or Brewster angles to be considered. Due to the small size of the presented cavity geometry, the setup can be implemented in conventional-size LC apparatuses. With a flow injection setup, a detection limit of 2.5 nM was obtained for Crystal Violet in ethanol, and the linear dynamic range of the system is at least 2 orders of magnitude. The method has the potential to become a powerful alternative for commercial LC UV/visible absorbance detectors.