RESUMO
The manuscript explores some advantages and limitations of laser based optical spectroscopy, aimed at achieving robust, high-reproducibility 13C16O2 and 12C16O2 ratio determinations on the VPDB-CO2 δ13C scale by measuring the absorbance of line pairs of 13C16O2 and 12C16O2. In particular, the sensitivities of spectroscopic lines to both pressure (P) and temperature (T) are discussed. Based on the considerations and estimations presented, a level of reproducibility of the 13C16O2/12C16O2 ratio determinations may be achieved of about 10-6. Thus one may establish an optical spectroscopic measurement technique for robust, high-precision 13C16O2 and 12C16O2 ratio measurements aimed at very low uncertainty. (Notably, creating such an optical instrument and developing technical solutions is beyond the scope of this paper.) The total combined uncertainty will also include the uncertainty component(s) related to the accuracy of calibration on the VPDB-CO2 δ13C scale. Addressing high-accuracy calibrations is presently not straightforward - absolute numerical values of 13C/12C for the VPDB-CO2 scale are not well known. Traditional stable isotope mass-spectrometry uses calibrations vs CO2 evolved from the primary carbonate reference materials; which can hardly be used for calibrating commercial optical stable isotope analysers. In contrast to mass-spectrometry, the major advantage of the laser-based spectrometric technique detailed in this paper is its high robustness. Therefore one can introduce a new spectrometric δ13C characterisation method which, being once well-calibrated on the VPDB-CO2 scale, may not require any further (re-)calibrations. This can be used for characterisation of δ13C in CO2-in-air mixtures with high precision and also with high accuracy. If this technique can be realised with the estimated long-term reproducibility (order of 10-6), it could potentially serve as a more convenient Optical Transfer Standard (OTS), characterising large amounts of CO2 gas mixtures on the VPDB-CO2 δ13C scale without having to compare to carbonate-evolved CO2. Furthermore, if the OTS method proves to be successful, it might be considered for re-defining the VPDB-CO2 δ13C-scale as the ratio of selected CO2 spectroscopic absorbance lines measured at pre-defined T & P conditions. The approach can also be expanded to δ18O characterisation (using 16O12C18O and 16O12C16O absorbance lines) of CO2 gas mixtures and potentially to other isotope ratios of other gases.
RESUMO
Thermal lensing (TL) permits ultra-sensitive measurements of optical absorption of analytes in very small liquid volumes. We report the construction and use of a TL detector based on pulsed ultraviolet (UV) laser excitation (266 nm). We applied this detector to quantitate amino acids using capillary electrophoresis (CE) as a means of separation. Sixteen individual amino acids are readily detected, but the signal has a complex dependence on intensity caused by the combination of (1) one-photon absorption; (2) two-photon absorption (TPA); and (3) photodestruction of amino acid molecules in the focus of the laser beam. An aqueous solution containing tyrosine, tryptophan, and cysteine is electrophoretically separated and the individual amino acids are detected by UV TL. The estimated limit of detection is 7 microM for tyrosine, 2.5 microM for tryptophan and 33 microM for cysterine, which translates into 0.35 fmol for tyrosine, 0.125 fmol for tryptophan, and 1.65 fmol for cysteine in the 140pL detection volume. It is found that two-photon absorption of water and the formation of color centers in the fused silica walls of the flowcell can contribute a significant, drifting background signal, but this interference can be minimized by selecting an appropriate focus condition and excitation-detection geometry. We suggest that as UV laser sources become available, UV TL may become a method of choice for measuring the concentrations of many analytes in different separation formats in which the volume is highly limited.