Steady-state and transient intracapillary temperature gradients are measured by Raman microthermometry during capillary zone electrophoresis. The axial temperature gradient away from a heat sink extends for several millimeters, depending upon the contact of the capillary and heat sink. In free-air convection, small radial gradients, 2-4 degrees C from the center of the lumen to the wall, are observed at 0.85 kW/cm3. The temperature profile is adequately described by a parabola. With forced cooling, the center-wall temperature difference is less than 1 degrees C. The time to steady-state temperature after power-on is found to be 20 s. The measurements are compared to the results of heat transport calculations.

A practical methodology is described that allows measurement of spatial resolution and sensitivity of Raman spectroscopy in backscatter and transmission modes under conditions where photon migration dominates, i.e., with turbid or opaque samples. For the first time under such conditions the width and intensity of the point spread function (PSF) has been accurately measured as a function of sample thickness and depth below the surface. In transmission mode, the lateral resolution for objects in the bulk degraded linearly with sample thickness, but the resolution was much better for objects near either surface, being determined by the diameter of the probe beam and collection aperture irrespective of sample thickness. In other words, buried objects appear to be larger than ones near either surface. The absolute transmitted signal decreased significantly with sample thickness, but objects in the bulk yielded higher signals than those at either surface. In transmission, materials are sampled preferentially in the bulk, which has ramifications for quantitative analysis. In backscattering mode, objects near the probed surface were detected much more effectively than in the bulk, and the resolution worsened linearly with depth below the surface. These results are highly relevant in circumstances in which one is trying to detect or image buried objects in opaque media, for example Raman tomography of biological tissues or compositional and structural analysis of pharmaceutical tablets. Finally, the observations were in good agreement with Monte Carlo simulations and, provided one is in the diffusion regime, were insensitive to the choice of transport length, which shows that a simple model can be used to predict instrument performance for a given excitation and collection geometry.

Local temperatures inside a 75-microns-i.d. capillary under electrophoresis conditions are measured noninvasively with a Raman microprobe. The method is based on the temperature dependence of the water O-H stretch equilibrium between weakly bent and strongly bent hydrogen bonded species. With calibration against a known temperature standard, this technique is shown to be capable of obtaining spatially resolved intracapillary temperature measurements with an accuracy of +/- 1.0 degree C and a precision of +/- 0.1 degree C. Intracapillary temperatures ranging from 25 to 70 degrees C are observed over the range of buffer compositions and electric fields used. Differences between local temperatures and average capillary temperatures are observed at all operating conditions. The difficulty of an accurate theoretical description of heat dissipation under CE conditions is discussed.