The Interference pattern from side-illuminated capillary tubes has been exploited to develop a sensitive universal refractive Index (RI) detector suitable for nanollter on-column capillary separation techniques. A 2-fold benefit Is obtained by surrounding the capillary tube with a RI-matching fluid: the fringe pattern Is simplified, and thermal noise Is reduced. The key to RI detection In CE, provided that the instrument delivers sufficient sensitivity, has been found to be thermal stability. A thermal stability of AT = 2.0 X 10"* °C is achieved In a Peltier cooled RI cell having a highly symmetric design aimed to ensure fast thermal response from the thermoelectric system. The linear dynamic range extends to more than 3 orders of magnitude with a typical RMS noise level of 3 X 10-* RIU and baseline drifts of 2 X 10-* RIU h1 at 1 Hz. This Is about 1 order of magnitude above the calculated shot noise limit for these tube dimensions. These noise levels correspond to an angular deflection of the selected fringe of 100 nrad, which has been obtained by using a large position-sensitive photodiode. The optical geometric arrangement of the photodiode, with respect to the fringe width, has been optimized theoretically. A detailed description of the Instrument and Its noise sources is presented, and the technique Is demonstrated In CE In the analysis of underlvatlzed carbohydrates using a 50-jim tube.

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A novel refractive index detector suitable for capillary separations is presented and demonstrated in the capillary electrophoretic separation of metal cations in 10-itm-i.d. tubes. The principle of the detector is interferometric; it features a laser diode and, as the main optical element, a holographic optical element which performs several optical functions allowing the use of capillaries with inner diameters as small as 5 /*m. In contrast to the "off-axis" method, the use of holographic plates permits probing of the capillary through its center, where the optical path is larger and diffraction effects are smaller. With an i.d. = 10 /tm capillary the detector resolves 2 juRIU and covers a linear dynamic range of three to four decades. Joule heat effects preclude the detector to operate at its maximum sensitivity which is delivered in the absence of electrical fields. For this reason, the use of smaller inner diameter capillaries and buffers having low conductivities is recommended. Under field-amplified sample injection conditions, the detection limits for the studied cations are in the low nanomolar range. The performance of this detector is compared with indirect UV-absorption detection, which also helps to elucidate the mechanisms leading to refractive index changes in ionic solutions.Considering that holographic optical elements (HOEs) could greatly simplify the construction of optical instruments without compromising performance, their use is gaining acceptance in analytical instrumentation such as spectrographs.1-2 The present investigation represents the first application of HOEs to on-column refractive index (RI) detection for the analysis of transparent substances using capillary electrophoresis (CE).Optically dense compounds are most commonly detected by absorbance methods, and when the substances of interest * Author to whom correspondence should be addressed.(1) Tedesco,

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On-column thermooptical absorbance (TOA) detection in capillary electrophoretic separations of various nucleoside and mono-and diphosphate nucleotide mixtures absorbing at 257 nm is demonstrated in 20 fim i.d. capillaries. The analytes are optically pumped by a frequency-doubled argon ion laser and probed by a laser diode or by a He/ Ne laser beam guided to the detection volume by a holographic optical element Absorption detection limits of 2.2 //AU using time constants of 0.3 s and 20 mW of UV power are obtained over a linear dynamic range covering three to four decades. As higher pumping power is required to enhance the thermooptical sensitivity, photobleaching appears as a major problem in the quest for lower detection limits for some of the substances studied such as deoxyuridine and uridine. Concentration detection limits as low as 50 nM for adenosine monophosphate, corresponding to a mass detection limit of 0.4 finol, and separation efficiencies up to 320 000 theoretical plates are measured. A theoretical model, which translates the obtained TOA signals into absorbances, is proposed and describes the TOA effect for smaller capillaries rather well.Various on-column optical detection methods have been devised for capillary electrophoresis (CE). When the sample of interest contains a fluorescent chromophore that coincides with a lasing line, laser-induced fluorescence (UF) is undoubtedly the best choice. Using this method, concentration detection limits (LODs) as low as 10"10 M (0.2 am injected) for native bovine serum albumin (BSA) have been reported.* 1 However, as the number of naturally fluorescent samples is small, and the laser emission wavelengths are limited, this case should be considered as an exception rather than the rule.Concentration LODs in the 10-11 M range, or lower, are obtained in CE using precolumn labeling with fluorescent tags and LIF detection. However, direct derivatizations (samples are first derivatized at high concentrations and then diluted to the impressive low levels reported) below the 10"' M range have not been reported to date. Furthermore, derivatization procedures are not always available for the substance of interest or are often rather tedious and, when available, often lead to quantification problems because the extent of derivatization is not always

The fringe pattern observed in a far field after a laser beam illuminates a fused silica capillary immersed in a refractive-index matching material and filled with an analyte fluid is exploited to develop a sensitive optical detector for capillary chemical analysis. The inner capillary interface splits the laser beam into a reflected beam fan and a refracted beam fan, which, on overlapping in the far field, lead to interferences. The intensity and the position of the fringes for capillaries with 250 microm >/= i.d. (inner diameter) >/= 25 microm are well reproduced by the presented model. The calculation predicts the fringe pattern for various beam/i.d. geometric configurations and is used to optimize the performance of the nanoliter-picoliter refractive-index on-column detection studied. It is found that the best contrast corresponds to a capillary that is illuminated with a beam waist of omega(0) ~ i.d./12, which is off-center focused with an offset of s ~ i.d./2. For a given interference pattern, the fringes that are found to be more sensitive to Deltan are those that appear near the optical axis but still retain high intensity and contrast. The sensitivity increases approximately linearly with the fringe number, and the maximal fringe number increases proportionally with the i.d.

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