Detection volumes, separations

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

Hyphenation of chromatographic separation techniques (SFC, HPLC, SEC) with NMR spectroscopy as a universal detector is one of the most powerful and time-saving new methods for separation and structural elucidation of unknown compounds and molecular compositions of mixtures [171]. Most of the routinely used NMR flow-cells have detection volumes between 40... 

Figure 4.7 shows the structures of important carotenoids (all-E) lutein, (all-E) zeaxanthin, (all-E) canthaxanthin, (all-E) p-carotene, and (all-E) lycopene. Employing a self-packed C30 capillary column, the carotenoids can be separated with a solvent gradient of acetone water=80 20 (v/v) to 99 1 (v/v) and a flow rate of 5 pL min, as shown in Figure 4.8 (Putzbach et al. 2005). The more polar carotenoids (all-E) lutein, (all-E) zeaxanthin, and (all-E) canthaxanthin elute first followed by the less polar (all-E) p-carotene and the nonpolar (all-E) lycopene. Figure 4.9 shows the stopped-flow II NMR spectra of these five carotenoids. The chromatographic run was stopped when the peak maximum of the compound of interest reached the NMR probe detection volume.

The third point listed above is of utmost importance if the theoretically expected improvement in separation performance should be exploitable with a real instrument. Apart from the inherent contribution of the separation column itself (variance crcol2), overall band broadening (a2) is also caused by the sample injector (crin2), the finite length of the detection volume (adet2), and by any contributions of dead volumes (adv2) due to fittings etc. (see for example [22] ... 

More recently, the description of a small-bore liquid chromatography by Manz et al. [83] on a 5 x 5 mm silicon chip revived the interest of the analytical chemical community. The chip incorporated an open tubular column of 6x2 pm cross section (column volume 1.8 nl) and a conductometric detector with a detection volume of only 1.2 pi. Although this work was an important benchmark study, the actual functioning of the chromatograph was never demonstrated with separations. 

Altria et al. reported the CE separation and detection of radiopharmaceuticals containing mTc, a 7 emitter with a 6-hour half-life (2, see also 10). Their design involved passing a capillary tube (= 2 cm long) through a solid block of scintillator material and detecting the light emitted as technetium-labeled sample zones traversed the detection volume. Unfortunately, detection limits and detector efficiency were not reported. 

The CARS system used to measure temperature and species concentrations in the combustor zone is composed of a single-mode ruby-laser oscillator-amplifier with a repetition rate of 1 Hz and a ruby-pumped, near-infrared broad-band dye laser. The two laser beams are combined collinearly and focused first into a cell containing a nonresonant reference gas and then into the sample volume (approximately 30-u diam. x 2 cm) in the combustion region. The anti-Stokes beams produced in the sample and reference volumes are directed to spatially separated foci on the entrance slit of a spectrometer and detected by separate photomultiplier tubes. An optional means of detection is provided for the sample signal in the form of an optical multichannel analyzer (OMA), which makes it possible to obtain single-pulse CARS spectra. 

Alternatively, in the absence of a significant change in neither diffusion properties nor fluorescence brightness as a consequence of the reaction taking place, various cross-correlation approaches can be applied (Fig. 8.1c). For this purpose, cross-correlation FCS has been introduced correlating different detection channels, separated with respect to emission wavelength range [16] or spatial localization of the observation volume [18]. 

Figure 11.2. Separation of Cr(IIl) and Cr(VI) by valveless IC and JCP-MS detection. The separation is affected by both the volume of sample introduced to the column and the speed of the peristaltic pump. The eluent was 0.35 % (w/w) nitric acid adjusted to pH 1.6 with ammonium hydroxide. The column is a low-capacity anion exchanger ANX3202 with dimensions of 3.2 x 20 mm. Detection limits for Cr(IlI) and Cr(Vl) are both <0.1 ppb. Courtesy of Transgenomic, Inc., Omaha, NE.

Online coupling of HPLC with nuclear magnetic resonance spectroscopy (NMR) has proved useful for a wide range of applications. The shortcoming of suppression of eluent signals can be circumvented by use of capillary separation technique. In this mode detection cells with internal volumes in the nanoliter scale and miniaturized probe heads have been developed by Albert et al. in Tuebingen. The system can be used in either HPLC, CE, or CEC, and consists of a capillary inserted into a 2.5 or 2.0 mm NMR microprobe equipped with a Helmholtz coil. In experiments, a capillary mbe of 315 /rm can create a detection volume of 900 nl. The flow rate of the capillary... 

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