The Limits of Miniaturization

Columns can still be operated effectively down to 35 mm diameter when filled with an ordered packing. For plate columns having one bubble cap per plate, the limit is about 50 mm. At still smaller dimensions, wall effects are difficult to suppress. 

Experimental equipment for handling solids or dealing with extreme operating conditions is often not available. This often necessitates in-house developments and cooperation with specialist firms, university institutes, and research companies. 

Columns 30 mm diameter, packed column 35 nun diameter, structured ordered packings, dual-flow columns 50 nun diameter, bubble-cap plates 

Pumps 1 mL h jet pumps 10 mL h piston pumps 100 L h rotary pumps 

Measured quantity Measurement principle Tested minimal measurement range 

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The assertions that science and engineering of the 21st century will acquire a nano and angstrom character have proved to be a reality. The limits of miniaturization of separate elements (e.g., density of arranging crystals in microelectronics)... 

An impressive example of the impact of miniaturization on the explosion limit has been given for the oxyhydrogen reaction [18]. For a conventional reactor of 1 m diameter, explosive behavior sets in at 420 °C at ambient pressure (10 Pa). An explosion occurs at about 750 °C, when the reactor diameter is decreased to about 1 mm. A further reduction to 100 pm shifts the explosive regime further to higher pressures and temperatures. 

In the first two contributions electroanalytical techniques are described for application in bioanalysis and medicine. The increasing interest in this field is mainly due to the excellent selectivities and detection limits. In addition, the possibilities of miniaturization allow the development of in vivo analysis. 

The use of robotics can be adopted also in sample preparation steps, in particular on-line SPE [7], This necessity is particular evident when small quantity of starting materials is available and the target molecules are present at low concentration levels. With the advent of miniaturization and automated procedures for samples handling, treatments and analysis, the lost of analytes due to a laboratory steps can be reduced. The reduction of analyte losses and the possibility to analyze even a total sample (no loss) leads to lower limits of detection (and consequently lower limits of quantification). Smaller volumes bring to obtain adequate sensitivity and selectivity for a large variety of compounds. In addition, on-line SPE requires low solvent consumption without the need to remove all residual water from cartridges, since elution solvents are compatible with the separation methods. 

Research has been done showing that rapid pressnre-driven LC analysis can be done with little solvent consumption, demonstrating this as a viable process analytical tool. Using electrokinetic nanoflow pumps LC can be miniaturized to the point of being a sensor system. Developments in terms of sampling to enable sampling directly from a process stream, to the separation channel on a chip are critical for the application of miniaturized process LC. The components (valves and pumps) required for hydrodynamic flow systems appear to be a current limitation to the fnll miniatnrization of LC separations. Detection systems have also evolved with electrochemical detection and refractive index detection systems providing increased sensitivity in miniaturized systems when compared to standard UV-vis detection or fluorescence, which may require precolumn derivatization. 

Several successful attempts were done to transfer classical CEIA to a microchip-based format. This kind of miniaturization is a trend that can overcome the limitations of CE in high-throughput systems. On-chip CE offers both parallel analysis of samples and short separation times. Koutny et al. showed the use of an immunoassay on-chip (32). In this competitive approach fluorescein-labeled cortisol was used to detect unlabeled cortisol spiked to serum (Fig. 8). The system showed good reproducibility and robustness even in this problematic kind of sample matrix. Using serum cortisol standards calibration and quantification is possible in a working range of clinical interest. This example demonstrated that microchip electrophoretic systems are analytical devices suitable for immunological assays that can compete with common techniques. 

Even the lawn format still does not represent the final limit of miniaturization. The one well/one catalyst or one bead/one catalyst strategy, where catalyst identity is spatially coded, can be replaced by in situ synthesis combined with mass spectrometry [48]. The advantage of this strategy is the use of a mass spectrometer for the synthesis, reaction and analysis. The described electrospray ionization procedure helps to avoid the cleavage of chemical bonds, which would falsify the results. The synthesis step does not have to deliver clean and isolated products. Instead, after synthesis, the reactants are first separated by a quadrupole. In a second step, they are further reacted in an octapole and the reaction products are finally isolated in a second quadrupole and analyzed. Figure 3.15 describes the screening process in detail [49],... 

The luminescent ATP assay is the most sensitive HTS method available for measuring the viability of cell populations in microwell plates. The limits of detection determined in samples of eukaryotic cells serially diluted from a known concentration may fall below 10 cells per well (Figure 6.7). This enables miniaturization to a 1536-well format. 

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