Sampling picoliter

Titrations conducted with microliter or picoliter sample volumes require a smaller absolute amount of analyte. For example, diffusional titrations have been successfully conducted on as little as 29 femtomoles (10 mol) of nitric acid. Nevertheless, the analyte must still be present in the sample at a major or minor level for the titration to be performed accurately and precisely. 

In the last decade, capillary electrophoresis (CE) has become one of the most powerful and conceptually simple separation techniques for the analysis of complex mixtures. The main reasons are its high resolution, relatively short analysis times, and low operational cost when compared to high-performance liquid chromatography (HPLC). The ability to analyze ultrasmall volume samples in the picoliter-to-nanoliter ranges makes it an ideal analytical method for extremely volume-limited biological microenvironments. 

K. Hosokawa, T. Fujii, and I. Endo, Handling of picoliter liquid samples in a poly(dimethylsiloxane)-based microlluidic device, Anal. Chem. 71, 1781 1785 (1999). 

In the diffusive interfacial transport-refractive index (DIT-NDX) method, compositions are determined using precise refractive index data (8). Refractive index data valid to +/- 0.00005 are obtainable using the DIT apparatus vithin an area of 30 ym2 in a sample approximately 25-ym thick (0.75 picoliter volume). Data collection and analysis require 9 seconds. The accuracy, spatial resolution, and speed vith vhich refractive indices can be determined are thus superb. 

Application of a potential between reservoirs 1 (sample) and 4 (injection waste) electrokinetically pumps sample solution as indicated in Fig. 3. In this way, a geometrically defined 150 pm (90 pi) section of the separation channel can be filled [19]. If the injection potential is applied long enough to ensure that even the slowest sample component has completely filled the injection volume, a representative aliquot of sample can be analyzed (so-called volume defined injection). This is in contrast to electrokinetic sample injection in conventional capillaries, which is known to bias the sample according to the respective ionic mobilities [61]. These characteristic differences are shown schematically in Fig. 4. It should be noted that this picoliter sample injector is exclusively controlled by the application of electric fields and does not require any active elements with moving parts such as valves and external pumps. The reproducibility of the peak height of the injected sample plugs has been reported to be within 2 % RSD (relative standard deviation) and less [19,23]. 

Onnerfjord, P., Nilsson, J., Wallman, L. et al. (1998) Picoliter sample preparation in MALDI-TOF MS using a micromachined silicon flow-through dispenser. Anal. Chem., 70(22) 4755-60. 

Separation of simple mixtures [13,44] to more complicated biofluidic mixtures with CE-NMR and CEC-NMR have been reported [51,52], Several instrumental modifications and methodologies have been described to use CE/CEC-NMR as a diagnostic tool [44,53-55], Though still in its infancy, chip-based CE-NMR with microfabricated microcoils may be able to analyze picoliter volume samples [56,57]. 

A schematic diagram of a typical CE instrument is shown in Figure 5-6. As indicated, the capillary tube serves as an electrophoretic chamber that is connected to a detector at its terminal end and, via buffer reservoirs, to a high-voltage power supply. Improved heat dissipation permits the application of voltages in the range of 10 to 30 kV, which enhances separation efficiency and reduces separation time in some cases to less than 1 minute. Only a few microliters of the sample are required, with volumes in the picoliter to nanoliter range actually used. This minimizes distortions in the applied field caused by the presence of samples. 

FIGURE 11.1 (See color insert following page 210.) General overview of MALDI-IMS. (a) Fresh section cut from sample tissue, (b) Mounted section after matrix application using a robotic picoliter volume spotter, (c) Partial series of mass spectra collected along one row of coordinates U-axis). (d) Three-dimensional volumetric plot of complete dataset with selected m/z slices or ion images. Principal axes are x, y, and m/z. Color of each voxel is determined by ion intensity. (From Cornett, D. et al., Nat. Methods, 4, 828, 2007. With permission.)... 

Miniaturization of the platform is required to scale down the process of protein analysis, which involves handling a much smaller amount of the sample. It may soon be possible to scale down the size of the sample from a nanoliter to a picoliter, which reduces the amount of protein from fetomoles to attomoles. Miniaturization will require use of microfluidics to handle a miniscule of sample without any loss by absorption or by evaporation. In this way, it will be possible to analyze the protein sample on a protein chip. 

Based on the electroosmotic flow, metering of volumes down to the picoliter range can be achieved. While the sample liquid is injected and crosses an intersection point of two perpendicular channels, the electrodes... 

The aim of HTS and UHTS is cost-effectiveness and speed of compound scanning. Hence, the robotics system not only has to deliver fast and accurate liquid samples into the wells, it has to be miniaturized to conserve the volumes required of the valuable samples and expensive assay reagents. For the 1536 wells, the liquids being dispensed are in the nanoliter (10-9 L) to picoliter (Kh12 L) range. The ink jet technology provided the technological basis for liquid dispensation in HTS. 

If the sample concentration is small compared to the buffer concentration, then the current is carried mostly by the buffer, so minimal broadening occurs from conductivity changes. Because of the small volume, little convective diffrision occurs from heating. The system is fast, usually requiring only a few minutes the separation is excellent, up to 400,000 plates/meter and only a few nL of sample are required. Compounds with mobility differences of lO" cm /cm have been separated. One picoliter (pL) can be detected in favorable cases. This is truly a micromethod. In addition, it is easy to automate. The column can be purged in a few seconds, new buffer added, and another sample injected automatically. 

Detection is a continuing problem because the sample volume passing through the detector at any one time is so small, a few picoliters (pL). A sample concentration of 10 M is the normal limit, but one method has been demonstrated to detect 10 M materials uv spectroscopy, fluorescence, Raman, and electroconductivity are used for detection. 

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148) 802-805 Chancellor EB, Wikswo JP, Baudenbacher E (2004) Heat conduction calorimeter for massively parallel high throughput measuremeuts wit picoliter sample volumes. Appl Phys Let 85 2408-2410... 

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