Probe small volume probes

G.E. Martin, R.C. Crouch and A.P. Zens, Magn. Reson. Chem., 36 (1998) 551-557 for 1.7 mm sensitivity G. Schlotterbeck, A. Ross, R. Hoch-strasser, H. Senn, T. Kuhn, D. Marek and O. Schett, Anal. Chem., 74 (2002) 4464 for 1mm Bruker probe performance M.E. Lacey, R. Subra-manian, D.L. Olsen, A.G. Webb and J.V. Sweedler, Chem. Rev., 99 (1999) 3133-3152 General review of small volume probe performance. 

Fig. 6.59. Fluid sampling probes (a) normal sample probe (b) small volume probe (c) gas-sampling probe (d) water-wash probe for furnace...

Volume and nature of the transmitting liquid. The volume of transmitting liquid is another variable to be considered with ultrasonic baths and also when the sample reservoir is immersed in a thermostated bath in the case of probes. Small volumes of the transmitting liquid make direct impingement of the ultrasonic probe on the sample microcell redundant. With large volumes, US efficiency is decreased through scatter of the ultrasonic energy in the bulk solution. 

The limitations of SIMS - some inherent in secondary ion formation, some because of the physics of ion beams, and some because of the nature of sputtering - have been mentioned in Sect. 3.1. Sputtering produces predominantly neutral atoms for most of the elements in the periodic table the typical secondary ion yield is between 10 and 10 . This leads to a serious sensitivity limitation when extremely small volumes must be probed, or when high lateral and depth resolution analyses are needed. Another problem arises because the secondary ion yield can vary by many orders of magnitude as a function of surface contamination and matrix composition this hampers quantification. Quantification can also be hampered by interferences from molecules, molecular fragments, and isotopes of other elements with the same mass as the analyte. Very high mass-resolution can reject such interferences but only at the expense of detection sensitivity. 

Surface analysis by non-resonant (NR-) laser-SNMS [3.102-3.106] has been used to improve ionization efficiency while retaining the advantages of probing the neutral component. In NR-laser-SNMS, an intense laser beam is used to ionize, non-selec-tively, all atoms and molecules within the volume intersected by the laser beam (Eig. 3.40b). With sufficient laser power density it is possible to saturate the ionization process. Eor NR-laser-SNMS adequate power densities are typically achieved in a small volume only at the focus of the laser beam. This limits sensitivity and leads to problems with quantification, because of the differences between the effective ionization volumes of different elements. The non-resonant post-ionization technique provides rapid, multi-element, and molecular survey measurements with significantly improved ionization efficiency over SIMS, although it still suffers from isoba-ric interferences. 

Since the early 1980s, the study of mechanical properties of materials on the nanometre scale has received much attention, as these properties are size dependent. The nanoindentation and nanoscratch are the important techniques for probing mechanical properties of materials in small volumes. Indentation load-displacement data contain a wealth of information. From the load-displacement data, many mechanical properties such as hardness and elastic modulus can be determined. The nanoindenter has also been used to measure the fracture toughness and fatigue properties of ul-... 

Flowever, in order to probe the fracture toughness of thin films or small volumes using ultra low load indentation, it is necessary to use special indenters with cracking thresholds lower than those observed with the Vickers or Berkovich indenters (for Vickers and Berkovich indenters, cracking... 

The second type of test involves driving or pushing a porous probe into the soil and pouring water through the probe into the soil. With this method, however, the advantage of testing directly in the field is somewhat offset by the limitations of testing such a small volume of soil. 

There are two important drawbacks of such an approach (1) a polarity scale based on a particular class of probes, in principle, does not account, for example, sizes of probes, which should strongly effect the interactions (2) betain dyes do not fluoresce, which restrict essentially the field of application of this approach, because in many cases, absorption spectrum could not be measured accurately (small volumes of samples, study of cells, and single molecules spectroscopy). Therefore, polarity-sensitive fluorescent dyes offer distinct advantage in many applications. 

The RPA is a sensitive method for quantifying specific RNAs from a mixture of RNAs. This is achieved using a small-volume hybridization of an RNA probe to the RNA under study. Unhybridized probe and sample is then digested with RNAses and the protected probe fragment is visualized after denaturing gel electrophoresis. Commonly, the probe is radiolabeled for maximum sensitivity. Following is a method for RPA detection of R-luc-4 sites and F-luc mRNA. 

Smaller diameter probes reduce sample volumes from 500 to 600 pi typical with a 5 mm probe down to 120-160 pi with a 3 mm tube. By reducing the sample volume, the relative concentration of the sample can be correspondingly increased for non-solubility limited samples. This dramatically reduces data acquisition times when more abundant samples are available or sample quantity requirements when dealing with scarce samples. At present, the smallest commercially available NMR tubes have a diameter of 1.0 mm and allow the acquisition of heteronuclear shift correlation experiments on samples as small as 1 pg of material, for example in the case of the small drug molecule, ibu-profen [5]. In addition to conventional tube-based NMR probes, there are also a number of other types of small volume NMR probes and flow probes commercially available [6]. Here again, the primary application of these probes is the reduction of sample requirements to facilitate the structural characterization of mass limited samples. Overall, many probe options are available to optimize the NMR hardware configuration for the type and amount of sample, its solubility, the nucleus to be detected as well as the type and number of experiments to be run. 

Let us dwell on Figure 6.4 for a moment. The standards and sample solutions are introduced to the instrument in a variety of ways. In the case of a pH meter and other electroanalytical instruments, the tips of one or two probes are immersed in the solution. In the case of an automatic digital Abbe refractometer (Chapter 15), a small quantity of the solution is placed on a prism at the bottom of a sample well inside the instrument. In an ordinary spectrophotometer (Chapters 7 and 8), the solution is held in a round (like a test tube) or square container called a cuvette, which fits in a holder inside the instrument. In an atomic absorption spectrophotometer (Chapter 9), or in instruments utilizing an autosampler, the solution is sucked or aspirated into the instrument from an external container. In a chromatograph (Chapters 12 and 13), the solution is injected into the instrument with the use of a small-volume syringe. Once inside, or otherwise in contact with the instrument, the instrument is designed to act on the solution. We now address the processes that occur inside the instrument in order to produce the electrical signal that is seen at the readout. 

The method of introduction of the fluorophore into the membrane is also important. Many probes are introduced into preexisting vesicles, natural membranes, or whole cells by the injection of a small volume of organic solvent containing the fluorophore. For DPH, tetrahydrofuran is commonly used, while methanol is often employed for other probes. The amount of solvent used should be the absolute minimum possible to avoid perturbation of the lipids, since the solvent will also partition into the membrane. With lipid vesicles this potential problem can be avoided by mixing the lipids and fluorophore followed by evaporation of the solvent and codispersing in buffer. For fluorophores attached to phospholipids, this is the only way to get the fluorophore into the bilayer with natural membranes, phospholipid exchange proteins or other techniques may have to be employed. 

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