Micropipettes, Preparation

Apparatus. Prepared silica gel plates. Chromatographic tank (see Fig. 8.6). Drummond (or similar) micropipette. 

Procedure. Pour the developing solvent into the chromatographic tank to a depth of about 0.5 cm and replace the lid. Take a prepared plate and carefully spot 5 pL of each indicator on the origin line (see Section 8.6, under Sample application) using a micropipette. Allow to dry, slide the plate into the tank and develop the chromatogram by the ascending solvent for about 1 h. Remove the plate, mark the solvent front and dry the plate in an oven at 60 °C for about 15 min. Evaluate the R value for each of the indicators using the equation... 

Fig. 21.2. Two-microelectrode current-clamp technique used to observe, in single Ascaris body muscle cells in a body-flap preparation, the response to a controlled pulsed application of levamisole. One micropipette, to measure membrane potential, and another micropipette, to inject current, are inserted inside the area of the muscle cell known as the bag region. Levamisole is applied in a time- and pressure-controlled manner from a microcatheter placed over the bag region of the muscle. A second microcatheter is used to apply additional chemical agents (Martin, 1982).

Avermectin-sensitive sites in A. mum have been identified on pharyngeal muscle (Martin, 1996) using a two-micropipette current-clamp technique (Fig. 21.13A). Glutamate and avermectins produce hyperpolarization and an increase in Cl conductance when either bath-applied or pressure-ejected on to the pharyngeal preparation (Fig. 21.13B,C). These observations establish that one site of action of the avermectins is the pharyngeal muscle of nematodes and that the avermectins can inhibit feeding in nematodes. 

The micropipette tip containing solid phases is a relatively new sample preparation technique that permits handling of microliter to submicroliter amounts of liquid samples, using the techniques of SPE, dialysis, and enzyme digestion. Various phases (reversed-phase, affinity, size-exclusion, etc.) are packed, embedded, or coated on the walls of pipette, permitting liquid samples to be transferred without undue pressure drop or plugging (Fig. 2.5). 

Note While the first reaction mixture is being heated, you may wish to prepare the other mixtures and put them in the hot-water bath. Working carefully, you should be able to stabilize more than one micropipette with the clamp. If you choose to do this, make sure that your materials are clearly labelled. Also remember to keep a record of the time at which each micropipette was introduced to the hot-water bath. 

The preparation of carbon disk-shape UMEs is similar to that of the carbon fibre UME. The only difference is that the active part is sealed on epoxy resin. With this purpose, a micropipette tip (1 mL) is glued to the head-tip of a carbon fibre UME. The carbon fibre is maintained in vertical position with a metal hook and the micropipette tip (1 mL) is filled with epoxy resin. Once the resin is cured, the tip is cross-sectioned with a microtome or a blade. Then, the disk carbon UME is ready. 

Prepare separate tubes containing all of the sample chamber components of the desired assays of Series I through IV except for the addition of KCN solution where it is called for in Series III and IV. Add the components to the tubes in the order in which they are listed in the tables. Prevent plugging of micropipette tips used to transfer the heart particle suspension by cutting about 2 mm off the tips. 

The increased sensitivity which is the main feature of electrothermal atomisation methods introduces a number of difficulties connected with the handling and preparation of samples. Some practical guidance on the avoidance of errors through contamination and on the choice and use of micropipettes is set forth in the following subsections. The analyst must appreciate, however, that we are dealing with a technique of ultramicroanalysis, and any advice or experience that he can make use of on that subject will be entirely relevant here. 

The application system a micropipette or a microsyringe to place the solution of the mixture on the chromatoplate. Micropipettes (Fig. 32.12) are the more common and Box 32.2 gives the instructions for their preparation. 

The universal TLC facilities are utilized plates, adsorbents, microcapillaries, or micropipettes for sample application, development tanks, detection spray reagents, devices for spraying, and densitometers for quantification. Plates are either commercially precoated or handmade. Silica gel G (G, for gypsum as a binding substance), silica gel H (no binding substance) and, rarely, alumina and kieselguhr, form the thin-layer stationary phases. Complete sets of devices necessary for the preparation of handmade plates are commercially available. After the silica gel slurry is spread on the plates, they are left to dry in the air for at least 24 hr and shortly in an oven at 110°C. The plates are then ready for either direct use or for modification of the layer. From the great variety of precoated plates, which are commercially available and preferred nowadays, silica gel plates and plates with layers... 

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