Pipette micropipette

This method relies on the simple principle that the flow of ions into an electrolyte-filled micropipette as it nears a surface is dependent on the distance between the sample and the mouth of the pipette [211] (figure B 1.19.40). The probe height can then be used to maintain a constant current flow (of ions) into the micropipette, and the technique fiinctions as a non-contact imaging method. Alternatively, the height can be held constant and the measured ion current used to generate the image. This latter approach has, for example, been used to probe ion flows tlirough chaimels in membranes. The lateral resolution obtainable by this method depends on the diameter of the micropipette. Values of 200 nm have been reported. 

For dealing with smaller volumes of solution, micropipettes, often referred to as syringe pipettes, are employed. These can be of a push-button type, in which the syringe is operated by pressing a button on the top of the pipette the plunger travels between two fixed stops and so a remarkably constant volume of liquid is delivered. Such pipettes are fitted with disposable plastic tips (usually of polythene or polypropylene) which are not wetted by aqueous solutions, thus helping to ensure constancy of the volume of liquid delivered. The liquid is contained entirely within the plastic tip and so, by replacing the tip, the same pipette can be employed for different solutions. Such pipettes are available to deliver volumes of 1 to 1000 pL, and the delivery is reproducible to within about 1 per cent. 

In order to indicate the accuracy that can be obtained from a capillary pipette, one may refer to the so called Sahli pipette, which is approximately 80 mm from mark to tip. This pipette is used as a washout pipette and can measure samples which are readily reproducible to 1 part in 80, since it is a relatively simple thing for the eye to see 1 mm. On the other hand, if one places a bulb in the tube, and cuts the bore down at the mark by 30%, then this would decrease the error by a factor of 4. It is thus practicable to sample from a micropipette, which is used as a washout pipette, with greater accuracy than one can sample from a conventional macro pipette, which delivers 1 ml by blowout, or to deliver. Figures5 and 6 illustrate various designs of micropipets (13,14). 

Most electrochemical studies at the micro-ITIES were focused on ion transfer processes. Simple ion transfer reactions at the micropipette are characterized by an asymmetrical diffusion field. The transfer of ions out of the pipette (ejection) is controlled by essentially linear diffusion inside its narrow shaft, whereas the transfer into the pipette (injection) produces a spherical diffusion field in the external solution. In contrast, the diffusion field at a microhole-supported ITIES is approximately symmetrical. Thus, the theoretical descriptions for these two types of micro-ITIES are somewhat different. 

No steady-state theory for kinetically controlled heterogeneous IT has been developed for micropipettes. However, for a thin-wall pipette (e.g., RG < 2) the micro-ITIES is essentially uniformly accessible. When CT occurs via a one-step first-order heterogeneous reaction governed by Butler-Volmer equation, the steady-state voltammetric response can be calculated as [8a]... 

The use of micropipette electrodes for quantitative voltammetric measurements of ion transfer (IT) and electron transfer (ET) reactions at the ITIES requires knowledge of geometry of the liquid interface. For the micrometer-sized micropipettes, both the orifice radius and the thickness of the pipette wall can be measured microscopically. A typical error of the microscopic determination of a radius was estimated to be 0.5/am for a micropipette and 1 /am for a microhole [24]. 

Another parameter essential for quantitative applications of micropipettes is the internal ohmic resistance, R. It is largely determined by the solution resistance inside the narrow shaft of the pipette, and can be minimized by producing short (patch-type) pipettes. The micropipette resistance has been evaluated from AC impedance measurements. Beattie et al. measured the resistance of micropipettes filled with aqueous KCl solutions (0.01, 0.1, and 1 M) [18b]. The value obtained for a 3.5/am-radius pipette was within the range from 10 to 10 As expected, the tip resistance was inversely proportional to the concentration of KCl in the filling solution. In ref. 18b, the effect of pipette radius on the tip resistance was evaluated using a constant concentration of KCl. The pipette resistance varied inversely with the tip radius. The iR drop was found to be 4.5-8 mV for the pipette radii of 0.6 to 19/rm when 10 mM KCl was used. 

Since the mass-transfer coefficient at a micropipette is inversely proportional to its radius, the smaller the pipette the faster heterogeneous rate constants can be measured. Micrometer-sized pipettes are too large to probe rapid CT reactions at the ITIES. Such measurements require smaller (nm-sized) pipettes. Nanopipettes are also potentially useful as SECM tips (see Section IV.D) because they can greatly improve spatial resolution of that technique. The fabrication of nanopipettes was made possible by the use of a micro-processor-controlled laser pipette puller capable of puling quartz capillaries [26]. Using this technique, Wei et al. produced nanopipettes as small as 20 nm tip radius and employed them in amperometric experiments [9]. 

Studies of electron transfer (ET) at micro-ITIES are scarce. Solomon and Bard first observed the ET between TCNQ (in DCE) and ferrocyanide (in water) at a micro-ITIES supported by micropipettes [5]. The pipette was used as a SECM probe for electrochemical imaging. The current was controlled by the rate of the bimolecular ET reaction at the micro-ITIES... 

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). 

UMEs used in our laboratory were constructed by sealing of carbon fibre into low viscosity epoxy resin (see Fig. 32.4) [118]. This method is simple, rapid and no specialised instrumentation is required. Firstly, the fibres are cleaned with this aim. They are immersed in dilute nitric acid (10%), rinsed with distilled water, soaked in acetone, rinsed again with distilled water and dried in an oven at 70°C. A single fibre is then inserted into a 100- iL standard micropipette tip to a distance of 2 cm. A small drop of low-viscosity epoxy resin (A. R. Spurr, California) is carefully applied to the tip of the micropipette. Capillary action pulls the epoxy resin, producing an adequate sealing. The assembly is placed horizontally in a rack and cured at 70°C for 8h to ensure complete polymerization of the resin. After that, the electric contact between the carbon fibre and a metallic wire or rod is made by back-filling the pipette with mercury or conductive epoxy resin. Finally, the micropipette tip is totally filled with epoxy resin to avoid the mobility of the external connection. Then, the carbon fibre UME is ready. An optional protective sheath can be incorporated to prevent electrode damage. 

Micropipettes, 0.250 and 1.0 mL tips, and 1.5 mL tubes (Eppendorf, Germany) are also employed. The rest of volumetric material (flasks, pipettes, vessels...) is of analytical reagent grade. 

Figure 1 Diagram of the micropipette aspiration technique (modified from Lim et al., 2006). L is the length of extension into the pipette, Rc the inner radius of the pipette and AP the suction pressure.

Patch clamping requires that an electrode, housed within a micropipette, is attached to the cell to make an almost perfect seal with the cell membrane. This generates a very high resistance between the cell and the pipette wall, typically 10 CA2. The resulting transmembrane currents, measured between microelectrodes inside and outside the cell, generate extremely low noise so single channel events can be... 

Do not ingest radioisotopes. Specifically, never pipette radioactive solutions by mouth. Instead, use a Propipette bulb or a micropipetter to withdraw and dispense radioactive solutions. Use an appropriate ventilated hood when working with volatile radioactive compounds. 

The pipettes are all based on air displacement with a simple plunger and are provided with non-wettable plastic (usually polypropylene) disposable tips to contain the solution, preventing any contamination of the pipette itself. Most micropipettes have a double action plunger system, i.e. calibration and overshoot positions, which ensures that the sample is completely dispensed. There are many manufacturers offering a complete range of volumes in addition some have available a selection of pipettes with adjustable volumes. 

Correct operation of a micropipette is essential to enable volumes to be dispensed precisely. The plunger of the micropipette should be depressed slowly but firmly to the first stop position, and the tip just inserted in the solution to be tested. The plunger should be allowed to rise carefully over a period of 3—4 s. To inject the sample into the atomiser, the plunger is slowly (3—4 s) depressed fully to expel the sample completely. The pipette and tip should be completely removed from the tube before the plunger is gently released to avoid taking the sample back into the tip. 

For injection of the water sample into the atomiser, micropipettes are used these are now commercially available and commonly specified to a 1% accuracy. Pipette tips are known to be contaminated with Fe, Zn and Cd, thus they should be soaked in 10% nitric acid and then washed in distilled-deionised water and sample prior to use. Accurate, precise pipetting and the correct adjustment of the drying, ashing and atomisation programme are essential factors required for a successful flameless atomic absorption analysis. When pipetting the sample, the water droplet must be positioned reproducibly on the filament or in the furnace and it should be of an optimum size such that it does not run or spit during heating. If this happens, irreproducible absorption peaks may result. 

It is usual with electrothermal atomisers to pipette between 5 and 100 pi samples into the device using a micropipette. With petroleum samples dissolved in organic solvents this may be a problem. Due to the low surface tension of many of these solvents they do not pipette easily and often dry irregularly in the atomiser, both factors giving rise to poor reproducibility. The problem of poor drying characteristics may be overcome with many solvents by pipetting into a pre-heated atomiser at approximately 80°C. The solvent is removed immediately, leaving the analyte on a reproducible spot each time. This technique, however, requires some care so as not to melt or contaminate the pipette tip. 

Micropipettes are made in sizes from 0.1 pL to 500 pL (0.5 mL) in several designs. Some are simply capillaries that All completely by capillary action and are blown or rinsed out. Others are capillaries with a single calibration mark that are filled beyond the mark by capillary action and then drawn down to the mark by touching the tip carefully to absorbent paper. Very often micropipettes are essentially miniature measuring pipettes fitted with a pipette control, usually an uncalibrated hypodermic syringe or a threaded piston in a cylinder, which permits suction or pressure to be applied to adjust the level of the liquid with high precision. Micropipettes can be used with considerable precision if sufficient care is exercised, but the importance of droplets left on the outside of the tip, internal cleanliness, and uniformity of technique is even greater than with pipettes of ordinary size. 

A more recently developed force measurement technique, coined the liquid siu-face force apparatus (LSFA), brings a drop made from a micropipette close to a flat liquid/liquid interface [29-32]. A piezo electric drive is used to change the position of the micropipette while the deflection of the pipette and the radius of the drop are recorded with piezo motion. The drop radius and thus the film thickness between the two liquid/liquid interfaces are recorded using interferometry. The method requires a calibration of the interferometer, where the drop must come into contact with the other liquid interface. The distance resolution of the film is about 1 nm at a 50-nm separation and 5 nm at a separation of 10 nm. This is a very robust technique where the authors have proposed attaching a particle to the end of the pipette instead of a drop [29]. In comparing this method to AFM, the only drawback of the LSFA is the weaker distance resolution. It is important point out that both methods required a contact point for distance calibration. 

Before measuring a volume it is important to choose the most appropriate equipment in order to achieve the greatest accuracy. The volume and level of accuracy will help determine which piece of equipment should be used. The most common equipment includes various pieces of glassware (e.g. volumetric flask, measuring cylinder, burette, pipette), mechanical micropipette (or pipettor) and syringes (Figure 2.3). 

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