Volume Pipettes

Set the pipette to the desired volume and draw this up into the pipette. Dispense the solution into a suitable receiving vessel this might be an HPLC vial or another small glass or plastic vial. Using a similar type of pipette, transfer the diluents into the same vessel. Shake or invert to mix. 

You have been asked to dilute a liver extract 10 times using mobile phase as the solvent. Following an extraction procedure, you have a volume of 200 pL (solution A). Using a variable volume automatic pipette, draw up 100 pL of solution A and dispense this into a suitable glass or plastic vial. Using a second variable volume automatic pipette, transfer 900 pL of mobile phase into the same vial. Stopper the vial and shake or invert to mix. The sample solution has been diluted 10 times. This is often referred to as a 1 in 10 or 1 to 10 dilution. 

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Small volume pipettes are avoided since they may lead to significant errors in the final results. Similarly, typical sizes for volumetric flasks should be between 25 and 500 ml if possible. 

Filter samples for the stability and repeatability studies were prepared by spiking the filter with a known volume of the dye solution using fixed volume pipettes or volumetric syringes. The filters were then dried in a dessicator filled with anhydrous calcium sulfate and phosphorous pentoxide. The dessicator provided more rapid and uniform drying of the... 

Six filters for each level of concentration were spiked using variable volume pipettes. The following volumes 13, 25, 50, 100, and 200 yL of the 10 mg/mL solution yielded 0.13, 0.25, 0.50, 1.00 and 2.00 mg of sodium dichloroisocyanurate dihydrate on a filter, respectively. When the filter was dry, the "filter extraction" section was followed. The end-point volumes were converted to milligrams from the calibration curve and the percent recoveries were calculated. 

Pipettes may be categorized as fixed-, variable-, or selectable-volume (see Chapter 1). Selectable-volume pipettes allow the selection of a limited number of predetermined volumes. In general, pipettes with selectable volumes are used in systems that allow many different applications, whereas fixed-volume pipettes are usually used for specimens and reagents in instruments dedicated to performing only a small variety of tests. 

Once the compound of interest has been isolated from the sample matrix, it is often necessary to dilute the sample in order to analyse it within the validated assay range (see Chapter 8). A test injection may have to be performed in order to assess the sample concentration in the first instance. Once it has been established that the sample does require dilution, this can easily be performed using calibrated glass (larger volumes) or variable volume pipettes (smaller volumes). 

Fixed and variable volume pipettes find application in microbiology labs for preparation of dilutions as well as accurate dispensing of small (pL) volumes of reagents. Generally, fixed volume pipettes are used for routine laboratory purposes such as dilution, whereas variable volume pipettes are used in enzymatic assays requiring small-volume transfers. 

The simplest technique to apply samples spotwise is to use a fixed volume pipette that fills by capillary action and delivers its content when it touches the layer. In order to ensure that the layer is not damaged and the spots are precisely positioned, it is advisable to guide the pipette with respect to the lateral position and with reproducible constant pressure. This is possible with a Nanomat " (Fig. 2), a mechanized spotting device. 

Fixed volume pipettes of 100 nL and 200 nL are available in the form of platinum-iridium capillaries melted in a glass holder. Repeatability of the volume dispensed by such a nanopipette is very good (< 1 %), but the absolute volume accuracy is only about 5%. This means that for quantitative analysis, all samples and calibration standards on one plate must be applied with the same nanopipette unless calibration is by internal standard. 

Figure 2 Nanomat. The fixed volume pipette (nanopipette or glass capillary in holder) is held by a magnet. The applicator head is moved to the desired position in the precision notch system, then the head is pushed down manually. Contact pressure is determined solely by the friction of rest against the magnet.

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