Using pipettes

There are two main sorts of pipettes used for accurately dispensing volumes of solutions: volumetric and graduated. Volumetric pipettes are used to dispense fixed volumes of solutions, whereas graduated pipettes allow the dispensing of varying ranges of volumes.  Pipettes are manufactured in a range of styles, and it is important to understand the differences.

Blowout

Pipettes may be labelled ‘BLOWOUT’ which indicates that every drop of liquid in the pipette is counted as part of the volume, meaning that the last drop needs to be ‘blown out’ of the pipette to ensure the correct volume is measured. Non-blowout pipettes differ in that the final drop of liquid that remains in a pipette is accounted for with the graduations and should not be forced out of the pipette when measuring volumes.

Volume graduations

Pipettes vary with the graduations as to whether they run from 0 to the pipette volume from the end of the pipette to the top or vice-versa. To take a 5 mL pipette as an example, on pipettes that start with 0 mL at the tip (top image), if you wanted to measure 3 mL of liquid, you would draw the liquid to the 3 mL mark which would then dispense the correct volume. Whereas for a 5 mL pipette where the tip starts at 5 mL (bottom image) you would calculate the difference, therefore you would fill the pipette to the 2 mL mark in order to dispense 3 mL.

Fillers come in three main designs (for each image the pipette is inserted into the bottom of the filler):

1. Three valved bulb fillers (left). There are valves which control the operation of the pipette. One valve allows the air to be emptied from the bulb. A second valve uses the reduced pressure of the bulb to draw up liquid in the pipette. The third valve opens the pipette to atmosphere which will cause the column of liquid to fall in the pipette.

2. Wheeled fillers (centre). These allow liquid to be drawn up into a pipette by rolling the wheel which pushes up the piece of plastic from the filler and draws liquid up the pipette. There is usually a valve to allow the filler to be reset to enable larger volumes to be drawn up.

3. Electronic fillers (right) usually provide a switch to draw up and release liquid. 

Regardless of the pipette filler used, the same principles apply for accurately measuring and dispensing volumes of liquids using pipettes. The instructions below are for using the valved bulb fillers and will need minor modifications if using a different type of filler.

1. Insert the top of a pipette into a pipette filler. The pipette should only be inserted a short way into the pipette filler, only enough to form a seal (usually only a few mm). Pushing the pipette in too far is likely to damage the pipette filler by dislodging valves in the fillers.

2. Squeeze the valve allowing the bulb to be emptied of air and deflate the bulb.

3. Place the tip of the pipette into the liquid to be drawn up and carefully squeeze the fill valve which will draw the liquid up the pipette. The amount of pressure applied to the fill valve will vary the speed at which the liquid is drawn up. Continue to draw up liquid until the desired graduation mark is reached.

4. If excess liquid is drawn up, use the empty valve to slowly release some liquid until the correct quantity is reached.

5. Transfer the pipette from the liquid and move it to the target vessel. Keep the pipette vertical and position the two vessels as close to each other as practical.

6. Release the liquid by squeezing the empty valve. For non-blowout pipettes, keep squeezing the empty valve and touch the tip of the pipette to the liquid surface allowing the surface to draw the liquid from the pipette leaving only the drop in the tip behind. For blowout pipettes, ensure the remaining drop is added to the collection flask.