Air displacement pipette

Schematic diagram of an air displacement pipette. The "Digital volume indicator" is a dial display that indicates the digits (i.e. unrelated to electronic). The components vary between brands and different volume sizes have different components, for example the piston in a P2 is needle-like and can be separated with ease from the piston assembly whereas in a P10ML it is drum-like and over 1 cm in diameter and is enclosed in plastic.

These pipettes operate by piston-driven air displacement. A vacuum is generated by the vertical travel of a metallic or ceramic piston within an airtight sleeve. The upward movement of the piston, driven by the depression of the plunger, creates a vacuum in the space left vacant by the piston. To fill the vacuum, air from the tip rises, which is then replaced by the liquid that is drawn up into the tip and thus available for transport and dispensing elsewhere.

Sterile technique prevents liquid from coming into contact with the pipette itself. Instead, the liquid is drawn into and dispensed from a disposable pipette tip that is discarded after transferring fluid and a new pipette tip is used for the next transfer. Depressing the tip ejector button removes the tip, that is cast off without being handled by the operator and disposed of safely in an appropriate container. This also prevents contamination of or damage to the calibrated measurement mechanism by the substances being measured.

The plunger is depressed to both draw up and dispense the liquid. Normal operation consists of depressing the plunger button to the first stop while the pipette is held in the air. The tip is then submerged in the liquid to be transported and the plunger is released in a slow and even manner. This draws the liquid up into the tip. The instrument is then moved to the desired dispensing location. The plunger is again depressed to the first stop, and then to the second stop, or 'blowout', position. This action will fully evacuate the tip and dispense the liquid. In an adjustable pipette, the volume of liquid contained in the tip is variable; it can be changed via a dial or other mechanism, depending on the model. Some pipettes include a small window which displays the currently selected volume. The plastic pipette tips are designed for aqueous solutions, and are not recommended for use with organic solvents that may dissolve the plastics of the tips or even the pipettes.

Several different type of air displacement pipettes exist:

• adjustable or fixed
• volume handled
• Single-channel or multi-channel or repeater
• adjustable tip spacing
• conical tips or cylindrical tips
• standard or locking
• manual or electronic
• manufacturer

Volume

Three air-displacement pipettes that handle different volumes.

For optimal usage, every pipette supplier offers a broad range of different capacities. A small volume range of a pipette like 10–100 µL results in a much higher accuracy than a broad range from 0.1–1,000 µL per pipette.

With regard to the volume transferred, the smallest pipette that can handle the required volume should be selected. This is important because accuracy decreases when the set volume is close to the pipette’s minimum capacity. For example, if 50 µl are dispensed using a 5,000 µl pipette, the results will be rather poor. Using a 300 µl pipette will give better results, whereas using a 50 µl pipette would be ideal.[5]

Single-channel and multi-channel pipettes

Depending on the number of pistons in a pipette, there is a differentiation between single-channel pipettes and multi-channel pipettes. For manual high-throughput applications like filling up a 96-well microtiter plate most researchers prefer a multi-channel pipette. Instead of handling well by well, a row of 8 wells can be handled in parallel as this type of pipette has 8 pistons in parallel.

Adjustable tip spacing pipette transferring samples from a 384 well plate to a 96 well plate

Locking mechanism

Some air displacement pipettes can additionally feature a locking mechanism (referred to as "locking pipettes") to allow better changing of volume yet preserving accuracy. By locking the set volume while performing several identical pipetting actions, accidental changes to the pipette volume setting are avoided. The lock mechanism is typically a mechanical toggle close to the pipette setting controls that interferes with the setting mechanism to prevent movement. Some pipettes, however, feature dials for setting the individual volume digits that can only be adjusted when unlocked by depressing and twisting the plunger.[7]

Volume dials on EVOLVE manual pipette from INTEGRA Biosciences

For sustained accuracy and consistent and repeatable operation, pipettes should be calibrated at periodic intervals. These intervals vary depending on several factors:

• The skill and training of the operators. Skilled operators tend to operate the instrument more correctly and make fewer accuracy-robbing mistakes.
• The liquid dispensed by the pipette. Corrosive and volatile liquids tend to emit vapors which ascend into the pipette shaft even under proper operating conditions and may corrode the metal piston and springs, or the seals and o-rings that provide an air-tight seal between the piston and the surrounding sleeve.
• Proper and careful handling. Pipettes that are frequently dropped, are subjected to careless handling or horseplay, or that are not properly stored in a vertical position, will tend to degrade in accuracy over time.
• The accuracy required by the instrument. Applications requiring maximum accuracy also demand more frequent calibration. Instruments used for purely research applications or in educational settings generally require less frequent calibration.

Under average conditions, most pipettes can be calibrated semi-annually (every six months) and provide satisfactory performance. Institutions that are regulated by the Food and Drug Administration's GMP/GLP regulations generally benefit from quarterly calibration, or every three months. Critical applications may require monthly service, while research and educational institutions may need only annual service. These are general guidelines and any decision on the appropriate calibration interval should be made carefully and include considerations of the pipette in question (some are more reliable than others), the conditions under which the pipette is used, and the operators who use it.

Calibration is generally accomplished through means of gravimetric analysis. This entails dispensing samples of distilled water into a receiving vessel perched atop a precision analytical balance. The density of water is a well-known constant, and thus the mass of the dispensed sample provides an accurate indication of the volume dispensed. Relative humidity, ambient temperature, and barometric pressure are factors in the accuracy of the measurement, and are usually combined in a complex formula and computed as the Z-factor. This Z-factor is then used to modify the raw mass data output of the balance and provide an adjusted and more accurate measurement.

The colorimetric method uses precise concentrations of colored water to affect the measurement and determine the volume dispensed. A spectrophotometer is used to measure the color difference before and after aspiration of the sample, providing a very accurate reading. This method is more expensive than the more common gravimetric method, given the cost of the colored reagents, and is recommended when optimal accuracy is required. It is also recommended for extremely low-volume pipette calibration, in the 2 microliter range, because the inherent uncertainties of the gravimetric method, performed with standard laboratory balances, becomes excessive. Properly calibrated microbalances, capable of reading in the range of micrograms (10⁻⁶ g) can also be used effectively for gravimetric analysis of low-volume micropipettes, but only if environmental conditions are under strict control. Six-place balances and environmental controls dramatically increase the cost of such calibrations.

• 
  Oxford Single-Channel Pipettes
• 
  Different volumes of micropipettes.