Air displacement pipette

Oxford Single-Channel Pipettes

Piston-driven air displacement pipettes are micropipettes, which are tools to handle volumes of liquid in the microliter scale pipettes. They are more commonly used in biology and biochemistry, but they are also used by chemists, though less commonly due to the risk of solvent damage to the pipettes and tips.

In 1972, several people of the University of Wisconsin–Madison (mainly Warren Gilson and Henry Lardy) enhanced the fixed-volume pipette, developing the pipette with a variable volume.^[1]

Operation

These pipettes operate by piston-driven air displacement. A vacuum is generated by the vertical travel of a metal or ceramic piston within an airtight sleeve. As the piston moves upward, driven by the depression of the plunger, a vacuum is created in the space left vacant by the piston. Air from the tip rises to fill the space left vacant, and the tip air is then replaced by the liquid, which 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 changed between transfers. 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.

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.

Schematic 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.

Models

Several different type of air displacement pipettes exist:

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

Adjustable or fixed volume

The most common type of pipettes can be set to a certain volume within its operational range and are called adjustable. These pipettes commonly have a label with their volume range like "10 - 100 µl". These limits are indeed the limits as overwinding these limits would result in damage of the pipetting system. The fixed volume pipette cannot be changed. As there are less moving parts, the mechanism is less complex, resulting in more accurate volume measurement.

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.

Tips

For the pipetting process there are two components necessary: The pipette and disposable tips. The tips are plastic-made tools for single-use. In general, they are made of Polypropylene. Depending on the size of the pipette, the user needs specific tip sizes like:

10 µL
100 µL
200 µL
1000 µL
other non-standard sizes, such as 5 mL or 10 mL.

The majority of tips have a color code for easy spotting like natural (clear) for low volumes (0.1 - 10 µL), yellow (10 - 200 µL), or blue (100 - 1000 µL). The corresponding pipette has the same color code, printed on the pipette.

For special applications, there are filter-tips available. These tips have a little piece of foam plastic in the upper conus to prevent sample aerosols contaminating the pipette.

In general, all tips are stored in 8x12 boxes for 96 pieces in an upright position. The spacing of tips in these boxes is usually standardised for multichannel pipette compatibility from a number of different suppliers.

Commonly available pipette volumes:

Name	Min. volume (µL)	Max. volume (µL)	Colour on Gilson	tip size (µL)
P2	0.2	2	Orange	10
P10	1	10	Red	10
P20	2	20	Lemon	200
P100	20	100	Salmon	200
P200	50	200	Yellow	200
P1000	200	1000	Blue	1000
P5000	1000	5000	Purple	5000
P10000	1000	10000	Sky	10000

Two major tip systems exist, called conical or cylidrical, depending on the shape of the contact point of the pippetes and the tip.^[2]

Single-channel, multi-channel, electronic pipettes, and repeaters

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.

To improve the ergonomics of pipettes by reducing the necessary force, electronic pipettes were developed. The manual movement of the piston is replaced by a small electric motor powered by a battery. Whereas manual pipettes need a movement of the thumb (up to 3 cm), electronic pipettes have a main button. The programming of the pipette is generally done by a control wheel and some further buttons. All settings are displayed on a small display. Electronic pipettes can decrease the risk of RSI-type injuries.

Repeaters are specialized pipettes, optimized for repeated working steps like dispensing several times a specific volume like 20 µL from a single aspiration of a larger volume. In general, they have specific tips which do not fit on normal pipettes. Some electronic pipettes are able to perform this function using standard tips.

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.

Accuracy

Certain considerations should be observed to ensure maximum accuracy and repeatability:

Operator consistency is paramount to repeatable operation. The necessity of operator practice and development of good pipetting practices and habits is absolute. Light guided pipetting aides are used to help reduce errors and speed up liquid handling protocols.
When drawing up liquid the tip should be dipped 3 to 5 mm below the surface of the liquid, always nearly completely vertical.
When dispensing the pipette should be held at a 45 degree angle, and the tip placed against the side of the receiving vessel. Glass vessels are preferred; the surface tension of the glass provides additional torsion that results in complete evacuation of the tip.
The tip must never be wiped off or blotted in any way, even from the exterior, while liquid is in the tip. These actions tend to attract and thus bleed off some of the liquid, resulting in decreased accuracy and repeatability.
A dry tip should always be pre-wetted by drawing up and dispensing the chosen volume a minimum of three times. This action reduces the surface tension on the inside walls of the tip and also provides the proper level of inter-tip humidity, which reduces evaporation of the sample liquid.
Most pipettes are calibrated "to deliver" (TD) and not "to contain" (TC). If they are TD pipettes they should not be rinsed after they have delivered their contents. If the pipette were calibrated TC it should be rinsed to obtain the correct amount of material. If the fluid to be measured is quite viscous or sticky (such as glycerol solutions) the pipette must be calibrated and in this case the outside of the tip must be carefully wiped with a lint free tissue to remove the adhering liquid - while being careful to not touch the opening of the pipette tip, which may require some practice. Accuracy in delivering liquids with high or low viscosity may require a "positive displacement" pipettor, which is quite distinct from an air displacement pipettor.
For maximum accuracy, and especially necessary when calibrating the pipette, relative humidity in the ambient environment should be maintained between 50% and 75%, and in no case should the humidity be allowed to dip below 50%. This limits the rate of sample evaporation which can cause significant errors, especially at lower volumes.

The importance of operator skill cannot be overstated. A high-quality, well-calibrated pipette in the hand of an uninterested or untrained operator is an unreliable instrument. Additionally, there are four factors that can reduce the accuracy and repeatability of even highly skilled operators, and these factors must be counteracted if optimal accuracy is to be achieved:

Heat from the operator's hand is absorbed through the handle of the instrument and transferred to the metallic components inside. If the pipette is operated continuously for a prolonged period of time this heat buildup becomes significant, causing the internal components to expand and changing the interplay between components. This reduces the consistency, accuracy, and repeatability of the instrument. The volume dispensed is dependent on the sizes of the piston and the springs that cause its travel. As these change in size the volume dispensed changes also. This effect is more pronounced in low-volume instruments. Additionally, the expansion of a metallic component that interacts with a non-metallic one that does not expand as readily in the presence of heat may cause the instrument to seem to stick, hang up, or react more slowly. Pipettes with thin handles are particularly susceptible to this phenomenon. Plumper handles are both more ergonomic and less likely to suffer from heat transfer problems. The best technique for maximum accuracy is to employ multiple pipettes and rotate them often, storing them between uses in a stand that holds them vertically.

Operator fatigue is an often-overlooked but crucial component when seeking maximal accuracy and repeatability. Repetitive motions cause stress in human joints and muscles. Even a well-trained and experienced operator will see a decrease in accuracy and repeatability as length of time on the job increases. It is for this reason that pipette calibration service providers that are dedicated to excellence limit the number of pipettes that can be calibrated by an individual technician to a maximum daily number. Each pipette, and each customer, deserves a high level of care in the treatment of the instrument. Additionally, some dedicated professionals train themselves to pipette ambidextrously, allowing them to reduce arm and finger strain by alternating hands. Another solution is choosing an electronic pipettor that significantly reduces hand fatigue. Once the operating button is touched the pipettor operates always the same way producing user independent accuracy and precision.

Long-term pipette operation can lead to repetitive strain injuries (RSI), such as carpal tunnel syndrome. These disorders may cause significant reductions in accuracy and repeatability by altering the proper pipetting techniques that are crucial to achieving optimal accuracy. Preventive measures include learning to pipette with both hands and alternating their usage, taking frequent breaks while pipetting, and choosing the most ergonomic pipette available. Instruments with plumper handles are generally superior in this regard. On the other hand, electronic pipettors which operate with a light touch reduce RSI significantly.

Letting the pipette "rest" for at least one minute after a volume change is made. This does not apply to single-volume instruments, also called set volume or fixed volume pipettes. A change in the dispensed volume of an adjustable pipette involves modifying the internal tensioning of a spring that governs the piston's travel distance. Springs subjected to changing tensioning behave more smoothly and consistently when they are allowed to enjoy an interval of rest to settle into their new configuration. A pipette that is left idle for at least one minute after a volume adjustment will perform more accurately than one that is pressed into service prematurely. This is especially important when calibrating a pipette.

Calibration

A set of Gilson micropipettes

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 colormetric method uses precise concentrations of colored water to affect the measurement and determine the volume dispensed. A spectrophotomer 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 gravimetic 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.

Sources

Bruce Alberts, Molecular biology of the cell, 5th ed
guide to pipetting

References

↑ Zinnen, Tom (June 2004). "The Micropipette Story". The Board of Regents of the University of Wisconsin System. Archived from the original on 26 December 2009. Retrieved 14 December 2009.
↑ http://www.rainin.com/pdf/tr9901.pdf