Piston-driven air displacement pipettes are micropipette

Micropipette

A micropipette is one of two different instruments used in science laboratories.-History:The first micropipette was patented in 1960 by Dr Hanns Schmitz . The founder of the company Eppendorf, Dr...

s, which are tools to handle volumes of liquid in the microliter scale and are the most accurate and precise pipette

Pipette

A pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...

s. They are more commonly used in biology and biochemistry, but 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

University of Wisconsin–Madison

The University of Wisconsin–Madison is a public research university located in Madison, Wisconsin, United States. Founded in 1848, UW–Madison is the flagship campus of the University of Wisconsin System. It became a land-grant institution in 1866...

 (mainly Warren Gilson and Henry Lardy

Henry A. Lardy

Henry A. Lardy was a biochemist and professor emeritus in the Biochemistry Department at the University of Wisconsin-Madison. He was elected to the National Academy of Sciences in 1958...

) enhanced the fixed-volume pipette, developing the pipette with a variable volume.

Operation

These pipettes operate by piston

Piston

A piston is a component of reciprocating engines, reciprocating pumps, gas compressors and pneumatic cylinders, among other similar mechanisms. It is the moving component that is contained by a cylinder and is made gas-tight by piston rings. In an engine, its purpose is to transfer force from...

-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.

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

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

Disposable

A disposable is a product designed for cheapness and short-term convenience rather than medium to long-term durability, with most products only intended for single use. The term is also sometimes used for products that may last several months to distinguish from similar products that last...

 tips.
The tips are plastic

Plastic

A plastic material is any of a wide range of synthetic or semi-synthetic organic solids used in the manufacture of industrial products. Plastics are typically polymers of high molecular mass, and may contain other substances to improve performance and/or reduce production costs...

-made tools for single-use. In general, they are made of Polypropylene

Polypropylene

Polypropylene , also known as polypropene, is a thermoplastic polymer used in a wide variety of applications including packaging, textiles , stationery, plastic parts and reusable containers of various types, laboratory equipment, loudspeakers, automotive components, and polymer banknotes...

.
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

Color code

A color code is a system for displaying information by using different colors. Color codes are often difficult for color blind and blind people to interpret....

, printed on the pipette.

For special applications, there are filter-tips available. These tips have a little piece of foam

Foam

-Definition:A foam is a substance that is formed by trapping gas in a liquid or solid in a divided form, i.e. by forming gas regions inside liquid regions, leading to different kinds of dispersed media...

 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.

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

Microtiter plate

A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...

 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 as 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

Electric motor

An electric motor converts electrical energy into mechanical energy.Most electric motors operate through the interaction of magnetic fields and current-carrying conductors to generate force...

 powered by a battery

Battery (electricity)

An electrical battery is one or more electrochemical cells that convert stored chemical energy into electrical energy. Since the invention of the first battery in 1800 by Alessandro Volta and especially since the technically improved Daniell cell in 1836, batteries have become a common power...

. 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

Wheel

A wheel is a device that allows heavy objects to be moved easily through rotating on an axle through its center, facilitating movement or transportation while supporting a load, or performing labor in machines. Common examples found in transport applications. A wheel, together with an axle,...

 and some further buttons. All settings are displayed on a small display

Display

Display may refer to:* Display , an American thoroughbred racehorse* Display , a form of animal behaviour* Display advertising, type that typically contains text, i.e., copy, logos, images, location maps, etc....

. Electronic pipettes can decrease the risk of RSI

RSI

-Business:*RADARSAT International, a subsidiary of MacDonald Dettwiler and Associates*Red Sector Incorporated, a demo and "warez" group*Resource Software International Ltd., a communication management call accounting software company* Resin Systems Inc....

-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 at a 90 degree angle.
• When dispensing the pipette should be held at a 45 degree angle, and the tip placed against the side of the receiving vessel. Glass
  Glass
  Glass is an amorphous solid material. Glasses are typically brittle and optically transparent.The most familiar type of glass, used for centuries in windows and drinking vessels, is soda-lime glass, composed of about 75% silica plus Na2O, CaO, and several minor additives...
  
   vessels are preferred; the surface tension
  Surface tension
  Surface tension is a property of the surface of a liquid that allows it to resist an external force. It is revealed, for example, in floating of some objects on the surface of water, even though they are denser than water, and in the ability of some insects to run on the water surface...
  
   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
  Humidity
  Humidity is a term for the amount of water vapor in the air, and can refer to any one of several measurements of humidity. Formally, humid air is not "moist air" but a mixture of water vapor and other constituents of air, and humidity is defined in terms of the water content of this mixture,...
  
  , which reduces evaporation
  Evaporation
  Evaporation is a type of vaporization of a liquid that occurs only on the surface of a liquid. The other type of vaporization is boiling, which, instead, occurs on the entire mass of the liquid....
  
   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
  Heat
  In physics and thermodynamics, heat is energy transferred from one body, region, or thermodynamic system to another due to thermal contact or thermal radiation when the systems are at different temperatures. It is often described as one of the fundamental processes of energy transfer between...
  
   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
  Fatigue (physical)
  Fatigue is a state of awareness describing a range of afflictions, usually associated with physical and/or mental weakness, though varying from a general state of lethargy to a specific work-induced burning sensation within one's muscles...
  
   is an often-overlooked but crucial component when seeking maximal accuracy and repeatability. Repetitive motions cause stress
  Stress (medicine)
  Stress is a term in psychology and biology, borrowed from physics and engineering and first used in the biological context in the 1930s, which has in more recent decades become commonly used in popular parlance...
  
   in human joint
  Joint
  A joint is the location at which two or more bones make contact. They are constructed to allow movement and provide mechanical support, and are classified structurally and functionally.-Classification:...
  
  s and muscle
  Muscle
  Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...
  
  s. 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
  Repetitive strain injury
  Repetitive strain injury is an injury of the musculoskeletal and nervous systems that may be caused by...
  
   (RSI), such as carpal tunnel syndrome
  Carpal tunnel syndrome
  Carpal Tunnel Syndrome is an entrapment idiopathic median neuropathy, causing paresthesia, pain, and other symptoms in the distribution of the median nerve due to its compression at the wrist in the carpal tunnel. The pathophysiology is not completely understood but can be considered compression...
  
  . 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
  Spring (device)
  A spring is an elastic object used to store mechanical energy. Springs are usually made out of spring steel. Small springs can be wound from pre-hardened stock, while larger ones are made from annealed steel and hardened after fabrication...
  
   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

For sustained accuracy and consistent and repeatable operation, pipettes should be calibrated

Calibration

Calibration is a comparison between measurements – one of known magnitude or correctness made or set with one device and another measurement made in as similar a way as possible with a second device....

 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
  Corrosion
  Corrosion is the disintegration of an engineered material into its constituent atoms due to chemical reactions with its surroundings. In the most common use of the word, this means electrochemical oxidation of metals in reaction with an oxidant such as oxygen...
  
   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

Food and Drug Administration

The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...

'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.

Additional Sources

Gilson website
Rainin website
Starlabs
Eppendorf
Biohit website
Pipette Calibration
RV Instruments
Apricot Designs website

See also