Pipette

This article is about the laboratory tool. For the band, see The Pipettes.

A selection of pipettes

A pipette (also called a pipet, pipettor, or chemical dropper) is a laboratory tool used to transport a measured volume of liquid. Measurement accuracy varies greatly depending on the style.

History

The first micropipette was patented in 1960 by Dr Hanns Schmitz (Marburg/ Germany). The founder of the company Eppendorf, Dr. Heinrich Netheler, inherited the rights and started the commercial production of micropipettes within the sixties. The adjustable micropipette is a Wisconsin invention developed through interactions among several people, primarily inventor Warren Gilson and Henry Lardy, a professor of biochemistry at the University of Wisconsin-Madison.

Use and variations

Scientists using pipettes in a laboratory.

Pipettes are commonly used in molecular biology, analytical chemistry, and medical tests. Pipettes come in several designs for various purposes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes. Many pipette types work by creating a partial vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw up and dispense liquid.

Pipettes that dispense between 1 and 1000 μl are termed micropipettes, while macropipettes dispense a greater volume. Two types of micropipettes are generally used: air-displacement pipettes and positive-displacement pipettes. In particular, piston-driven air-displacement pipettes are micropipettes which dispense an adjustable volume of liquid from a disposable tip. The pipette body contains a plunger, which provides the suction to pull liquid into the tip when the piston is compressed and released. The maximum displacement of the plunger is set by a dial on the pipette body, allowing the delivery volume to be changed. Larger capacity tubular pipettes, such as volumetric or graduated pipettes, are used by temporarily attaching a pipetting dispenser. Pipetting syringes typically handle volumes in the 0.5 mL to 25 mL range, for aliquot transfer and incremental dispensing in titrations, with a positive displacement method of operation.

Piston-driven air displacement pipettes

Single-Channel Pipettes designed to handle 1-5ml and 100-1000µl with locking system

Main article: Air displacement pipette

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. The liquid around the tip moves into this vacuum (along with the air in the tip) and can then be transported and released as necessary. These pipettes are capable of being very precise and accurate. However, since they rely on air displacement, they are subject to inaccuracies caused by the changing environment, particularly temperature and user technique. For these reasons this equipment must be carefully maintained and calibrated, and users must be trained to exercise correct and consistent technique.

The micropipette was invented and patented in 1960 by Dr. Hanns Schmitz of Marburg, Germany. Afterwards, the co-founder of the biotechnology company Eppendorf, Dr. Heinrich Netheler, inherited the rights and initiated the global and general use of micropipettes in labs. In 1972, the adjustable micropipette was invented at the University of Wisconsin-Madison by several people, primarily Warren Gilson and Henry Lardy.

Several different type of air displacement pipettes exist:

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

Positive displacement pipette

These are similar to air displacement pipettes, but are less commonly used and are used to avoid contamination and for volatile or viscous substances at small volumes, such as DNA. The major difference is that the disposable tip is a microsyringe (plastic), composed of a plunger which directly displaces the liquid.

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  Positive displacement pipette

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  The chuck which will be used to move the plunger

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  An early pipette

Pipetting syringe

Pipetting syringes are hand-held devices that combine the functions of volumetric (bulb) pipettes, graduated pipettes, and burettes. They are calibrated to ISO volumetric A grade standards. A glass or plastic pipette tube is used with a thumb-operated piston and PTFE seal which slides within the pipette in a positive displacement operation. Such a device can be used on a wide variety of fluids (aqueous, viscous, and volatile fluids; hydrocarbons; essential oils; and mixtures) in volumes between 0.5mL and 25mL. This arrangement provides improvements in precision, handling safety, reliability, economy, and versatility. No disposable tips or pipetting aids are needed with the pipetting syringe.

Adjustable micropipette

Main article: Air displacement pipette

Pipettes are used to accurately measure and dispense small volumes of liquid. The capacity of a micropipette can range from less than 1 µl to 1000 µl (1 ml), while macropipettes can measure volumes greater than 1ml.

There are four standard sizes of micropipettes: P10 (0.5uL-10uL), P20 (2uL-20uL), P200 (20uL-200uL), and P1000 (200uL-1000uL). P10 micropipetts use white disposable tips, P20 and P200 micropipetts use the same yellow disposable tips, and P1000 micropipetts use blue disposable tips.

Micropipettes brands include Gilson, ErgoOne, Eppendorf, Hamilton, Rainin, Drummond, BrandTech, Oxford, Hirschmann, Biohit, Labnet, Nichiryo, Gilson, Corning, VistaLab, Thermo, Jencons, Vertex, Handypett, and Pricisexx.

Glass micropipette

Borosilicate glass micropipette pulled with a Flaming/Brown micropipette puller P-97

These are used to physically interact with microscopic samples, such as in the procedures of microinjection and patch clamping. Most micropipettes are made of borosilicate, aluminosilicate or quartz with many types and sizes of glass tubing being available. Each of these compositions has unique properties which will determine suitable applications.

Glass micropipettes are fabricated in a micropipette puller and are typically used in a micromanipulator.

Pipette calibration

Pipette recalibration is an important consideration in laboratories using these devices. It is the act of examining or adjusting the quality of being near to the true value of a measuring device by comparison with a standard. Pipette calibration is essential to ensure that the instrument is working according to expectations and as per the defined regimes or work protocols. Pipette calibration is considered to be a complex affair because it includes many elements of calibration procedure and several calibration protocol options as well as makes and models of pipettes to consider.

Factors to consider while calibrating pipettes

There are numerous things to consider during pipette calibration, either if the laboratory performs the process themselves or if undertaken by a third party. It includes the following;

• Operator training applications
• Accuracy and precision of liquid volumes
• Other external factors

Vacuum assisted pipette

Serological pipettes are used for large volumes and for sterile work, but require a pipette dispenser

Non-piston-driven vacuum assisted pipettes are hollow narrow cylinders which work like a straw and require the use of some kind of additional suction device. Originally pipettes were made of soda-lime glass, but currently many are made of borosilicate glass which is tougher and more chemically resistant. Disposable and single use pipettes are often made of polystyrene. All of these are commonly used in chemistry, mainly with aqueous solutions. There are two types. One type, the volumetric(or bulb)pipette, has generally a large bulge with a long narrow portion above with a single graduation mark as it is calibrated for a single volume. Typical volumes are 10, 25, and 50 mL. The second type, the graduated pipette, is straight-walled and as the name implies has graduation marks along most of its length.

The pipette is filled by dipping the tip in the fluid, then drawing up the liquid by using a pipette filler to create a partial vacuum above the fluid. The surface of the fluid inside the pipette is generally concave and this is called the fluid meniscus. The fluid level is read at the center of the meniscus and by aligning it with the graduation marks while holding the pipette at eye level. Liquid is dispensed by releasing the vacuum created by the pipette filler ; slow release for gradual dispensing and complete removal for fast dispensing. With safe fluids the finger may be used on the open end of the pipette to control the vacuum. While moving the pipette to the receiving vessel, care must be taken not to shake the pipette because the column of fluid may "bounce". Complete emptying of the pipette is accomplished by either blowing the pipette out with air from the filler (blow-out pipettes), or touching the tip against the side of the receiving vessel, according to pipette type.

Micro Pipette calibration

Micro Pipettes are one of the most important tools for various procedures in a laboratory. The accuracy and precision of pipette decides the quality of test result perform in particular laboratory.

Micro Pipettes assist lab technician in handling liquids with precise measurement. It is a superior edition of medicine dropper or pasteur pipette or serological pipette and works on the equal principle of making equivalent vacuum for drawing up the liquid. To ensure maximum accuracy, Micro pipettes require calibration. This process is known pipette calibration.

Irrespective of brand or expense of pipette, every micro pipette manufacturer recommends checking the calibration at least every six months, if used regularly. Companies in the drug or food industries are required to calibrate their pipettes quarterly (every three months). Schools which are conducting chemistry classes can have this process annually. Those studying forensics and research where scores of testing is common place will perform monthly calibrations.

Microfluidic pipette

A Microfluidic pipette, housed in a manifold holder. The colored solutions highlight the sample wells of the PDMS pipette.

A recent introduction into the micropipette field integrates the versatility of microfluidics into a positionable pipette platform. These are used to create a recirculation zone, allowing for constant control of a micrometer sized environment at the tip of the pipette. Currently these pipettes are made of polydimethylsiloxane (PDMS) which is molded from a physical master. Interfacing of these pipettes allows for multiple solutions to be loaded and switched on demand, along with concentration control and solution pulsing. Invented by Alar Ainla in Owe Orwar's group at Chalmers University of Technology in Sweden.

Volumetric pipettes

Main article: Volumetric pipettes

Volumetric pipettes allow the user to measure a volume of solution extremely accurately (accuracy of four significant figures) and then add it to something else. They are commonly used to make laboratory solutions from a base stock as well as prepare solutions for titration. They are typically marked to indicate one single volume in a particular size pipette (as are volumetric flasks). Many different sizes are available.

Graduated pipettes

Graduated pipettes use a series of marked lines (as on a graduated cylinder) to indicate different calibrated volumes. These also come in a variety of sizes. These are used much like a burette, in that the volume is found by calculating the difference of the liquid level before and after liquid is dispensed. Historically, the accuracy of a graduated pipette was not as good as that of a volumetric pipette(accuracy of 3 significant fig); however, with improved manufacturing methods, the accuracies listed by the manufacturer can equal volumetric pipettes.

Two types of graduated pipettes exist:

• Mohr pipettes or drain-out pipettes have a 0ml mark before the start of the conical end, which is a dead volume.
• serological or blow-out pipettes have no 0ml mark as that corresponds to an empty pipette.

Graduated pipettes have +/- tolerances that range from 0.6% to 0.4% of the nominal volume when measured at 20C.Graduated pipettes are manufactured according to ISO specifications for accuracy and the arrangement of the graduations. A grade pipettes are more accurate than B grade pipettes and Volumetric pipettes are the most accurate of all.

Pipette Dispensers

Various methods exist to handle the liquids inside a pipette. Before the advent of more sophisticated pipette dispensers, it was common practice to "mouth pipette" i.e. to aspirate fluid into the pipette by applying suction with one's mouth. Mouth pipetting is now considered unsafe due to the possibility of accidentally ingesting or inhaling toxic chemicals or pathogens. The main three types of pipette dispenser are the bulb filler, pipette pump and the electronic controller.

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  Bulb fillers are the simplest cylindrical pipette controllers, employing pinch-valves.

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  Pipette pumps allow more accurate handling of the volume inside a cylindrical pipette.

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  Electronic dispensers are expensive (200-300GB£), but allow easy, accurate handling.

Pipette accessories

An example of pipettes manipulated by an anthropomorphic robot (Andrew Alliance)

• Pipette fillers are used to fill the pipette easily, avoiding the need for mouth pipetting.
• Pipette dispensers are battery-operated and are designed to be used with disposable pipette tubes. These pipettes cannot be calibrated and their accuracy is determined by that of the printed graduations on the disposable tubes.
• Light-guided pipetting systems are pipetting accessories which are computer based. They use LCD monitors or LED arrays to light up source and destination wells in microplates or vials for accurate well to well pipetting. Some of these systems use text to speech to alert the operator during plate or volume changes when pipetting lab protocols.
• Pipette tips. The pipettors and injection molded plastic disposable tips form together a pipetting system.
• Pipette robots. The pipettes can be also exploited by means of anthropomorphic robots (right picture) that are capable of manipulating the pipettes as humans would do.

Pasteur pipette

Dispensable pipettes.

Pasteur pipettes, also known as teat pipettes or droppers, are plastic or glass pipettes used to transfer small amounts of liquids, but are not graduated or calibrated for any particular volume. Transfer pipettes, also known as Beral pipettes, are similar to Pasteur pipettes. However, they are made from a single piece of plastic and their bulb can serve as the liquid-holding chamber.
A commercial variant of the pasteur pipette is the disposable pipette which is often made of plastic and intended to be used to administer medicine into the eye or ear of a patient (see image).

Forward and backward pipetting

There are two ways to pipet defined volumes with graduated pipettes, forward and backward. Forward is when the liquid is drawn as high as needed into the pipette and emptied at the destination. Backward is when you fill the pipette to its uppermost graduation and release a measured amount into the destination. The latter is more accurate for viscous mediums, although the loss of substance remaining in the pipette is an obvious disadvantage. The graduation printed onto a pipette corresponds to the intended use, as seen below. Upper: backward (10 at tip) Lower: forward (zero at tip).

The smallest pipette

A zeptoliter pipette has been developed at Brookhaven National Laboratory. The pipette is made of a carbon shell, within which is an alloy of gold-germanium. The pipette was used to learn about how crystallization takes place.

External links

• Helpful Hints on the Use of a Volumetric Pipet by Oliver Seely
• The History of the Pipette (YouTube video)
• The History of the Pipette according to the US Patent Database
• The History of the Pipetting (Spanish)
• The History of the Pipetting (Italian)

Pipettes in the news

• Gilson/Rainin Lawsuit