Piston-driven air displacement pipettes are a type of micropipette, which are tools to handle volumes of liquid in the microliter scale. They are more commonly used in biology and biochemistry, and less commonly in chemistry; the equipment is susceptible to damage from many organic solvents.
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:
Micropipettes can take a minimum volume of 0.2 µL and maximum volume of 10,000 µL (10 mL). [2] [3] They thus are used for smaller-scale transfers than equipment such as graduated pipettes, which come in 5, 10, 25 and 50 mL volumes.
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.
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. [4] Warren Gilson founded Gilson Inc. based on this invention.
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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]
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, 1,000 µL, other non-standard sizes, such as 5,000 µL (5 mL) or 10,000 µL (10 mL). The majority of tips have a color code for easy spotting like natural (colorless) for low volumes (0.1–10 µL), yellow (10–100 µL), or blue (100–1,000 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 8 × 12 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.
| Name | Min. volume (µL) | Max. volume (µL) | Color on Gilson[ clarification needed ] | 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 | 500/1000 | 5000 | Purple | 5000 | |
| P10000 | 1000 | 10000 | Sky | 10000 | |
Two major tip systems exist, called conical or cylindrical, depending on the shape of the contact point of the pipettes and the tip. [6]
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.
Some manufacturers offer adjustable tip spacing pipettes. These allow to transfer multiple samples in parallel between different labware formats.
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. [7] [8]
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.
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. [9]
For sustained accuracy and consistent and repeatable operation, pipettes should be calibrated at periodic intervals. These intervals vary depending on several factors:
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.
A viscometer is an instrument used to measure the viscosity of a fluid. For liquids with viscosities which vary with flow conditions, an instrument called a rheometer is used. Thus, a rheometer can be considered as a special type of viscometer. Viscometers can measure only constant viscosity, that is, viscosity that does not change with flow conditions.
A micrometer, sometimes known as a micrometer screw gauge, is a device incorporating a calibrated screw widely used for accurate measurement of components in mechanical engineering and machining as well as most mechanical trades, along with other metrological instruments such as dial, vernier, and digital calipers. Micrometers are usually, but not always, in the form of calipers. The spindle is a very accurately machined screw and the object to be measured is placed between the spindle and the anvil. The spindle is moved by turning the ratchet knob or thimble until the object to be measured is lightly touched by both the spindle and the anvil.
In measurement technology and metrology, calibration is the comparison of measurement values delivered by a device under test with those of a calibration standard of known accuracy. Such a standard could be another measurement device of known accuracy, a device generating the quantity to be measured such as a voltage, a sound tone, or a physical artifact, such as a meter ruler.
A burette is a graduated glass tube with a tap at one end, for delivering known volumes of a liquid, especially in titrations. It is a long, graduated glass tube, with a stopcock at its lower end and a tapered capillary tube at the stopcock's outlet. The flow of liquid from the tube to the burette tip is controlled by the stopcock valve.
A pipette is a type of laboratory tool commonly used in chemistry and biology to transport a measured volume of liquid, often as a media dispenser. 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. Measurement accuracy varies greatly depending on the instrument.
A syringe is a simple reciprocating pump consisting of a plunger that fits tightly within a cylindrical tube called a barrel. The plunger can be linearly pulled and pushed along the inside of the tube, allowing the syringe to take in and expel liquid or gas through a discharge orifice at the front (open) end of the tube. The open end of the syringe may be fitted with a hypodermic needle, a nozzle or tubing to direct the flow into and out of the barrel. Syringes are frequently used in clinical medicine to administer injections, infuse intravenous therapy into the bloodstream, apply compounds such as glue or lubricant, and draw/measure liquids. There are also prefilled syringes.
A hygrometer is an instrument which measures the humidity of air or some other gas: that is, how much water vapor it contains. Humidity measurement instruments usually rely on measurements of some other quantities such as temperature, pressure, mass and mechanical or electrical changes in a substance as moisture is absorbed. By calibration and calculation, these measured quantities can lead to a measurement of humidity. Modern electronic devices use the temperature of condensation, or they sense changes in electrical capacitance or resistance to measure humidity differences. A crude hygrometer was invented by Leonardo da Vinci in 1480. Major leaps came forward during the 1600s; Francesco Folli invented a more practical version of the device, while Robert Hooke improved a number of meteorological devices including the hygrometer. A more modern version was created by Swiss polymath Johann Heinrich Lambert in 1755. Later, in the year 1783, Swiss physicist and Geologist Horace Bénédict de Saussure invented the first hygrometer using human hair to measure humidity.
A torque wrench is a tool used to apply a specific torque to a fastener such as a nut, bolt, or lag screw. It is usually in the form of a socket wrench with an indicating scale, or an internal mechanism which will indicate when a specified (adjustable) torque value has been reached during application.
In various contexts of science, technology, and manufacturing, an indicator is any of various instruments used to accurately measure small distances and angles, and amplify them to make them more obvious. The name comes from the concept of indicating to the user that which their naked eye cannot discern; such as the presence, or exact quantity, of some small distance.
A liquid handling robot is used to automate workflows in life science laboratories. It is a robot that dispenses a selected quantity of reagent, samples or other liquid to a designated container.
An eye dropper, also called Pasteur pipette or simply dropper, is a device used to transfer small quantities of liquids. They are used in the laboratory and also to dispense small amounts of liquid medicines. A very common use was to dispense eye drops into the eye. The commonly recognized form is a glass tube tapered to a narrow point and fitted with a rubber bulb at the top, although many styles of both plastic and glass droppers exist. The combination of the pipette and rubber bulb has also been referred to as a teat pipette. The Pasteur pipette name is from the French scientist Louis Pasteur, who used a variant of them extensively during his research. In the past, there was no equipment to transfer a chemical solution without exposing it to the external environment. The hygiene and purity of chemical compounds is necessary for the expected result of each experiment. The eye dropper, both glass and plastic types, can be sterilized and plugged with a rubber bulb at the open end of the pipette preventing any contamination from the atmosphere. Generally, they are considered cheap enough to be disposable, however, so long as the glass point is not chipped, the eye dropper may be washed and reused indefinitely.
A volumetric pipette, bulb pipette, or belly pipette allows extremely accurate measurement of the volume of a solution. It is calibrated to deliver accurately a fixed volume of liquid.
Alcoholic spirits measures are instruments designed to measure exact amounts or shots of alcoholic spirits.
A graduated pipette is a pipette with its volume, in increments, marked along the tube. It is used to accurately measure and transfer a volume of liquid from one container to another. It is made from plastic or glass tubes and has a tapered tip. Along the body of the tube are graduation markings indicating volume from the tip to that point. A small pipette allows for more precise measurement of fluids; a larger pipette can be used to measure volumes when the accuracy of the measurement is less critical. Accordingly, pipettes vary in volume, with most measuring between 0 and 25.0 millilitres.
Forward pipetting is a technique to dispense a measured quantity of liquid by means of air displacement pipette. The technique is mainly recommended for aqueous solutions, such as buffers, or diluted acids or alkalis. In case of solutions with a high viscosity or a tendency to foam, reverse pipetting is more suitable.
Reverse pipetting is a technique to dispense a measured quantity of liquid by means of air displacement pipette. The technique is mainly recommended for solutions with a high viscosity or a tendency to foam: as it reduces the risk of splashing, foam or bubble formation. Reverse pipetting is more precise in dispensing small volumes of liquids containing proteins and biological solutions compared to forward pipetting, which is mostly used for aqueous solutions, such as buffers, diluted acids or alkalis.
A media dispenser or a culture media dispenser is a device for repeatedly delivering small fixed volumes of liquid such as a laboratory growth medium like molten agar or caustic or volatile solvents like toluene into a series of receptacles. It is often important that such dispensers operate without biological or chemical contamination, and so must be internally sealed from the environment and designed for easy cleaning and sterilization before use. At a minimum, a media dispenser consists of some kind of pump connected to a length of discharge tubing or a spout. Dispensers used in laboratories are also frequently connected to microcontrollers to regulate the speed and volume of the medium as it leaves the pump.
Gas blending is the process of mixing gases for a specific purpose where the composition of the resulting mixture is specified and controlled. A wide range of applications include scientific and industrial processes, food production and storage and breathing gases.
Heinrich Schnitger was a German physician. He is considered the inventor of the piston stroke Micropipette, a laboratory device for dispensing small amounts of liquid.
Positive displacement pipettes are a type of pipette that operates via piston-driven displacement. Unlike an air displacement pipette, which dispenses liquid using an air cushion in the pipette tip, the piston in a positive displacement pipette makes direct contact with the sample, allowing the aspiration force to remain constant.