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Additional recommended knowledge
Use and variations
Pipettes are commonly used in chemistry and molecular biology research as well as 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. A pipette works by creating a 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 of liquid.
The original pipette is made of glass. It is more commonly used in chemistry, with aqueous solutions. There are two types. One type has a large bulb, and is calibrated for a single volume. Typical volumes are 10, 25, and 50 mL. Alternatively, it is straight-walled, and graduated for different volumes such as 5 mL in 0.5 mL increments. The single volume pipette is usually more accurate, with an error of ± 0.1 or 0.2 mL.
The pipette is filled by dipping the tip in the volume to be measured, and drawing up the liquid with a pipette filler past the inscribed mark. The volume is then set by releasing the vacuum using the pipette filler or a damp finger. While moving the pipette to the receiving vessel, care must be taken not to shake the pipette because the column of fluid may "bounce".
Piston-driven air displacement pipettes
These are the most accurate and precise pipettes. They are more commonly used in biology, though they are commonly used by chemists as well. The plastic pipette tips are designed for aqueous solutions, and are not recommended for use with organic solvents which may dissolve the plastic.
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. Instead, the liquid is drawn into and dispensed from a disposable pipette tip which is changed between transfers. Depressing the tip ejector button removes the tip, which 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.
Certain considerations should be observed to ensure maximum accuracy and repeatability:
The importance of operator skill cannot be overstated. A high-quality, well-calibrated pipette in the hand of a 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:
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 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 nanograms (10-9 g) can also be used effectively for gravimetric analysis of low-volume micropipettes.
The choice of calibration providers is also a significant factor in the quality of the calibration service provided. The easiest way to choose a competent laboratory is to look for one that is certified to a national or international standard, such as ISO/IEC 17025. These labs have proven adherence to rigid regulations and protocols and are certified by accredited institutions authorized to perform such certification by the body issuing the standard in question. These standards require the laboratory to provide demonstrated adherence to certain practices that tend to establish their competence and impartiality. These practices include implementation of a comprehensive quality system, standardized and published procedures for all laboratory operations, use of only accepted or properly validated test and/or calibration methods, proper personnel training and supervision, provision for handling nonconforming work, transparency of laboratory operations, document and records control, provision for handling complaints, prevention of conflicts of interest, independence of laboratory operations, personal and institutional integrity, rigorous uncertainty analysis, and a commitment to continual improvement of all aspects of laboratory operations and measurements. In short, retaining the services of certified laboratories relieves the retaining organization of the large and expensive burden of verifying the competence of the provider.
Other pipette types
The smallest pipette
A zeptoliter pipette has been developed at Brookhaven National Laboratory
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Pipette". A list of authors is available in Wikipedia.|