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Pipette calibration - Trust your assay results

A pipette is a laboratory instrument used to transport a measured volume of liquid. It works by creating a vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw up and dispense liquid.

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. Accuracy and precision is of utmost importance in any pipetting job, however there are many sources of error affecting pipette calibration which in turn affects the performance of the pipette.

Simple, reliable pipette calibration maximizes liquid handling quality. For sustained accuracy and consistent and repeatable operation, pipettes should be calibrated at periodic intervals.

Accuracy and precision

Inaccuracy is the numerical difference between the average of a set of measurements and the nominal value. Imprecision means variation between repeated measurements.

Low imprecision means that calibrator can perform very consistent calibration (taking into consideration that pipette is in good shape).

COMPONENTS OF QUALITY CALIBRATION

Components of quality calibration can be divided into three parts: Operator, Equipment and Environment. Impact of these components on pipetting results are as follows:

Leaky piston / cylinder system (1-50%),
uneven piston movement (0.5%), uneven pipetting rhythm (1.5%), immersion depth (1.0%), liquid/air temperature variations (0.3%), changes in relative humidity (3.0%), failure to pre-wet tips (2.0%), failure to "touch off" (3.0%), poorly sealed pipette tips (0.5-50%), reusing pipette tips (4.0), variations in air pressure (0.2%), differences in liquid density (1.0%), differences in vapor pressure (2.0%)

OPERATORS TECHNIQUE

Operator's pipetting technique plays the most important role in pipettor calibration. The misconception with pipetting is that people consider it to be a very easy job. But usually this is not the case, especially if the operator has never been trained for pipetting techniques and calibration. Pipetting technique must be paid attention to, especially with mechanical pipettors. The way operators handle plunger movement can make a big difference.

Good pipetting recommendations are as follows:

• Pipettes should be held vertical during aspiration interval
• Holding a pipette at 30 degrees angle off vertical position can cause 0.7% more liquid to be aspirated
• Tips should always be pre-wetted
• Immersion depth should be between 2-3mm
• Plunger release should be smooth, methodical and consistent
• The "Touching off" technique should be employed when aspirating and dispensing

The graph above shows the difference between experienced and inexperienced operators.

EQUIPMENT

A) Tips

• To prevent error, tips usage should be as recommended by the manufacturer
• They should be manufactured from high quality non-wettable virgin grade polypropylene (clean, hydrophobic surface)
• They should have perfectly centered opening with smooth interior & straight even sides
• They should be uniform in construction with a good tip cone match for perfect seal & tip ejection
  Also, choose as small a tip as possible, to reduce the air dead space and ensure that the manufacturing facility is covered by a quality system.
  
  Defects in tips

B) Balance and accessories

• Pipetting error may occur if balance does not have enough digits. In other words, readability is not sufficient enough (Electronic balance with resolution 0.001 mg is better recommended, however for calibrating pipettes above 10ul the balance with resolution 0.01mg is acceptable)
• Balance is old or not calibrated
• Evaporation is not controlled
• Container

Some containers force liquid into a concave configuration while other force it into a convex configuration.

Glass transfer containers are recommended, since they force liquid into a concave configuration and help to eliminate the effects of hydrostatic pressure

• Viscosity of transfer liquid

If liquid other than de-ionized water (ISO 3696) is used, it will impact calibration results.

ENVIRONMENT

A) Barometric Pressure: Pressure is reduced in accordance with elevation.
B) Relative Humidity: Accepted guidelines for pipette volume delivery specify that relative humidity should be between 45%-75%. Humidity traps can be employed in order to create a "micro-climate" inside the balance, humidity should be monitored in the room and inside the evaporation trap
C) Temperature: Volume delivery performance is usually defined between 20-25 °C. Stabilisation period of two hours before calibration is recommended in order to reduce the effects of thermal expansion on pipette, sample and tips. Pipette, tip and liquid must always be kept at the same temperature.

ISO 8655 - 2 Specification With Maximum Permissible Errors For Nominal Volumes & Pipette Calibration. Under average conditions GLP certified labs should check for pipettes calibration every six months to provide satisfactory performance.

Performance testing

It is important to understand the difference between calibration and performance testing because most often the performance testing is thought to be calibration, which it is not. Calibration is the adjustment of the pipette or the precision instrument to give out the selected volume. Performance testing is the actual testing procedure, verification of the pipette by using the gravimetric, photometric or other test methods. In other words, it is the check that the calibration of the pipette is correct.

Usually, when arriving from the production line, the pipette fulfils the type specifications given by the manufacturer. The performance test confirms this. In case the results are incorrect the pipette will need calibration. If calibration is needed, the pipette will be performance tested again. After approved results, a quality control certificate with test results is printed for each individual pipette.

Gravimetric analysis for pipette calibration

In gravimetric calibration you are measuring the weight of water a pipette dispenses at a given dial setting. This weight must then be converted into a volume by using the physical property of water density.

Volume (ul ) = Weight of water dispensed ( mg ) / density of water (mg/ml)

Gravimetric calibration is one of the most accurate ways to calibrate a pipette and is the accepted standard for the calibration of pipettes.
The technique 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.

There is a clear difference between the performance level that the pipettor must have when it leaves the factory (controlled testing conditions) and the performance that is possible to reach by the user in various non-standard conditions (environmental conditions are not optimum or controlled). In addition to varying conditions, pipetting techniques may not be adhered to and the pipetted fluid is seldom purified water.

Conclusions

The trend today is to look for reassurance that the purchased products fulfill laboratory requirements and meet high end-user expectations. This often leads to testing or measurement of characteristics against a standard or specification. To be able to test accurately and reliably, the testing laboratory should have the full competence to perform calibration according to international standards. There are two sources of uncertainties in the calibration of pipettors, namely the uncertainty of the pipettor and the uncertainty of the gravimetric test method and its related apparatus. An accredited calibration service can differentiate between these uncertainties and give out the combined uncertainty of the measurement. The calibration certificate given and signed by the accredited calibration service separates the uncertainties of the balance and the test method and, thus, eliminates the sources of error. As a result, the customer can have full confidence using the pipettors in their tests.

(Article contributed by Transasia Bio-Medicals E-mail: transasia@transasia.co.in)