Pipettes Calibration Facts
On the surface, the process of calibrating a pipette appears to be a simple procedure; however several important factors can greatly influence the accuracy of a pipette. Among those factors are: the calibration technique, balance resolution, how the water density is determined , the calibration/refurbishing procedure , and the parts replaced .
There are numerous ways to calibrate a pipette including measuring of radio nuclide, color change reactions, air pressure/vacuum testing, and gravimetric.
Gravimetric - 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.
Radio nuclide – The principle behind radio nuclide calibration is that if a fluid containing a known amount of radioactivity per unit volume is used one can determine the volume of a pipette by the equation:
Volume ( ul ) = ( Disintegrations per minute (dpm ) / counter efficiency for the isotope ) / dpm/ml in the standard solution
Although this technique can be highly accurate the obvious drawbacks of using radioactivity make it an impractical calibration technique.
Color change reactions – In color change calibrations, a chemical reaction producing color is used to calibrate a pipette. Using a test tube or microtiter plate, a known and equal amount of one reactant is added to each well, then the pipette to be calibrated is used to dispense the second reactant into the well. Since chemical reactions are dependant upon the concentration of each reactant, the more reactant that is pipetted into the well the more intense the color that will develop in that well. The color intensity is then converted into a volume by using a standard curve of color intensity versus volume of reactant.
Methods based on color change are secondary calibration techniques, ie. you must transform data using a standard curve, and as such can be inaccurate, inconsistent and highly susceptible to error.
Air pressure/vacuum – This technique is not suited to calibrate pipettes, in fact the technique is not quantitative and only serves as a qualitative test for the condition of the seal inside the pipette.
Any service using this technique is not calibrating your pipette, they are merely testing if the pipette seal is worn or not!
Since gravimetric calibration is the most widely used standard, let’s look into factors that can affect the accuracy of the calibration.
Balance – Typically when calibrating a pipette you should use a balance that has at least a four times higher resolution than the quantity you are testing against. As an example if a P20 is being calibrated at 2 ul where the accuracy tolerance is +/- .012 ul, the balance used should have a resolution of at least .03 mg. When a 4 to 1 resolution is not possible (either because the technology does not exist or conditions prevent measure to that level resolution), the resolution ratio should be stated on any calibration document.
A company calibrating your pipettes should be using a 5 place balance for pipettes whose maximum volume is 10 ul or greater and a 6 or 7 place balance for pipettes less than 10 ul.
Water density is dependant upon temperature and barometric pressure. Because we are using the water density to convert the weight of water dispensed by a pipette into the volume of water dispensed by a pipette, it is extremely important that the water density is determined properly and accurately. Most services use air temperature instead of water temperature to determine the water density, which will produce an incorrect value for the water density. The reason for this is that typically the water being used to test your pipette is in an open container, be it a beaker or trough. As such, the water in the vessel is being cooled by the evaporation of water into the air from the surface of the water. This evaporative cooling typically produces water temperatures that are 1 or more degrees centigrade cooler than the air temperature.
A calibration service should measure the temperature of the water being used to calibrate your pipette and the barometric pressure at the instant that pipette is being calibrated.
Your pipette should always be calibrated to manufacturer’s specifications and over the entire range it was designed to operate. A Gilson P200 should be calibrated at its extremes of 50 and 200ul, and at the midpoint 100ul. Many services do not calibrate either to manufacturer’s specifications or over the entire range the pipette was designed to operate.
A calibration service should always calibrate your pipettes to manufacturer’s specifications and over it’s entire operational range (minimum, maximum and midpoint design volumes) unless the user specifies otherwise.
Typically the piston inside a pipette is made of highly polished steel or ceramic. In either case, abrasives should never be used to clean these surfaces. Some companies use either abrasives or steel wool to clean pistons both of which will damage the piston of the pipette.
A calibration service should only use non-abrasive cleaning methods to refurbish the internal components of your pipette.
Replacing parts on your pipette will not effect the pipette’s calibration. This statement is patently false; no two parts produced will be identical, there will always be a variation in a part’s dimension. It is therefore extremely likely that changing a part such as a seal, piston or shaft will alter the calibration of your pipette.
A pipette’s calibration should always be checked when a part is replaced.
Pipettes should only be serviced if they are not working correctly. Typically a pipette can appear to be functioning properly and may in fact be in calibration even though the piston may be contaminated with salts and other laboratory chemicals.
As a rule a pipette should be serviced at least once a year to minimize or prevent damage to internal components such as pistons and springs.