Leave the distilled water out for about 15 minutes to ensure temperature consistency. Use the thermometer to measure the temperature of the distilled water. Record. Place the empty, clean beaker on the balance. Tare the beaker’s weight. Ensure the pipettes are clean, unclogged and ready to function. When filling the pipette up to the volume that will be calibrated (1ml, for example) with the distilled water, make sure you don’t create air bubbles in the flask or in the pipette. Dispense the distilled water in the beaker using the pipette. Use the balance to measure the distilled water’s weight. Repeat 5-10 times at least, recording each measurement. Use the formula V = w * Z to calculate the volume dispensed by the pipette. W is the weight of the distilled water. Z is the “conversion factor based on the density of the water” and V is the calculated volume of dispensed water. The temperature of the water determines its density, which is why it is important to keep the water temperature as constant as possible.
Under a constant temperature and atmospheric pressure, the density of distilled water is constant. The
volume of water can be determined by weighting dispensed water. The calibration of pipette is carried out by gravimetric method. When determining the volume of water, the accuracy of measurements is effected by ambient temperature, atmospheric pressure and relative humidity. These factors are usually combined to give the Z factor, used in calculation of volume of water. Then the calculated volume of water is compared with the theoretical volume to determine the accuracy and precision of the pipette.
It is a process of examining and/or adjusting the pipettes to the true value by comparison to the standard which is set by the manufacturer of that said pipette. The standard in this case is the pipetting range set by the pipette manufacturer to be accurate and precise at low volume and high volume of the pipette. Pipette calibration table of Z factors for distilled water at 1 atm pressure Source: Wisconsin State Laboratory of Hygiene Steps Involved in Pipette Calibration.
Determine the water temperature and record it. Place a beaker filled with distilled water into analytic balance and close the door of balance waiting for equilibrium of inner vapor. Place a plastic medicine cup on the pan and adjust the weight to zero. Put a tip onto the pipette and set the volume which is to be tested. Pre-rinse the tip: aspirate and dispense the setting volume three times and press the push – button on the second stop to remove any remaining liquid. Press the push-button to the first positive stop. Hold the pipette vertically, immerse the tip so that 1-4 mm in the liquid and release the push button slowly and smoothly to aspirate the liquid. Wait one second and withdraw the tip from the liquid. Wipe any droplets away from the outside of the tip using a kimwipe.
The Verification Procedure certifies both pipette accuracy and precision. Environmental conditions, test equipment, and other qualifications previously described in this document should be implemented to assure the validity of the test results. After pre-rinsing the tip, record ten individual weighings per elected volume. For variable volume pipettes, three volume settings are selected per pipette model based on the pipette’s useful volume range (nominal, approximately 50 % and minimum volume or 10% of the nominal volume). For fixed volume pipettes (Pipetman F) only the nominal volume is used.
Pipette calibration is necessary to prevent inaccuracies in order to attain better results, performance, and longevity of the pipette itself.
The Clinical and Laboratory Standards Institute (CSLI) recommends that pipettes (single and multi-channel) and automated liquid handlers be calibrated every 3 to 6 months. A minimum of two volumes must be tested (nominal and lowest setting) with ten replicates at each volume.
The most common way to check your pipette accuracy is by weighing water. The density of water is 1 g/mL. This means that every microliter (µL) should weigh 0.001 g. In other words, if your pipette is accurate, the amount of water you dispense will equal the amount the water weighs.
Calibration involves checking the reading given on the balance against standard known-mass calibration weights. Most electronic balances in an educational setting require Span Calibration, which involves calibrating the balance at two points on its scale. These are usually zero and maximum capacity.