A pipette is only as good as the user and the calibration checks that are carried out routinely to check for accuracy and precision. You will receive your new pipette with a calibration certificate, but this will need to be verified for continual use and the pipette kept in tip top condition if results are to be meaningful. In our experience the success of an experiment or a manufacturing process can be highly dependant on these parameters especially where small volumes are involved, i.e. immuno-assay conjugate volumes, addition of cofactors in IgG Cardiolipin assay manufacturing whereby the variation in less than a tenth of a microlitre will have a large impact on the results obtained when the immuno-assay kit is used to determine cardiolipin antibody levels. This could lead to erroneous results for the patient and wrong diagnoses.
Environmental Conditions for Checking Pipette Calibration
The test should be carried out in a draught-free room with a stable environment. The test room should have humidity and temperature control so that the atmospheric conditions of the environment where the procedure will take place and the temperature of the equipment used are stable and homogeneous before and during the procedure. The temperature of the pipettes being verified and the distilled water (grade 3, ISO 3696) used in the gravimetric test should have stabilised before the procedure commences. The pipettes, water and test apparatus should have been placed in the test room at least 2 hours before starting the tests. Ideally, verification takes place under the following conditions:
1) Temperature (t)
ISO 8655 recommends that the gravimetric tests take place where the ambient and water temperature (t) are stable (± 0.5 °C) between 15 °C and 30 °C. The recommended range between 20 °C and 23°C with a constant temperature (± 0.5 °C) between the beginning and the end of gravimetric tests. It is recommended to put water and pipettes at least 2 hours in the calibration room to reach an equilibrium with the room conditions. Z-factor is used to convert mass into volume according to temperature and pressure.
2) Relative humidity (RH)
ISO 8655 states that the RH must be greater than 50%. However, it is recommended that a humidity range of between 50% and 75% be maintained throughout the verification procedure. In all cases, the evaporation rate will be evaluated for volumes 50 µL.
3) Barometric pressure
Tests should take place at 1013 ± 25 hPa. The barometric pressure in the test room should be recorded to the nearest 0.5 kPa. Z-factor is used to convert mass into volume according to temperature and pressure.
Consistent pipetting technique needs to be taken into consideration and the person carrying out the tests should be a competent and experienced pipette user. Inexperienced pipette users can cause substantial variations in the results obtained so this factor has to be taken into account. A steady rhythm is important with tip depth, speed and smoothness all being variables that need to be controlled.
The pipette should be clean before testing and fitted with a new tip that is designed fro the pipette being tested. An ill fitting tip will cause erroneous results and possibly leakage.
Test Equipment Required:
Balance – Balances should be serviced, calibrated and certified for pipette calibration. The sensitivity of the balance should be consistent with the accuracy required. This can be quantified as one tenth of the deviation to be assessed ( ISO 8655-6, Table 1.). Many balances now have built in programs for pipette calibration like the Ohaus EX semi micro balances.
The balance table on which the balance is placed must be equipped with a marble surface plate that is independent of the perimeter, to avoid transmitting vibrations. For the same reason, the table must not be in contact with a wall. Avoid placing the balance near to a window or near to a door to avoid too long a response time for the balance and irregular evaporation caused by drafts. Use a calibrated thermometer to measure the water temperature at the beginning and at the end of each test series. Use a thermometer with a maximum uncertainty of measurement of 0.2 °C. The hygrometer shall have a standard maximum uncertainty of 10% and the barometer a standard uncertainty of less than or equal to 0.5 kPa.
Special containers are used to receive water from the test pipette during weighing. Controlling evaporation during the gravimetric test is essential. To minimize evaporation, you can use custom-designed cylindrical flat-bottomed weighing containers made of nonporous plastic.
The liquid used for testing must be distilled or deionized water grade 3 (degassed) conforming to ISO3696 at room temperature. To avoid fluctuations in water temperature, use a large container as a water reservoir. The reservoir should contain sufficient water for all of the tests.
• Always follow the same pre-established control protocol
• Only trained personnel should perform instrument control and calibration
• Instrument should always be cleaned and in perfect working condition
• All temperatures (instrument, room, water) should be stabilised (between 21-25°C)
• Use analytical balance with digit number in relation with the measured volume (5 digits for volumes over 25 µL, 6 digits under 25 µL)
• Use a humidity trap when controlling volumes below 25 µL
• Perform the control measurements without interruption
• Use pipette tips recommended by the manufacturer
• Do not recycle tips after control
1 Place clean reservoir on analytical balance and zero the balance Pre rinse two or three times on nominal (maximum) volume to equal liquid/tip/air cushion temperature.
2 Set instrument to desired volume (always start by minimal volume on adjustable models)
3 Take new pipette tip and rinse twice to pre-wet internal tip surface
4 Aspirate slowly first sample. Do not slide along reservoir wall. If water remains on external tip wall, start operation again or use a new tip.
5 Empty completely the tip on the balance reservoir, sliding tip briefly along reservoir wall so that no liquid remains in the tip
6 Register sample weight after balance stabilisation, then zero the balance
7 Repeat actions 4 to 6 as many times as defined in SOP (i.e.10 x)
8 Calculate mean weight and transform into volume ( multiply by 1/water density at given temperature and atmospheric pressure)
9 Do the necessary statistical evaluation (E for inaccuracy, CV for imprecision)
10 Compare inaccuracy and imprecision with values recommended by the manufacturer or defined in the internal SOP. If necessary, perform calibration according to manufacturer’s instructions (or send the instrument back to the authorized dealer) Please note: Set instrument on minimal volume before performing any calibration (modification of the calibration settings) and check resulting value on both min and max volume.
Refer to this document for calculation guidelines.
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