🔬 Pipette Calibration (Volumetric)
In short
Gravimetric determination of systematic and random errors of piston-operated pipettes according to ISO 8655. The test is performed according to PN-EN ISO 8655-6:2022; Volume range: 1 µL – 10 000 µL. The procedure comprises 10 steps (about 3 h 5 min–3 h 10 min in total); it is used for: Periodic calibration of pipettes in accredited laboratories (ISO/IEC 17025), Pipette calibration in pharmaceutical laboratories (GMP/GLP), Pipette verification in diagnostic laboratories (ISO 15189).
At a glance
- Standard: PN-EN ISO 8655-6:2022
- Category: Metrology
- Procedure steps: 10
- Total time of stages: 3 h 5 min–3 h 10 min
- Volume range: 1 µL – 10 000 µL
- Permissible systematic error: ±0.8% (100 µL) to ±5% (1 µL) according to ISO 8655-2
- Permissible random error (CV): ≤0.3% (100 µL) to ≤5% (1 µL) according to ISO 8655-2
Overview
Calibration (verification) of piston-operated pipettes by the gravimetric method is a key quality assurance procedure in analytical laboratories. Standard PN-EN ISO 8655-6 defines the reference measurement method — weighing water dispensed by the pipette on a microanalytical balance with conversion of mass to volume taking into account water density, temperature and atmospheric pressure.
For each pipette, two types of errors are determined: systematic error (es) — difference between the average dispensed volume and the nominal volume, and random error (CV) — coefficient of variation of the measurement series. Standard ISO 8655-2 specifies the limit values of es and CV for fixed and variable volume pipettes in the range 1 µL – 10 000 µL.
Calibration is performed at 3 volumes (nominal, 50% nominal, 10% nominal for variable pipettes) with series of 10 measurements for each volume. Environmental conditions must be strictly controlled: temperature 20±2°C, humidity >50%, no drafts. Pipette tips must be new (disposable) — replaced after each series.
In Poland, the leading manufacturer of pipette calibration stations is RADWAG, offering SDKP and SDKP DUAL stations integrated with PIPETY software. Pipette calibration services are provided by RADWAG, HTL, Eppendorf Service and accredited measurement laboratories.
Method principle
The gravimetric method consists of dispensing distilled water with a pipette into a weighing vessel placed on a microanalytical balance. The mass of the dispensed water portion is converted to volume using the formula V = (m × Z), where m — water mass, Z — correction factor taking into account water density at measurement temperature, atmospheric pressure and air density. Based on a series of 10 measurements, systematic error (es = V_average − V_nominal) and random error (CV = s / V_average × 100%) are calculated.
Applications
- Periodic calibration of pipettes in accredited laboratories (ISO/IEC 17025)
- Pipette calibration in pharmaceutical laboratories (GMP/GLP)
- Pipette verification in diagnostic laboratories (ISO 15189)
- Control of multichannel pipettes (8/12/96-channel)
- Calibration of pipettes after service/repair
- Validation of electronic and automated pipettes
Key parameters
| Parameter | Value |
|---|---|
| Volume range | 1 µL – 10 000 µL |
| Permissible systematic error | ±0.8% (100 µL) to ±5% (1 µL) according to ISO 8655-2 |
| Permissible random error (CV) | ≤0.3% (100 µL) to ≤5% (1 µL) according to ISO 8655-2 |
| Number of measurements | 10 dispensings for each test volume |
| Environmental conditions | 20 ± 2°C, humidity > 50%, no drafts |
| Water temperature | Thermal equilibrium with environment (±0.5°C) |
Standard
- Standard number
- PN-EN ISO 8655-6:2022
- Title (PL)
- Urządzenia tłokowe do objętościowego odmierzania cieczy — Część 6: Metoda grawimetryczna wyznaczania błędu pomiaru
- Title (EN)
- Piston-operated volumetric apparatus — Part 6: Gravimetric reference measurement procedure
Step-by-step procedure
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Acclimatization
Acclimatize pipettes, tips, water and station for min. 2 h in calibration room. Record T, RH, pressure.
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Balance preparation
Turn on microanalytical balance min. 60 min before measurement. Perform internal adjustment. Check repeatability.
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Weighing vessel preparation
Place weighing vessel with lid (evaporation trap) on balance pan. Add a small amount of water. Zero the balance.
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Pre-pipetting
Attach new tip. Perform 5 cycles of aspiration and dispensing of water (pre-pipetting) to wet the internal walls of the tip.
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Measurement series
Dispense water into weighing vessel. After each dispensing record the balance indication. Perform 10 consecutive dispensings. Between dispensings do not wait longer than 10 s.
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Volume change
For variable pipettes repeat the series of 10 measurements for 50% and 10% of nominal volume. Change tip when changing volume.
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Mass to volume conversion
Calculate volume of each dispensing: V = m × Z, where Z — correction factor (ISO 8655-6 tables or calculation from water density, air and pressure).
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Error calculation
Determine systematic error es = V_average − V_nominal and random error CV = (s / V_average) × 100%. Compare with ISO 8655-2 limits.
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Conformity assessment
If es and CV are within limits — pipette compliant. If not — pipette requires adjustment (calibration screw adjustment) and re-calibration.
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Documentation
Prepare calibration certificate with results, uncertainties, environmental conditions and next calibration date.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Microanalytical balance | RADWAG MYA 11.4Y, Mettler Toledo XPR2U, Sartorius Cubis II 2.7S | 30 000–90 000 PLN |
| Calibration station | RADWAG SDKP / SDKP DUAL with PIPETY software | 80 000–150 000 PLN (complete set) |
| Evaporation trap | Weighing vessel with lid and capillary | 500–2 000 PLN |
| Thermohygrometer | Testo 176 H1, Pt100 water temperature sensor | 2 000–5 000 PLN |
| Barometer | Druck DPI 142, Vaisala PTB330 | 3 000–8 000 PLN |
| Disposable tips | Eppendorf epT.I.P.S., Brand, HTL — original for given pipette model | 50–300 PLN / 1000 pcs |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Distilled / deionized water | — | Class 2 according to ISO 3696, degassed, in thermal equilibrium with environment |
| Wetting solution | — | Optionally — surfactant to reduce surface tension at very small volumes |
Health and safety (OHS)
- Avoid skin contact with tips (grease contamination changes wettability)
- Do not touch weighing vessel with bare hands — use tweezers
- Ensure stable environmental conditions — closed windows, no forced air conditioning
- Water must be degassed — air bubbles disturb results
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 8655-6:2022 — “Piston-operated volumetric apparatus — Part 6: Gravimetric reference measurement procedure”.
How does this method work?
The gravimetric method consists of dispensing distilled water with a pipette into a weighing vessel placed on a microanalytical balance. The mass of the dispensed water portion is converted to volume using the formula V = (m × Z), where m — water mass, Z — correction factor taking into account water density at measurement temperature, atmospheric pressure and air density.
What is the measuring range and accuracy?
Volume range: 1 µL – 10 000 µL; Permissible systematic error: ±0.8% (100 µL) to ±5% (1 µL) according to ISO 8655-2; Permissible random error (CV): ≤0.3% (100 µL) to ≤5% (1 µL) according to ISO 8655-2; Number of measurements: 10 dispensings for each test volume.
How long does the test take?
The times given for the individual stages add up to approximately 3 h 5 min–3 h 10 min. The procedure comprises 10 steps.
What equipment is required?
Microanalytical balance, Calibration station, Evaporation trap, Thermohygrometer, Barometer, Disposable tips. Indicative cost of the core instrument (Microanalytical balance): 30 000–90 000 PLN.
Where is this test used?
Periodic calibration of pipettes in accredited laboratories (ISO/IEC 17025); Pipette calibration in pharmaceutical laboratories (GMP/GLP); Pipette verification in diagnostic laboratories (ISO 15189); Control of multichannel pipettes (8/12/96-channel); Calibration of pipettes after service/repair; Validation of electronic and automated pipettes.
What safety precautions apply?
Avoid skin contact with tips (grease contamination changes wettability); Do not touch weighing vessel with bare hands — use tweezers; Ensure stable environmental conditions — closed windows, no forced air conditioning; Water must be degassed — air bubbles disturb results.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 8655.