Pipette calibration: the ISO 8655 gravimetric method, step by step

Pipette calibration means measuring how much liquid a pipette really delivers and how much that amount varies, then comparing both with a limit. The reference method is gravimetric: you dispense distilled water onto an analytical balance, convert each weight into a volume with the Z factor (which accounts for the density of water and air buoyancy), and calculate the systematic error (how far the mean is from the set volume) and the random error (how much the deliveries scatter). Under ISO 8655-6:2022 that means three volumes, at least ten weighings each, a tip change at each volume, a controlled room and a balance chosen for the pipette’s size (ISO 8655-6, 5.2 and 8.1; tip change: Sartorius white paper).

Once the pipette, tips and water have equilibrated, a lab tech can do a quick version of the same test in about fifteen minutes with fewer readings. It is a useful check, but it is not a calibration to ISO 8655 unless every requirement of Part 6 is met (ISO 8655-6, clause 4). This page covers both, with a worked example and a free calculator.

Can you calibrate a pipette yourself?

You can check it and, with the manufacturer’s tool, adjust it. Whether the result is a calibration to ISO 8655 depends on how you do it:

The middle column is based on Sartorius’s summary of Part 7, which allows routine tests with fewer volumes and at least four measurements per volume, and alternative gravimetric procedures with relaxed conditions (Sartorius, ISO 8655 white paper; SelectScience, 2022). Part 2 asks users to set up routine testing as described in Part 1 (ISO 8655-2, 9.1.3). For a calibration, the uncertainty of the result is required; for a test it is optional (Sartorius white paper).

A quick gravimetric check

1. Let everything reach room temperature: pipette, tips and a beaker of distilled water, for at least two hours. Record the water temperature and, if you can, the air pressure.
2. Use a balance fine enough for the volume (see the balance table below). Put a small weighing vessel on it with a little water in the bottom and close the doors.
3. Pre-wet the tip by aspirating and dispensing the test volume five times.
4. Dispense and weigh at least four times, ideally ten, taring between deliveries and keeping a steady rhythm.
5. Convert each weight to volume by multiplying the reading in mg by Z (about 1.003 µL/mg; see the Z table), then calculate the mean, the systematic error and the CV with the calculator below.
6. Compare with the manufacturer’s specification for that volume. If the pipette is outside it, take it out of use and have it calibrated and, if needed, serviced.

ISO 8655:2022 in brief

ISO 8655 is the international standard for piston-operated volumetric apparatus: pipettes, burettes, dilutors, dispensers and syringes. The second edition was published in 2022 and has nine parts, two more than the 2002 series (European Instruments, 2023; SelectScience, 2022). A Part 10, with user guidance and requirements for competence and training, followed in 2024 (ISO 8655-10:2024, catalog entry):

Part 6 is the one you follow to calibrate by weighing; Part 2 says what the results must meet. ISO 8655 describes how to calibrate; ISO/IEC 17025 sets the requirements for the laboratory that does it (Sartorius, ISO 8655 flyer).

What changed from the 2002 edition

The change to the error limits matters most for variable pipettes. Under the 2002 tables a 100–1,000 µL pipette had to deliver within ±8 µL at every setting: ±0.8 % at 1,000 µL, but ±8 % at 100 µL (Thermo Fisher, Finnpipette specifications; Troemner). The 2022 edition sets limits at the lower volumes directly, so users no longer have to derive them (Eppendorf, 2023).

What did not change: the ten measurements per volume. The 2002 limits were already expressed for “a tenfold measurement”, and the 2022 text still asks for at least ten (Troemner; ISO 8655-6:2022, 8.1.2). Eppendorf compared both editions and found the limits at nominal volume unchanged for single-channel pipettes above 100 µL, at 0.8 % systematic and 0.3 % random error, while several small-volume limits changed, most of them tightened (Eppendorf, 2023).

What you need to calibrate a pipette

• Analytical balance

  Resolution, repeatability and uncertainty in use chosen from ISO 8655-6 Table 1 for the pipette's nominal volume

  The table is set so that the balance's expanded uncertainty in use is below one quarter of the pipette's permissible systematic error. Calibrate the balance where it is used (see scale calibration) and check it between calibrations with calibrated weights.

  ISO 8655-6, 5.2; DKD-R 8-1, §8.2

• Thermometers, hygrometer, barometer

  Water: 0.1 °C resolution, 0.2 °C uncertainty. Air: 0.1 °C, 0.3 °C. Humidity: 1 %, 5 % RH. Pressure: 0.1 kPa, 1 kPa (k = 2)

  All traceable to the SI, plus a timer with 1 s resolution.

  ISO 8655-6, 5.1 and Table 2

• Test liquid

  Distilled or deionized water, grade 3 or better (ISO 3696), within ±0.5 °C of the air

  Grade 3 means a conductivity below 5 µS/cm. Never tap water.

  ISO 8655-6, clause 6; DKD-R 8-1, §8.3; NIST SOP 14

• Test room

  Draft-free, (20 ± 3) °C, 45–80 % RH, temperature stable within ±0.5 °C during the test

  Pipette, tips, water and equipment in the room for at least 2 hours before you start; often longer.

  ISO 8655-6, 7.2

• Weighing vessel and evaporation control

  A vessel with a little water in the bottom; a lid or an evaporation trap for small volumes

  Evaporation matters most below 50 µL.

  ISO 8655-6, 5.4, 7.3 and 8.1.3

• Tips and adjustment tool

  Tips approved for the pipette, at least two per test volume; the manufacturer's adjustment tool

  Record the tip type: the result is valid for that pipette and tip combination only. See also how to write a calibration procedure.

  DKD-R 8-1, §5.3; ISO 8655-2, 5.3

Which balance?

The table is keyed to the nominal volume of the pipette, not to the volume being tested, so a 1,000 µL pipette tested at 100 µL can use a 0.1 mg balance (ISO 8655-6, 5.2 and Table 1). Resolution is only the first requirement: the balance must also meet the repeatability and the expanded uncertainty in use of Table 1 (for example 0.006 mg and 0.012 mg in the smallest range), which you take from its calibration certificate. Not every certificate states an uncertainty in use (Sartorius white paper). The 2002 column is from a balance maker’s summary of the old table, which was set by the volume tested rather than the nominal volume (Radwag). When a single-channel balance is used only for multichannel pipettes, those two values are doubled, and a multichannel balance measures all channels in parallel (ISO 8655-6, Table 1, notes a and d; SelectScience).

Test volumes, number of measurements and tips

A fixed-volume pipette is tested at its nominal volume. A variable-volume pipette is tested at least at three volumes: the nominal volume, 50 % of it, and the lower limit of the usable range or 10 % of nominal, whichever is greater. Each volume gets ten measurements or more (ISO 8655-6, 8.1.1 and 8.1.2), and the tip is changed at least once per volume (Sartorius white paper; SelectScience).

Multichannel pipettes are tested channel by channel: a 12-channel pipette at three volumes means 360 weighings (Sartorius). On a single-channel balance, all channels aspirate together and only the channel being measured dispenses into the weighing vessel; on a multichannel balance, all channels are measured in parallel (Sartorius white paper; SelectScience).

Step-by-step gravimetric procedure

The sequence follows ISO 8655-6 and the as-found and as-left logic of any calibration. Write your choices into your calibration procedure.

1. 1

   Identify and inspect

   Record the pipette ID, model, volume range and tip type. Check for cracks, a bent or dirty shaft, leaks, an unintended change of the volume setting and a secure tip fit.

   Damaged or leaking: record it, test as found only if useful, then service (DKD-R 8-1, §5.3)

2. 2

   Equilibrate

   Leave the pipette, tips, water and equipment in the test room for at least 2 hours within the room conditions (ISO 8655-6, 7.2 and 8.2).

3. 3

   Record the conditions

   Water temperature at the start and end of the measurements; air temperature, pressure and humidity (ISO 8655-6, 8.1.4).

4. 4

   Set the volume and pre-wet

   Select the test volume and do not change it during the series. Fit a tip and aspirate the test liquid five times to saturate the air inside the pipette (ISO 8655-6, 8.2 and 8.3.2).

5. 5

   Run the test cycle

   Forward pipetting only. Ten deliveries per volume, a steady rhythm, the balance zeroed or read before each delivery, the tip changed at least once per volume. Keep the cycle time short and regular (ISO 8655-6, 7.4 and 8.3.1; tip change: Sartorius white paper).

6. 6

   Correct and calculate (as found)

   Apply the evaporation correction, convert each reading to volume with Z, and calculate the mean, the systematic error and the CV. Compare them with your limits.

   Outside the limits: record as found and open an out-of-tolerance report

7. 7

   Adjust if needed

   Following the manufacturer's instructions, with its tool. Mark the pipette so the change from the initial adjustment is visible, and record it (ISO 8655-2, 5.3).

8. 8

   As left

   Repeat the full test after any adjustment or repair and record it as a separate set. If nothing was adjusted, the as-found results are also the as-left results.

9. 9

   Uncertainty, record and label

   Evaluate the measurement uncertainty (required for a calibration), complete the record or certificate and label the pipette.

Technique matters as much as the balance. Hold the pipette vertically and immerse the tip only a few millimeters: DKD-R 8-1 suggests 2–3 mm and a 1 s wait for 1–100 µL, 2–4 mm for 100–1,000 µL, and 3–6 mm with a 3 s wait above 1,000 µL. Withdraw the tip slowly without touching the wall of the reservoir (DKD-R 8-1, §7.3), and dispense with the tip touching the wall of the weighing vessel, in the same way every time (the tip requirements of ISO 8655-2, 7.1.1, assume deliveries made this way). Handle the pipette as little as possible: hand warmth makes the delivered volume drift down over a series (ISO 8655-2, 6.2; DKD-R 8-1, §8.9.3). If the manufacturer specifies a different delivery procedure, you may follow it and document it (ISO 8655-6, 8.1.5).

From mass to volume: the Z factor

The balance gives a mass. To turn it into a volume you divide by the density of water and correct for air buoyancy, because the balance was adjusted with steel weights of about 8,000 kg/m³ while water displaces far more air. EURAMET cg-19 and NIST SOP 14 use the same equation (cg-19, equation 1; NIST IR 7383, SOP 14, equation 1):

V = m × 1 / (ρ_W − ρ_A) × (1 − ρ_A / ρ_B)

where ρ_W is the water density at the water temperature, ρ_A the air density and ρ_B the density of the balance’s reference weights (8.0 g/mL). Everything after m is the Z factor, in µL/mg. ISO 8655-6 allows either the general formula or Z (ISO 8655-6, 9.3).

The water density comes from the Tanaka formula, with a small correction for dissolved air; the air density from the simplified CIPM formula, valid from 600 to 1,100 hPa, 15 to 27 °C and 20 to 80 % RH (cg-19, equations 2 to 4; NIST IR 7383, GLP 10). Water temperature matters most: Z changes by about 0.0002 µL/mg per °C near room temperature. Pressure matters less: at 900 hPa, about 1,000 m above sea level, Z is only about 0.0001 lower. Our calculation reproduces the published values for air-saturated water at 20 °C (998.204 kg/m³) and DKD’s worked example (100.059 mg at 21.7 °C gives 100.38 µL) (NIST IR 7383, GLP 10, Table 2; DKD-R 8-1, Appendix B).

Evaporation

Water evaporates from the weighing vessel while you work, so the balance reading creeps down and every delivery looks smaller than it was. ISO 8655-6 treats this as a known error, most important below 50 µL: keep the test cycle short and regular, reduce evaporation with the vessel geometry, a lid or an evaporation trap, keep water in the bottom of the vessel to raise the humidity, then determine the loss (during the cycle or in a separate study), correct the results for it and include the correction’s uncertainty (ISO 8655-6, 7.3, 7.4 and 8.1.3). In one balance maker’s test, an evaporation trap cut the loss from a 14.5 mm vessel from 0.015 mg to 0.003 mg in ten minutes (Radwag). That is why NIST’s general gravimetric procedure is not meant for micropipettes unless extra evaporation corrections are made (NIST IR 7383, SOP 14, §2).

Humidity, temperature and altitude

Air-displacement pipettes are sensitive to the air cushion between the piston and the liquid. A tiny amount of evaporation into that cushion displaces a large volume, which is why pre-wetting helps and why low humidity lowers the measured volumes (DKD-R 8-1, §8.4 and §8.7.2). A temperature difference between the water, the pipette and the air is one of the largest contributions in DKD’s uncertainty budgets, so the guideline asks for less than 0.5 K, and 0.2 K for the smallest uncertainty (DKD-R 8-1, §8.7.1 and Appendix A). At altitude, the lower air pressure makes these pipettes deliver less, so results from different altitudes need a correction to be compared (DKD-R 8-1, §8.7.3; DKD-E 8-1, §5.2.4.3). ISO 8655-2 defines the adjustment of a pipette at 20 °C, 50 % RH and 101.3 kPa (ISO 8655-2, 5.1).

Accuracy and precision: systematic and random error

ISO 8655 judges a pipette on two numbers at each volume (Sartorius white paper; DKD-R 8-1, §2.1 and §8.8):

• Systematic error, e_s = V̄ − V_s: the mean of the delivered volumes minus the selected volume, in µL or as a percentage of V_s. It describes trueness, which labs usually call accuracy: whether the pipette delivers the right amount on average (VIM 2.14).
• Random error, the standard deviation s of the deliveries (with n − 1), usually given as the coefficient of variation CV = 100 × s / V̄. It describes precision: how much the deliveries scatter.

A systematic error can usually be adjusted out. A large random error cannot: an imprecise pipette usually needs repair or replacement, or the method needs a closer look (Troemner). The piston seal, leaks and the fit of the tip are among the things DKD-R 8-1 asks you to check before calibrating (DKD-R 8-1, §5.3).

Worked example: a 100–1,000 µL pipette, as found and as left

Pipette PIP-014 is fictitious: a single-channel, air-displacement pipette tested at 1,000, 500 and 100 µL, ten deliveries each, on a 0.1 mg balance, with water at 21.4 °C, air at 21.6 °C, 1009 hPa and 51 % RH, so Z = 1.00314 µL/mg. The lab’s acceptance limits (also fictitious, set from a manufacturer-style specification, not taken from ISO 8655-2) are ±0.6 % and CV 0.2 % at 1,000 µL, ±0.8 % and 0.3 % at 500 µL, and ±1.5 % and 0.6 % at 100 µL.

• As found, the pipette delivered about 1.1 % too little at 1,000 µL and 1.0 % too little at 500 µL: both outside the systematic limit. At 100 µL the mean was inside its limit (−1.4 % against ±1.5 %), but the CV of 0.75 % was not: the deliveries scattered too much.
• The technician serviced the pipette (seals cleaned and replaced), then adjusted the volume with the manufacturer’s tool to bring the mean back.
• As left, all three volumes were inside both limits, with systematic errors between +0.1 % and +0.6 %.

Because the as-found result failed, everything pipetted with PIP-014 since its last good calibration was delivered about 1 % short at the upper volumes. Our guide to out-of-tolerance results walks through the impact assessment.

Maximum permissible errors

ISO 8655-2 sets maximum permissible errors for the pipette and its tip together, for the systematic and the random error. In the 2022 edition they are given at 100 %, 50 % and 10 % of the nominal volume, with separate tables for multichannel pipettes and for positive-displacement pipettes with disposable capillaries (ISO 8655-2:2022, 9.2 to 9.5; Eppendorf, 2023). At the nominal volume of a single-channel air-displacement pipette between 100 and 5,000 µL, the limits are 0.8 % for systematic error and 0.3 % for random error (CV), the same as in 2002, according to Eppendorf’s comparison of the two editions (Eppendorf, 2023; Troemner for the 2002 values). For the 50 % and 10 % limits, check Table 1 of ISO 8655-2:2022 itself: we have not been able to confirm them from two independent sources, so we don’t quote them.

You may also compare results with the manufacturer’s specification or your own limits, which are usually tighter than the ISO limits; to state compliance with ISO 8655, they need to be equal to or stricter than the ISO values (Eppendorf, 2023; SelectScience). A simple comparison of the error with the limit ignores the uncertainty of the calibration; if your results sit close to the limit, agree a decision rule (see guard banding).

Measurement uncertainty

A calibration states the uncertainty of each mean volume. ISO 8655-6 points to ISO/TR 20461 for an example budget (ISO 8655-6, 5.1 and 9.6), and DKD-R 8-1 and EURAMET cg-19 publish free ones. The balance is rarely the main contribution. In DKD’s budget for a 1,000 µL pipette, the largest terms are the handling contribution (0.40 µL), the temperature difference between water, pipette and air (0.25 µL), repeatability (0.21 µL) and humidity (0.20 µL), against 0.015 µL for the balance; the expanded uncertainty is 1.2 µL, or 0.12 % (k = 2) (DKD-R 8-1, Appendix A). DKD sets a minimum handling contribution of 0.07 % of nominal volume for fixed-volume and 0.1 % for variable and multichannel pipettes, and cg-19 also uses at least 0.1 % for piston pipettes (DKD-R 8-1, §8.9; cg-19, 6.3.7.2). Our uncertainty calculator combines a budget like this, and the TUR calculator checks the ratio between your uncertainty and the pipette’s tolerance.

Check your own numbers

Enter the conditions and the balance readings of one volume. The calculator converts them with Z and gives the mean, the systematic error, the standard deviation and the CV. It is pre-filled with the as-found readings at 1,000 µL from the example above.

The math is checked against published values: NIST’s water density table, a published Z table, DKD-R 8-1’s worked example and EURAMET cg-19’s flask example. It does not calculate the measurement uncertainty.

What to record on a pipette calibration certificate

ISO 8655-6 lists what the report must contain (ISO 8655-6, clause 10, as summarized in the Sartorius white paper):

• Identification of the pipette and of the tips or other exchangeable parts, and whether it delivers (Ex) or contains (In).
• Conditions under which the test was done: temperatures, pressure, humidity.
• Results: the volume of each delivery, the number of measurements per volume, and the systematic and random errors. Report them as found and as left when the pipette was adjusted or repaired; Sartorius notes that the 2022 edition calls for an as-found calibration or test before maintenance or repair.
• Tolerances the results were compared with, if any, and the expanded uncertainty of each mean volume, for each volume and channel, when required.
• Method: a reference to ISO 8655-6:2022, any deviation from it, and the formula used to convert weights into volumes; the reference temperature and expansion coefficient if a thermal correction was made.
• Who and when: date and operator.

Our guides to calibration certificates and calibration records explain each field. Print the status sticker with the free calibration label generator.

How often, and in-house vs a calibration service

There is no single interval for every pipette. Sartorius, summarizing ISO 8655:2022, lists the factors to weigh: the risk of the application, how often the pipette is used and by how many people, the liquids and their vapors, the permissible errors you need, the manufacturer’s information and your liquid handling process (Sartorius, ISO 8655 flyer). Routine tests should run at shorter intervals than full calibrations (Sartorius white paper). Older manufacturer documents written for the 2002 edition say ISO 8655 asks for a yearly calibration (Eppendorf SOP, 2019, §4); we could not confirm that from the 2022 text, so treat any figure as a starting point and adjust it from your results. Our calibration intervals guide explains how.

Recalibrate, or at least recheck, after a pipette is dropped or repaired, or when a routine check fails. A calibration before and after maintenance or repair is recommended (Sartorius white paper), and aggressive chemicals speed up the drift (Eppendorf SOP, 2019, §4).

In-house or a service?

On lab forums, users report service prices of roughly $15 to $25 per single-channel pipette and $45 to $50 per multichannel in the US (r/labrats). Treat that as anecdote; quotes vary with the scope.

How to check a calibration service

The certificate is your evidence, so read it (how to read a calibration certificate) and ask:

• How many volumes and measurements per volume? A calibration of a variable-volume pipette to ISO 8655-6 is three volumes × ten measurements per channel (ISO 8655-6, 8.1). Fewer is a routine test under Part 7, which is fine if that is what you ordered and it is stated.
• Are as-found results reported before any adjustment or repair? Without them you cannot assess past results.
• Which tips were used? The result is valid for that tip type only.
• Is the measurement uncertainty stated, and against which limits was the pipette judged: ISO 8655-2, the manufacturer’s or yours?
• Do the numbers look real? Ten readings should show some scatter, and the time on site should be plausible for the number of weighings: 30 per single-channel pipette, 360 for a 12-channel one.

When a pipette fails

Take it out of use, label it, and assess the impact on results produced since its last good calibration, as in the example above. If repeated checks show drift, shorten the interval. See out of tolerance: what to do when a calibration fails.

The photometric method

ISO 8655-8:2022 describes a photometric reference procedure with the same status as the gravimetric one (European Instruments; SelectScience). Its scope covers apparatus up to 5,000 µL nominal volume (ISO 8655-8:2022, scope). Instead of weighing water, the pipette dispenses a dye solution, and its absorbance, which is proportional to the dye concentration at a fixed optical path, gives the delivered volume. The dyes must be well characterized and must behave like water in the pipette (Troemner). It is less sensitive to the environment than weighing, but not immune: temperature equilibrium and cleanliness still matter (Troemner). Commercial photometric systems exist; ISO 8655-7 covers other alternative procedures.

Common mistakes when calibrating a pipette

Related guides

• How to write a calibration procedure: the general structure this procedure follows.
• Pipette calibration hub: everything on pipettes in one place.
• Accuracy vs precision: the concepts behind systematic and random error.
• How to calibrate a scale: the balance is your reference standard.
• Uncertainty calculator and TUR calculator.
• Out of tolerance: what to do when a calibration fails.
• Calibration intervals.
• Calibration label generator.