To ensure accurate pipette measurements, perform calibration regularly and follow these steps: 1. Gather materials and record water temperature. 2. Pre-rinse pipette tips and weigh dispensed water. 3. Calculate volume using the Z-factor and average results. If accuracy falls outside 99–101%, consult with supervisors and refrain from use. Calibrate every 3–6 months for reliable results.

Performing your own pipette calibration may sound scary, but in this article, we walk you through how to do it in simple, easy steps.

## The Importance of a Well-Calibrated Pipette

A pipette is used to precisely measure and transfer small volumes of liquid in the laboratory in experiments that demand high accuracy.

However, the sources of error in pipette measurements could arise from device failure or due to misuse by the operator. Any discrepancy in volumes dispensed may affect the outcomes and reproducibility of an experiment such as qPCR results.

It is, therefore, necessary to check pipette calibration every few months to ensure accuracy by dispensing the right volumes. This is why pipette calibration is considered a fundamental part of good laboratory practice (GLP).

In a previous article, we discussed tips for preserving pipette accuracy—today, we will learn how to perform pipette calibration.

## Principles of Pipette Calibration

At a temperature of 20°C and one atmosphere of pressure, water density stays constant at 1 g/mL. This means that the volume of water can be determined by weighing dispensed water.

However, changes in temperature and barometric pressure can influence the accuracy of measurements. The effect of pressure is negligible in practice, but the temperature should be taken into consideration. This is done using the Z-factor or ‘Z correction factor’, which is applied in the calculation of the volume of water.

Comparing the calculated volume with the theoretical volume allows you to determine the accuracy of the pipette.

Table 1 shows the Z-factors for distilled water at various temperatures

Temperature (°C) Z-factor
151.0019
161.0020
171.0023
181.0025
191.0027
201.0029
211.0031
221.0033
231.0035
241.0037
251.0039

Pipette calibration table of Z-factors for distilled water at 1 atm pressure. Adapted from Wisconsin State Laboratory of Hygiene

## Steps Involved in the Calibration of Your Pipette

Note: You should check the calibration of your pipette using two different volumes.

1. Take distilled water in a beaker and record its temperature.
2. Gather your pipette and the correct tips based on both the small and large volumes that the pipette can dispense.
3. Place a weigh boat on a balance that can accurately weigh in the microgram range, and set it to zero after closing the balance door.
4. Pre-rinse the tip by aspirating and dispensing the set volume three times, and push it fully to remove any remaining liquid.
5. Aspirate the calibration volume without bubble formation and dispense the liquid slowly into the weigh boat. Then, record the weight on the balance and repeat the process ten times.
6. Calculate the dispensed volume by using the equation:
V = W x Z
where,
W = weight of the water
Z = Z-factor (see Table 1)
V = calculated volume of dispensed water.
7. Next, determine the mean value from ten trials.
8. Finally, calculate accuracy by using the equation:
A = 100 x Vavg/V0
where,
A = accuracy of the pipette
Vavg = average calculated volume
V0 = theoretical volume you tried to dispense.
9. If the accuracy value lies in the 99–101% range, the pipette is considered normal and calibrated.

## Caution

Large changes in the testing environment, such as temperature, humidity, and atmospheric pressure, may impact evaporation during calibration, which could eventually lead to an unreliable test.

It is also important to adjust the pipettes to the calibration environment for more than an hour before starting the calibration.

## What if My Pipette is Not Well-Calibrated?

If your pipette accuracy is out of the 99–101% range, stop using the pipette and speak with your lab manager or supervisor. There may be existing arrangements available to get your pipette serviced and calibrated either in-house or with a company to ensure it’s performing correctly.

You may need to arrange to use an alternative pipette in the meantime. Ensure you check the accuracy of this pipette before embarking on new experiments.

## How Often Should My Pipette Be Calibrated?

As a minimum, it’s a good idea to check the calibration of your pipette every 3-6 months. If you are deep-cleaning your pipette, it’s a good idea to check after reassembly too.

The more regularly you check your pipette calibration, the sooner you can pick up issues that might affect your results.

Make sure you record when you last checked your pipette so that if you do have an issue in the future, you know the timescale of experiments that might have been affected.

For more tips, tricks, and hacks for getting your experiments done, check out the Bitesize Bio DIY in the Lab Hub.

And while you’re here, why not download our handy pipette cleaning guide and share it with your lab buddies, or stick our pipette care poster up in your lab? They’re free!

## FAQs

Q. How do changes in temperature and barometric pressure affect the accuracy of pipette measurements, and how are these factors accounted for during pipette calibration?

A. Changes in temperature and barometric pressure can affect the accuracy of pipette measurements. These factors are accounted for during pipette calibration by considering the Z-factor or ‘Z correction factor’, which you apply to the calculation of the volume of dispensed water.

This correction factor varies with temperature, as shown in Table 1, and ensures accurate measurements despite environmental changes.

Q. Are there any specific guidelines or recommendations for adjusting pipettes to the calibration environment, especially regarding factors, such as temperature, humidity, and atmospheric pressure?

A. Adjusting pipettes to the calibration environment is crucial for accurate results.

Large changes in temperature, humidity, and atmospheric pressure can impact evaporation during calibration, leading to unreliable tests.

Allow the pipettes to adjust to the calibration environment for more than an hour before starting the calibration process. This ensures that the pipettes are acclimated to the conditions, minimizing potential errors during calibration.

Q. If a pipette’s accuracy falls outside the 99–101% range after calibration, what are the potential consequences of using it, and what steps should be taken to rectify the situation?

A. If a pipette’s accuracy falls outside the 99–101% range after calibration, stop using the pipette for accuracy-critical tasks.

There may be arrangements available to service and recalibrate the pipette, either in-house or with a company specializing in calibration services. Speak to a technician or your supervisor about this.

In the meantime, use an alternative pipette you know is accurate to conduct new experiments. Using a poorly calibrated pipette can lead to inaccurate results, compromising the integrity of experiments and research outcomes.

Originally published May 2018. Reviewed and updated on October 2021 and April 2024.

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