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Pipette calibration and the ISO 8655 standard

Sami Koivisto
2 July, 2006  

Sami Koivisto
MSc(Tech) EUR ING
Technical subcommittee
CEN/TC 332
European Committee for Standardization

As a concept, calibration can be understood in several ways  and the interpretation can vary between different devices.  When associated with pH meters, calibration means adjusting  the device to give a specific value with a defined standard  solution. With balances the same word means only the  determination of the difference between the reference weight  mass and the reading obtained from the device display. The  understanding of these concepts can vary even within the  same organisation. It is important that the concepts used in  discussion are understood. In this article the following  definitions apply:

  • Calibration is the determination of the difference between  the mean volume of the measurement series and the selected  volume on the pipette display.
  • Adjustment is the alteration of pipette settings for the  actual volume to correspond to the selected volume.

This means that during calibration no physical changes are  made to the device itself and the process gives only the  measurement result. During the adjustment phase the device  is physically altered to deliver a different volume. To  confirm the effect of adjustment the device needs to be  recalibrated.

Calibration in practice
In practice, calibration means the functional verification  of the pipette. This begins at the factory, where the  pipettes are adjusted and calibrated for quality assurance.  It is recommended that the clients perform an inspection of  the devices before they are released for operator use. This  is due to the different operating conditions, which can have  an effect on the calibration results. However, very often  the pipettes are not calibrated until the first maintenance  session.

The calibration procedures are usually performed according  to a standard method and only the functionality of the  pipette is verified. The standard check is always performed  using water. Since pure water is seldom used in laboratory  applications, the actual amount of liquid can differ from  the standard calibration value. The result obtained in  calibration is bound to time, place, ambient conditions, the  liquid used and the operator. Therefore, in addition to the  reference calibration it is suggested that a calibration is  performed using the liquid to be transferred. This should be  done in laboratory conditions by each operator using the  pipette to verify that a desired volume amount is obtained.  The pipetting method and the equipment setting (pipette + tip) demanded by the application should also be included in  the test.

However, adjustment is not recommended for liquids other  than water due to the insufficient or approximate  correlation data (mass to volume conversion). The operator  should also bear in mind that pipettes will be used to  transfer different liquid types with deviating properties.  Adjustments would have to be done each time a liquid is  changed. Water is officially accepted as a standard  calibration liquid due to thorough research and statistical  results. This gives reliable reference values for the  devices. The follow-up of the mechanical functioning of the  pipette is therefore easier for the user and better accepted  by official parties.

The calibration values obtained using liquids other than  water can be referred to the obtained reference values and  necessary corrective actions can be taken accordingly. For  example the statistical deviation of these results can be  added to the uncertain calculations used by accredited  laboratories.

International standard ISO 8655 for POVA devices
In January 2003 the new EN ISO 8655 standard for  piston-operated volumetric apparatus (POVA) was published as  a result of the work of joint technical subcommittees ISO TC  48/SC1 and CEN/TC 332/WG1. This standard replaced the  well-known DIN 12650 standard, used since 1997. The previous  DIN standard was mainly directed at manufacturer use, but  the present ISO standard is also directed at service houses  and end-users. ISO 8655 contains seven parts covering  different kinds of piston-operated liquid transfer devices. The sections that relate  to piston pipettes are:

  • Part 1: Terminology, general requirements and user  recommendations.
  • Part 2: Piston pipettes.
  • Part 6: Gravimetric methods for the determination of  measurement error.
  • Part 7: Nongravimetric methods for the assessment of  equipment performance.

Part 1 contains valuable general information for all parties  including vocabulary. Part 2 describes the structure and  operation principle of a piston pipette and also contains  the acceptance tolerances for calibration – according to  nominal volumes. The actual gravimetric testing procedure is  described in Part 6. The demanded calibration conditions are  also stated. Part 7 describes alternative calibration  methods and the terms when these methods can be used to  fulfil the standard requirements. Present methods include  the photometric method (horizontal direction) and the  titrimetric method.

Coverage of ISO 8655
The standard states the acceptance specifications, test  methods and testing conditions for pipettes. It also gives  some information about the basic pipetting errors affecting  the procedure. The standard is directed to manufacturers as  a basis for product type testing and quality control,  including, when appropriate, the issuance of manufacturer’s  declarations of conformity. The standard also addresses the  needs of test houses and other bodies as a basis for  independent certification of conformity, and the needs of  equipment users to enable routine performance checking.

The test method described in ISO 8655-6 can be applied in  every calibration procedure regardless of the ambient  conditions. However, when the functionality of the equipment  or the conformance to official acceptance limits are  concerned, only the conditions mentioned in the standard are  acceptable. The acceptance limits in the standard do not  apply to liquids other than water or pipetting methods  different from the forward pipetting technique.

Manufacturer’s acceptance specifications versus ISO 8655  acceptance specifications
One of the puzzling questions about ISO 8655 is the  difference between the manufacturer’s acceptance limits and  acceptance specifications. The specifications in the  standard are designed to serve all parties from  manufacturers to end-users. Since the acceptance limits are  the same for everyone, the manufacturers continue to use  their internal quality control limits to demonstrate the  performance capabilities of the pipettes. The manufacturer’s  acceptance limits are usually much stricter than the  specifications mentioned in the standard, especially where  adjustable pipettes and small volumes are concerned.

The standard specifications are wider than the corresponding manufacturer’s specifications since there is no actual need for more accurate or precise dosing in applications where POVA devices, such as pipettes, are used. Although the sensitivity of laboratory applications has increased, in many cases the required level of measurement accuracy can still be more than 10-fold compared with device acceptance limits. Naturally, it is good to minimise known error sources, but most are not caused by the device itself. Knowing and understanding the most critical error factors demands both theoretical and practical knowledge from the operator.

The main difference between the manufacturer’s acceptance limits and standard specifications concerns the variable volume pipettes. Manufacturers give volume-bound specifications for certain test volumes. These volumes are usually the nominal volume of the pipette (maximum volume) and the minimum volume or 10% of the nominal volume. Sometimes additional specifications, such as the middle volume (50% of the nominal value), are also published. The standard, however, gives specific values that are bound to the nominal volume values of the pipettes. All other  selectable volumes have the same absolute acceptance limit values as the model specific nominal volume. For example, a pipette model of 100–1000µl has an acceptance value of 8µl at the maximum volume for systematic error (inaccuracy). This is 0.8% of the nominal volume. At the setting of 100µl, the acceptance limit is the same (8µl). However, the relative value is 8%. The limits are always defined according to the absolute error values.

Special requirements of ISO 8655
ISO 8655 standard describes many requirements that end-users and independent test-house operators should follow to fulfil the standard. These requirements can be classified under different categories.

Use of pipettes
To ensure safe and efficient use of the device the standard  requires the user to follow instructions provided by the  manufacturer. Special care should be taken to prevent the  entry of liquid into the pipette interior. The user is also  responsible for ensuring that used pipettes and pipette tips  are resistant to the transferred liquids.

Calibration performed by the user
To achieve the needed reliability of volume measurement, users should establish their own acceptance maximum error limits for both systematic (inaccuracy) and random  (imprecision) error. These requirements are determined by the applications where pipettes are used. Conformity to the defined acceptance limits should be tested by the user at  regular intervals as part of their own quality protocol. The required time interval should be based on the frequency of use, number of operators using the device, nature of the  liquids and finally the demands of the applications. The  time interval can be, for example, three months but no more than one year, and the defined protocols should be recorded into the user’s quality system. In case pipette tips different from those of the manufacturer are used, the operator should confirm the suitability and functionality of the tips with the pipette.

Maintenance of the pipette
Many laboratories send their pipettes to other test houses  or the manufacturer for maintenance and calibration  procedures. In these cases it is the responsibility of the  user to perform pipette decontamination. Nondecontaminated devices should not be sent for servicing.

Other uses of the standard method
When the standard method is used by the manufacturer for  purposes other than type testing, such as quality control or  functional verification as part of the after-sales service,  the standard allows the manufacturer to define:

  • The number of needed test volumes.
  • The number of measurements used per volume.
  • For multichannel pipettes, the number of channels tested.

According to these definitions, many manufacturers use their own testing protocols in quality assurance. Both one- and two-test volumes are used. The results are determined using four or more measurement values per volume. Results from multichannel pipette calibration can be reported for two or more individual channels. These choices are mainly based on the reference measurements and extensive statistical experience of the manufacturer. The present quality systems require increasing attention to the equipment performance and to traceable reference calibration records. This can be  achieved by using a standardised calibration method such as the procedure described in the ISO 8655 standard. It is  recommended that pipettes are also adjusted according to a standard procedure.

[[HHE06_fig1_L36]]

Although the best way to assure the functional performance  of the pipette is to follow a standard procedure, the real  situation within the laboratory can be different. To ensure reliable results, it is recommended that pipettes are calibrated in a “real” laboratory environment in addition to undertaking traceable standard quality control. This, however, demands both good theoretical and practical  knowledge from the operators.

Further reading
International standard EN ISO 8655. Piston-operated volumetric apparatus. Geneva: ISO; 2002.
Technical report. ISO/TR 20461. Determination of uncertainty for volume measurements made using the gravimetric method, Geneva: ISO; 2000.