In the calibration certificate of a pipette, measurement uncertainty is the datum that establishes whether that instrument is fit for use in the analytical context in which it operates. It is not an ancillary parameter: it is the quantitative synthesis of all the error contributions that characterise the pipette’s performance under the conditions in which it was calibrated.
A superficial reading of the certificate leaves the central question unresolved: is the stated uncertainty compatible with the process limits or with the laboratory’s internal specifications? Without this verification, the calibration certificate fulfils a documentary function but not a metrological one.
Why uncertainty is the most critical parameter in a pipette calibration certificate
The metrological performance of a pipette is determined by two distinct components, both quantified in the calibration certificate and both necessary to assess the instrument’s suitability for the process.
The first is the systematic error (SE), expressed in µL or as a percentage of the nominal volume: it measures the constant, repeatable deviation between the volume dispensed by the pipette and the set nominal volume. A positive systematic error means the pipette consistently dispenses a volume greater than the set value; a negative one means it dispenses a smaller volume.
This component is correctable, in principle, through instrument adjustment — but only if it has been quantified by an accredited calibration.
The second is the random error, expressed as the coefficient of variation CV% across the number of repeated measurements specified by ISO 8655-6. CV% does not measure the average error of the pipette: it measures how much its values vary from one measurement to the next. A pipette with a high CV% produces poorly repeatable results, regardless of the systematic error. In quantitative analysis, an out-of-tolerance CV% compromises the quality of the analytical result even when the systematic error is within the permitted limits.
The expanded uncertainty U stated in the certificate incorporates both components, propagated according to the criteria of the GUM (JCGM 100). It is not the sum of SE and CV%: it is the result of a quadrature combination of the standard uncertainties associated with each contribution, multiplied by the coverage factor k. Reading only the systematic error or only the CV% without considering the expanded uncertainty provides only a partial picture of the pipette’s actual performance.
How to read the expanded uncertainty in the calibration certificate
In the calibration certificate of a pipette issued by a laboratory accredited to ISO/IEC 17025, the expanded uncertainty is reported in the form:
U = ± Y µL (k = 2, confidence level 95.45 %)
The value Y expresses the half-width of the interval within which the true volume dispensed by the pipette lies, at the stated confidence level. The coverage factor k = 2 corresponds to a normal distribution and a confidence level of 95.45 %: in other words, if the calibration were repeated a large number of times, the actual volume would fall within the interval [measured value − U, measured value + U] in 95.45 % of cases.
A certificate that reports the uncertainty without specifying k and the confidence level does not comply with the requirements of ISO/IEC 17025. Equally, an uncertainty expressed only as a percentage without an absolute value in µL is insufficient for a correct metrological compatibility check — particularly for single-channel pipettes with a low nominal volume, where the difference between ±0.5 % and ±0.5 µL can be substantial.
Systematic error and CV%: what they indicate in practical terms
Systematic error according to ISO 8655
ISO 8655-6, the technical standard of reference for the verification of piston-operated volumetric pipettes, defines the maximum acceptable limits for systematic error and coefficient of variation as a function of the nominal volume of the pipette and the calibration volume. The limits are expressed both as absolute values (µL) and as percentages, and apply to new pipettes or following adjustment; in continuous use, organisations set internal criteria that may be more stringent.
The systematic error stated in the certificate must be compared with the ISO 8655-6 limits to verify the instrument’s conformity, and then with the laboratory’s internal specifications or the process limits to verify its operational suitability. The two comparisons are not equivalent: an instrument may comply with ISO 8655-6 and yet be unsuitable for a process requiring tighter tolerances.
CV% and measurement repeatability
CV% is particularly critical in processes where the pipette is used for serial dilutions, preparation of calibration curves, or dispensing steps in which uncertainty accumulates across multiple stages. In such cases, even a CV% nominally conforming to ISO 8655-6 limits may generate a cumulative uncertainty incompatible with the requirements of the analytical method.
Correct assessment requires propagating the pipette’s uncertainty through the method’s steps and comparing the result with the total permissible uncertainty defined in the method validation. This calculation is not required by the calibration certificate, but it is the laboratory’s responsibility; its absence is detectable during an audit as a gap in the metrological management of the analytical process.
How to compare the stated uncertainty with process tolerances
The comparison between the expanded uncertainty stated in the certificate and the operational tolerances is the step that transforms the certificate from a mere archive document into a decision-making tool. The procedure to follow is similar to that described for other measuring instruments in a GMP context: the metrological coverage ratio criterion of 4:1 is applied, whereby the expanded uncertainty of the calibration must be at most one quarter of the permissible process tolerance.
Applied example: a dilution process requires that the dispensed volume does not deviate by more than ±2.0 µL from the nominal value. The pipette calibration certificate must report U ≤ ±0.5 µL (k = 2) to meet this criterion. If the certificate reports U = ±1.2 µL, the pipette is not metrologically adequate for that process, regardless of whether it complies with the ISO 8655-6 limits.
When process tolerances are not explicitly defined, the laboratory manager is required to derive them from the requirements of the analytical method — quantification limits, method confidence intervals, repeatability requirements — and to document the metrological rationale justifying acceptance of the instrument. The absence of this documentation constitutes a non-conformity during an audit.
Signals indicating an out-of-tolerance pipette and how to proceed
Certain operational signals indicate that the pipette may be operating outside its metrological limits even before the calibration interval has expired:
- unexpected variability in analytical results: replicates of the same analysis show a dispersion greater than that expected from the method, not attributable to other factors.
- progressive drift in quality controls: the values of internal QC samples tend towards one extreme of the acceptance interval over time.
- visible physical anomalies: damage to the piston, wear of the seals, variations in the aspiration mechanism — all signals that metrological performance may have degraded.
- change of operator or environmental conditions: significant variations in laboratory temperature or humidity affect the viscosity of the liquid and therefore the dispensing accuracy of the pipette.
When these signals are present, the pipette must be withdrawn from use for critical measurements and sent for unscheduled calibration at a laboratory accredited to ISO/IEC 17025, without waiting for the scheduled calibration interval to expire. The unscheduled calibration must be documented in the instrument register, with a statement of the cause that prompted it.
If the calibration reveals an out-of-tolerance instrument, a metrological non-conformity is raised, with assessment of the impact on the data produced during the previous period. The procedures for managing this situation — including the retrospective assessment of measurements taken with the non-conforming instrument — are the same as those applicable to any measuring instrument in a GMP context, as described in the article on calibration certificate expiry management and metrological non-conformities.
The Altiorlab team is available to provide technical consultancy for the verification of the metrological compatibility of pipette calibration certificates in use, and for the definition of acceptance criteria within the quality management system.
