Kenya Bureau of

Standards

Inter-Lab comparison KEBS-FF-6-2023

Volume comparison at 10µl and 1000µl – Calibration of micropipettes

Report

Pilot
Miheso, KEBS-Kenya

November 2023
 Kenya Bureau of
Standards
Contents

1. Introduction..........................................................................................................................................3
2. Participant Laboratories........................................................................................................................3
3. The transfer standard............................................................................................................................4
4. The measurement procedure.................................................................................................................4
4.1 Experimental method................................................................................................................4
4.2 Water characteristics.................................................................................................................5
4.3 Equipment.................................................................................................................................5
4.4 Ambient conditions of the measurements.................................................................................6
5. Measurement results.............................................................................................................................6
5.1 Stability of the micropipettes....................................................................................................6
5.2 Results of the participants Labs................................................................................................7
5.3 Uncertainty correction...............................................................................................................9
6. Determination of the key comparison reference value, uncertainty, consistency and degree of
equivalence..............................................................................................................................................10
6.1 Micropipette M40098F...........................................................................................................11
7. Uncertainty calculation.......................................................................................................................15
8. Conclusions........................................................................................................................................18
9.References............................................................................................................................................18

Kenya Bureau of
Standards
Introduction

In October2023, it was agreed to start a bilateral inter-Comparison in volume measurements in the range of the
Micro-litre. This comparison has the main purpose of comparing the results and methods of calibration for 100
L micropipette single channel will allow the participating laboratories to test their agreement of results and
uncertainties despite the use of different equipment and calibration methods.
KEBS the Pilot Laboratory, prepared the protocol, performed the initial and final measurements of one
micropipette and collected the results of the participants.
The Volume and Flow Laboratory of the Kenya Bureau of Standards (KEBS - National Metrology Laboratory
(NMI), performed the initial and final measurements of the other two micropipettes and prepared the report.
One 100 100 µl micropipettes (transfer package) was tested, all supplied by KEBS.
2 participants agreed to participate in this comparison, and all supplied results.
The comparison started in October 2023 and finished in December 2023

1. Participant MIs
Each participant had 4 weeks to receive the micropipettes, perform the measurements and send the instruments
to the next participant according to the following schedule:

Table 1 – Time schedule

Report results
Laboratory Country Responsible
date
KEBS Kenya B. Miheso Oct 2023
AVIOTEQ Kenya Mr. Juma Oct 2023

2. Transfer standard
The chosen instruments were single channel variable micropipettes of low value, 100-10 L (see Figure 1). Kebs
Kenya supplied the micropipette marked with the serial number M40098E with corresponding tips.
The micropipettes used for this comparison are essentially made of plastic material with a thermal expansion
coefficient of 2,4 × 10-4 /ºC [1].

Figure 1- Fixed micropipette of 100-10 L

3. Measurement procedure

4.1 Experimental method

The gravimetric method was used by all participating NMIs, to determine the amount of water that the
micropipettes deliver at the reference temperature of 20 ºC, based on ISO 8655[2] and ISO 4787 [3], with
equation (1):

( )
1 ρ
V 20=( I I −I E )× × 1− A ×[ 1−γ (t−20) ]
ρW −ρ A ρB
(1)

Where:
V20/L: volume at reference temperature, 20 ºC
mg: weighing result of the recipient full of liquid
E/mg: weighing result of the empty recipient
W/(mg/L): water density at the calibration temperature, using Tanaka density formula [4]
A/(mg/L): air density
B/(mg/L): density of the masses used during measurement (substitution) or calibration of the balance
°C-1 cubic thermal expansion coefficient of the material of the piston pipette
t/°C: water temperature during the calibration process
During the comparison, the participants were not allowed to adjust, clean or re-grease the micropipettes.

4.2 Water characteristics

The water used by the participants NMIs had different characteristics. A summary is found in Table 2.

Table 2 – Water characteristics

NMI Type of water Density Formula Conductivity (S/cm)

KEBS Dionized Tanaka 0,6

AVIOTEQ Distilled Tanaka -

Not all participants sent the information regarding the water characteristics except Kebs. All participant that
presented values used de-ionized water. All participants used Tanaka formula as the reference for water density.

4.3 Equipment

The majority of the participants described the equipment used in the calibration and respective traceability filling
a form that was sent with the protocol. The summary of these characteristics is presented in the following table:
 Table 3 – Equipment characteristics

Equipment Type Resolution

Balance Comparator (0,001 - 0,1) mg
Weights F1, E2 -
Water thermometer Digital (0,0001 - 0,1) ºC
Air thermometer Digital (0,001 - 0,1) ºC
Barometer Digital (0,001– 0,1) hPa
Hydrometer Digital (0,1 – 0,5) %

The last three instruments were used to calculate the air buoyancy effect.

4.4 Ambient conditions during the measurements

The ambient conditions were described by all participants when using the 2 micropipettes. The values given in
Table 4 refer to micropipette KJ19345. For the other micropipettes the values are very similar.

Table 4 - Ambient conditions

Air Temperature Pressure Relative humidity Water Temperature

NMI
(ºC) (hPa) (%) (ºC)
KEBS 23.25 834.5 62 22.4
AVIOTEQ 19.9 - 52.75 20.5

The majority of the laboratories presented values that are in agreement with what was proposed in the protocol:
humidity higher than 40 % and ambient temperature between 17 ºC - 25 ºC, but others laboratories declared
values that are outside of the specification, mainly the relative humidity.
From this table it can also be seen that there are some differences in atmospheric pressure due to the altitude.

4. Measurement results
5.1Stability of the micropipettes

The measurements of the micropipette with serial number M40098E was performed by Kebs during the
comparison in order to verify the stability of the standards.
 Table 5 - Stability of the instruments

Micropipette Measurement Date Volume (L) Uncertainty (L) ΔV(L)

KEBS 1 Oct 2023 100.55 0.49
M40098E 0,01
KEBS 2 Dec 2023 100.54 0.48

For both micropipettes the two results obtained by Kebs, are consistent with each other and are within the
presented uncertainty, proving that the micropipettes had a stable volume during the entire comparison.
The second result of kebs will be considered for the determination of the reference value for the comparison.

5.2Results of the participants

The results for the two micropipettes with serial number J10599F and M40098E, of the participating laboratories
are included in Table 6.

Table 6 – Volume measurement results

NMI M40098E
V/L U/L

KEBS 100.54 0.48

AVIOTEQ 99.45 1.55

5.4Uncertainty for “process-related handling contribution”

The “process-related handling contribution” uncertainty should always be included in the determination of the
measurement uncertainty according to the Guideline DKD-R 8-1[5]. This contribution value encompasses the
influences on the dispensed volume which occur due to handling of the devices during the calibration of
micropipettes. The DKD – R 8-1 guideline recommends to include a value of 0,07 % of the nominal volume of
the micropipettes as the standard uncertainty for “process-related handling contribution”. This value was added
to the uncertainty budget of all participants in order to have a more realistic uncertainty result.
In Table 10 are presented the final results obtained after all corrections were applied.

Table 10 – Final volume measurement results (corrected)

LAB M40098E
V/L U/L

KEBS 100.55 0.48

AVIOTEQ 99.52 1.56

5. Determination of the lab Comparison Reference Value, Uncertainty, consistency

and degree of equivalence
To determine the Reference Value of this Comparison (KCRV)the weighted mean (3) was selected, using the
inverses of the squares of the associated standard uncertainties as the weights [7], according to the instructions
given by the BIPM:
x 1 / u2 ( x1 )+. ..+ x n / u2 ( x n )
y=
1 /u 2 ( x 1 )+. . ..+1/ u2 ( x n )
(3)

To calculate the standard deviation u(y) associated with the volume y [7] equation (4) was used:

u( y )=
√ 1
1 /u ( x 1 )+. ..+1 /u 2 ( x n )
2

(4)

The expanded uncertainty of the reference value is U(y)=2×u(y).

To identify an overall consistency of the results a chi-square test can be applied to all n calibration results [7].
( x 1− y )2 ( x n − y )2
χ 2obs= +.. .+
u 2( x 1) u 2( x n )
(5)
where the degrees of freedom are: = n -1
Pr { χ 2 ( ν )> χ 2obs }<0 , 05
The consistency check is regarded as failed if: .
The function CHIINV(0,05;n-1) in MS Excel was used and the consistency check fails if CHIINV(0,05;n-1) <
χ2obs.

If the consistency check did not fail then y was accepted as the KCRV xref and U(xref) was accepted as the
expanded uncertainty of the KCRV.
( x i− y )2
2
If the consistency check failed then the laboratory with the highest value of u ( x i ) is excluded from the next
round of evaluation and a new reference value, reference standard uncertainty and chi-squared values are
calculated again without the excluded laboratory. Finally when the consistency check passes, for each laboratory
results, xi the degree of equivalence di between each laboratory and the KCRV (xref) is calculated using the
following formulas [7]:
di = xI - xref (6)
U(di) = 2 × u(di) (7)
where u(di) is calculated from
u 2(di) = u 2(xi) – u 2(xref) (8)
Discrepancy values can be identify if it is obtained|di|> 2u ( d i ) .
To calculate the degrees of equivalence dij between the laboratories the following formulas were used [7]:
di,j= xi- xj (9)
U(di,j) = 2 × u(di,j) (10)
Where u(di,j) is calculated from
u 2(di,j) = u 2(xi) + u 2(xj) (11)
The factor 2 in equation (7 and 10) corresponds to a 95 % coverage under the assumption of normality.
The normalized error En,i describes the degree of equivalence of a laboratory related to the KCRV.
En,i was calculated for each reported value of the participant as follows,
En,i = di/U(di)(12)
If |E n ,i|≤1, the measurement is generally consider as acceptable and the measured values are consistent.

6.1 Micropipette M40098E

high low mean

KEBS 101.02 100.07 100.55
HOPE
LABS 101.08 97.96 100.46

Figure 2- Measurement results of micropipette M40098E with reference value and uncertainty

6. Conclusions
.
En results were observed with M40098E micro-pipette with KEBS recording 0.13 and Hope laboratory
recording -0.58 which is within the allowable limits

The comparison was organized by KEBS regarding micropipette calibration and 2 laboratories decided to
participate.
Two micropipettes showed a stable volume during the whole comparison. This was confirmed by the results
from Kebs.
The original results of all participant laboratories were corrected for the standard atmospheric pressure in order
to compare results under the same calibration conditions.
The reproducibility of the micropipette was added to the reference uncertainty.
There is a large variability in the uncertainty values presented by the participating laboratories, which means that
the uncertainty procedure is not yet harmonized, considering that for micropipettes the largest source of
uncertainty comes from the repeatability and not from the calibration method.

7. References

1. ASTM E 542:2000 - Standard practice for calibration of laboratory volumetric apparatus;

2. ISO 8655-1/2/6:2022, Piston-operated volumetric apparatus;
3. ISO 4787:2010; Laboratory glassware – Volumetric glassware – Methods for use and testing of capacity;
4. Tanaka, M., et. al; Recommended table for the density of water between 0 °C and 40 °C based on recent
experimental reports, Metrologia, 2001, Vol.38, 301-309;
5. Calibration guide DKD-R 8-1; calibration of piston pipettes, 2011;
6. Chistoph Spalti, Influence of altitude on the dispensed volume of a piston pipette with air cushion, study of
Spaelti-TS AG, 2011;
7. PTB/DKD Guidelines DKD R 8-1; calibration of piston-operating pipettes with air cushion, 2011
8. M.G. Cox, The evaluation of key comparison data, Metrologia, 2002, Vol. 39, 589-595.