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A calibration system was set up for calibrating multi-channel pipettes using the gravimetric method in accordance with the ISO 8655-6. The calibration system consisted of a display unit, a motor control unit and a measuring unit. The measuring unit included a single weighing cell, twelve individual containers for receiving the dispensed volume from multi-channel pipettes and transport rails to automatically convey the containers to the weighing cell one by one. This paper described the measurement traceability for volume measurements to the SI unit of mass by using the calibration system and the validation of the calibration system for volume measurements. The calibration system was shown to be capable of measuring the dispensed volume from 30 μl to 1000 μl with the measurement uncertainty of 0.7 μl to 2.9 μl.

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Journal of Physics: Conference Series

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Calibration of multi-channel pipettes using gravimetric method in

accordance with the ISO 8655-6

To cite this article: W M Leung et al 2018 J. Phys.: Conf. Ser. 1065 092004

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XXII World Congress of the International Measurement Confederation (IMEKO 2018)

IOP Conf. Series: Journal of Physics: Conf. Series 1065 (2018) 092004 IOP Publishing

doi:10.1088/1742-6596/1065/9/092004

1

Calibration of multi-channel pipettes using gravimetric

method in accordance with the ISO 8655-6

W M Leung1 , C F Yuen, H K L Chiu2 , L L Grue3

1 Electrical and Mechanical Engineer, Standards and Calibration Laboratory

2 Senior Electrical and Mechanical Engineer, Standards and Calibration Laboratory

3 Technical Metrologist, FORCE Technology, Denmark

E-mail: wmleung@itc.gov.hk

Abstract. A calibration system was set up for calibrating multi-channel pipettes using the

gravimetric method in accordance with the ISO 8655-6. The calibration system consisted of a

display unit, a motor control unit and a measuring unit. The measuring unit included a single

weighing cell, twelve individual containers for receiving the dispensed volume from multi-

channel pipettes and transport rails to automatically convey the containers to the weighing cell

one by one. This paper described the measurement traceability for volume measurements to

the SI unit of mass by using the calibration system and the validation of the calibration system

for volume measurements. The calibration system was shown to be capable of measuring the

dispensed volume from 30  l to 1000  l with the measurement uncertainty of 0.7  l to 2.9  l.

1. Introduction

Pipettes are widely used for measuring the volume in various fields such as health, chemistry, biology,

pharmaceuticals and genetics. In order to ensure that the measurement results are reliable, laboratories

would send their pipettes for calibration [1]. The gravimetric method according to the international

standard, ISO 8655-6 [2] is commonly used for determination of the measurement errors for pipettes

owing to the simplicity, accuracy and traceability.

Multichannel pipettes can increase the throughput by eliminating the time-consuming and repetitive

pipetting. Hence, the applications of multi-channel pipettes are ever increasing, as they can increase

the output and productivity instantly. According to the ISO 8655-6, the measurement method for

multi-channel pipettes is similar to that for single-channel pipettes in that they comprise a set of

single-volume measuring and delivering units that are all operated simultaneously by a single piston

operating mechanism. During the measurements, each channel is regarded as a single channel to be

tested and reported as such. Ideally, all channels of the multi-channel pipette should being

simultaneously dispensed to the corresponding containers and then the weighing instrument

simultaneously weighs the dispensed fluid volume, but that demands the weighing instrument with a

number of weighing cells, that might be costly. The multi-channel pipette calibration system set up in

the SCL had up to 12 containers but only had one weighing cell. Hence, the objectives of this study

were to verify the repeatability and the linearity of the calibration system by using standard weights,

and validate the calibration system by comparing the volume measurement results for six selected

multi-channel pipettes with that of FORCE Technology.

1 36/F., Immigration Tower, 7 Gloucester Road, Wanchai, Hong Kong.

XXII World Congress of the International Measurement Confederation (IMEKO 2018)

IOP Conf. Series: Journal of Physics: Conf. Series 1065 (2018) 092004 IOP Publishing

doi:10.1088/1742-6596/1065/9/092004

2

2. Details of the multi-channel pipette calibration system

The setup of the multi-channel calibration system was shown in Figure 1. Twelve glass containers

housed in twelve brackets where the multi-channel pipette was dispensing the test liquid were orderly

placed in the rack, as shown in Figure 2. Guide rail was provided on both side of the measuring unit

for conveying the containers to the weighing position on by one.

Figure 1. The measuring unit of the

multi-channel pipette calibration system. Figure 2. Twelve containers were placed

in the rack.

2.1. Performing the measurement

The distilled water from the multi-channel pipette were respectively dispensed into the corresponding

containers. The containers were then conveyed onto the weighing cell as simulated in Figures 3 and 4.

The orientation of the pipette was not recommended to be changed during the measuring cycle.

Figure 3. The container (number 3) was

shifted onto the weighing position. Figure 4. The container (number 10) was

shifted onto the weighing position.

2.2. Calculation of the dispensed volume

The delivered volume was determined by using the gravimetric method. That was achieved by

dividing the mass by the density of the distilled water, calculated from equation (1)





20

aw

am

m

02 1

ρρ

ρρ

ρ

m

Vttdwt 

























(1)

Where V20 = volume, at temperature of 20  C (in milliliters, ml),

m = (mL -mE ) the net balance reading (in grams, g); where mL the weighing result of the balance after pipetting,

m

E the weighing result of that before pipetting,

 m = reference density of weights used to adjust the balance (i.e. 8 g/ml),

 w = density of water at calculation temperature t  C, in g/ml;  a = density of air, in grams per milliliter, g/ml,

t = thermal expansion coefficient of the pipette (in litre per degree Celsius, l/ C

t

dw = temperature of the distilled water (in degree Celsius, C).

3. Verification of the repeatability and linearity of the calibration system

According to the ISO 8655-6, the weighing instrument used to calibrate pipette with nominal capacity

above 10 µl shall be of resolution of 0.01 mg. The repeatability and linearity shall be of 0.02 mg and

the standard uncertainty shall not be more than two to three times the resolution. The specification of

Measuring unit

Containers and

dispensed location

Display and motor

control unit

Slide rack cover

XXII World Congress of the International Measurement Confederation (IMEKO 2018)

IOP Conf. Series: Journal of Physics: Conf. Series 1065 (2018) 092004 IOP Publishing

doi:10.1088/1742-6596/1065/9/092004

3

the weighing instrument meets requirements of the ISO 8655-6. However, in real application, due to

various environment and human factors, the performance of weighing instrument might be different.

Also, the measurement results of the weighing instrument shall be metrologically traceable to the SI of

mass. Hence, the weighing instrument's performance was verified using standard weights.

3.1. Verification method

Ten stainless steel mass standards ranging in mass from 2 mg to 10 000 mg were selected. To

simulate the actual operation of the calibration system, the standard weights were placed onto the

containers. If the repeatability and linearity of each channel were verified one by one, a large number

of standard weights and a number of steps might be involved. To streamline the calibration process, a

comprehensive calibration procedure was proposed, as indicated in the table 1.

Table 1. The calibration steps for evaluating the linearity of the weighing unit of the

calibration syste

. (unit in mg)

Channel

Steps 1 2 3 4 5 6 7 8 9 10

1 2 10000 5 000 2 000 1 000 200 100 20

2 5 2 10000 5 000 2 000 1 000 200 100

3 10 5 2 10 000 5 000 2 000 1 000 200

4 20 10 5 2 10 000 5 000 2 000 1 000

5 100 20 10 5 2 10 000 5 000 2 000

6 200 100 20 10 5 2 10 000 5 000

7 1 000 200 100 20 10 5 2 10 000

8 2 000 1 000 200 100 20 10 5 2 10 000

9 5 000 2 000 1 000 200 100 20 10 5 2

10 5 000 2 000 1 000 200 100 20 10 5 2

11 10 000 5 000 2 000 1 000 200 100 20 10 5

12

10 000 5 000 2 000 1 000 200 100 20 10

3.2. Verification results

The repeatability (standard deviation of 10 measurement results) and linearity (the mass value minus

the reading of the weighing instrument) measurement results were shown in table 2. Based on the

standard deviation and zero offset of measurement; resolution of the system and the uncertainty due to

reference weights, the overall expanded uncertainty of measurement was estimated of 0.06 mg.

Table 2. The repeatability and linearity performance of the weighing unit of the calibration system.

(unit in g)

Nominal mass value 0.002 0.005 0.01 0.02 0.1 0.2 1 2 5 10

Repeatability 0.000 03 0.000 03 0.000 03 0.000 02 0.000 03 0.000 03 0.000 03 0.000 02 0.000 03 0.000 03

Linearity 0.000 01 0.000 00 0.000 00 0.000 01 0.000 00 0.000 00 0.000 00 -0.000 01 -0.000 06 -0.000 11

4. Comparison of the results for six multi-channel pipettes at SCL and at FORCE Technology

Six selected multi-channel pipettes ranged from 30  l to 1000  l were selected as unit under tests for

comparisons with that of FORCE Technology. The calibration system used for FORCE Technology

was different, which calibrated one channel at a time using precision balances of finer resolution.

4.1. Comparison results

The measurement results for the dispensed volume of the six multi-channel pipettes were compared

with that obtained from FORCE Technology. For comparison measurements, the E n value [3] is

calculated from equation (2).

2

Re

2

Re

fLab

fLab

nUU

XX

E



 (2)

The calculation results were shown in table 3. The results showed the comparability of the calibration

system between the SCL and Force Technology, despite the calibration system was different.

XXII World Congress of the International Measurement Confederation (IMEKO 2018)

IOP Conf. Series: Journal of Physics: Conf. Series 1065 (2018) 092004 IOP Publishing

doi:10.1088/1742-6596/1065/9/092004

4

Table 3. Results of E n calculation based on the maximum deviation obtained from the twelve

channels

er each calibration

oin

Nominal Volume FORCE Technolo

SCL

E

ratio (Unit = µl)

Measured delivered

volume

Expanded

measurement

uncertainty

Measured delivered

volume

Expanded

measurement

uncertainty

30 30.19 0.38 29.85 0.4 0.6

50 50.15 0.18 49.75 0.7 0.6

100 100.00 0.33 98.8 1.3 0.9

300 298.64 0.24 299.2 1.7 0.3

500 499.96 0.43 500.7 2.0 0.4

1000 999.37 0.80 1000.5 2.9 0.4

4.2. Uncertainty of measurements

The uncertainty of measurement for the measurement of the volume dispensed by the multi-channel

pipette had been carried out in accordance with principles in the Evaluation of Measurement Data –

Guide to the Expression of Uncertainty in Measurement, JCGM 100:2008. The uncertainty of

measurement in each measured delivered volume was calculated by combining in quadrature a number

of uncertainty components, in which the following four uncertainty components were the dominant

components for the calibration system at SCL:

 Uncertainty due to the repeatability of measurement,

 Uncertainty due to the effect of evaporation of the dispensed water,

 Uncertainty due to the effect of heat transferred from the operator.

The uncertainty of measurement for measuring the delivered volume ranged from 30  l to 1000  l by

using the calibration system was summarized in table 4.

Table 4. The uncertaint

of measuremen

for various dis

ensed volume ran

es.

Volume dispensed (µl) Uncertainty of measurement (µl)

30 to 50 0.70

Above 50 to 100 1.3

Above 100 to 300 1.7

Above 300 to 500 2.0

Above 500 to 1000 2.9

5. Conclusions

An assessment of the repeatability and linearity errors of the newly set up calibration system for

calibrating multi-channel pipettes was performed using ten mass standards in value from 2 mg to 10 g.

The repeatability and linearity of the calibration system were found in compliance with the stringent

criteria as indicated in the ISO 8655-6. The comparison results between the SCL and Force

Technology for the measured volume results of six selected multi-channel pipettes ranging from 30µl

to 1 000 µl further confirmed that the multi-channel pipette calibration system was suitable for the

intended use. This meets the aim of providing multi-channel pipette calibration services for the local

chemical / medical testing and certification laboratories in accordance with the international standard

of ISO 8655-5.

6. References

[1] Ismail Z., Hayu R., Sutanto H. and Hafid XXI IMEKO World Congress "Measurement in

Research and Industry" p 1059

[2] ISO 8655-6 2002 Piston-operated volumetric apparatus – Part 6 : Gravimetric methods for the

determination of measurement error

[3] W. Wöger, Remarks on the E n–Criterion Used in Measurem. Comp.: PTB-Mitteilungen 109

(1999) 24

• Maryam Sharafi Farzad
• Brian Møllegaard Pedersen
• Helle Smidt Mogensen
• Claus Børsting

Here, we present the development of an automated AmpliSeq™ (ThermoFischer, MA, USA) workflow for library building using the Biomek ® 3000 Laboratory Automation Workstation (Beckman Coulter Inc., CA, USA), in which the total volume of PCR reagents and reagents for library preparation are reduced by one-half. The automated AmpliSeq workflow was tested using 43 stain samples (blood, bone, muscle tissue, semen, swab, nail scrape and cigarette butts) collected from crime scenes. The sequencing data were evaluated for locus balance, heterozygous allele balance and noise. The performance of libraries built with the automated AmpliSeq workflow using one-half of the recommended reagent volumes were similar to the performance of libraries built with the recommended (full) volumes of the reagents.

• W. Wöger

To set up a criterion for deciding on the compatibility of two different measurements of the same measurand not only the results of measurement but also the associated uncertainties must be taken into account. In this note the socalled En-criterion is considered that without detailed justification was introduced in the field of laboratory accreditation [2]. It will be discussed here within the framework of [4]. A decision on compatibility using this criterion will be all the more reliable the smaller the uncertainties of the two measurements involved. The statement of the uncertainties must be carefully checked for correctness. Due to systematic effects common to both measurements but not treated correctly the stated compatibility not necessarily means that the results of measurement are reasonable estimates of the measurand. To easily apply the criterion standard uncertainties or expanded uncertainties with the same coverage factor k = 2 are needed. A generalized form of the criterion is given that includes the case of correlated measurements.

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