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Calibration of micropipettes: Test methods and uncertainty analysis

Article  in  Measurement · April 2007

DOI: 10.1016/j.measurement.2006.05.012

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Elsa Batista Leão Pereira Pinto

Instituto Português da Qualidade Universidade Federal do Rio Grande do Norte
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Eduarda Filipe Adriaan M. H. van der Veen

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 CALIBRATION OF MICROPIPETTES: TEST METHODS AND
UNCERTAINTY ANALYSIS

E. Batista1, L. Pinto1, E. Filipe1, A.M.H. van der Veen2

1
Instituto Português da Qualidade (IPQ)- Rua António Gião, 2 – Caparica – Portugal
2
Nederlands Meetinstituut (NMi) - Schoemakerstraat 97, 2628 VK Delft – the Netherlands

Abstract. Micropipettes or piston pipettes are used to make the most of volume

measurements in fields like health, chemistry, biology, pharmacy and genetics.

Laboratories must ensure that obtained results using these instruments are reliable, for

that is necessary to calibrate micropipettes using the correct method and estimate

uncertainties. The objective of this work is to present the best method to calibrate

micropipettes according to the describe measurement conditions. A model for the

calculation of the measurement uncertainty is also described.

Keywords: Micropipette, uncertainty, measurement, calibration

1. Introduction

Pipetting in the microliter range is now a current and necessary task for almost every

field of chemistry. New dispensing systems allow experiments to be simpler and more

automated, but at the same time new field like genetics put heavier demands on the

reliability of the results. It is therefore important to focus on calibration and uncertainty

related with this kind of equipment.

Volume measurement is critical in any analytic laboratory, but in particular in

highly sensitive tests (that use a very small amount of liquid) where a small mistake in

pipetting may cause a large error in the final result. In order to reduce and identify
possible errors in liquid handling it is necessary to calibrate micropipettes under similar

conditions as the ones used in the laboratory.

This paper has the main purpose to test and discuss several calibration

procedures and find the best method for calibrating micropipettes.

The methodology applied to evaluate all uncertainty components and a model

for evaluating the measurement uncertainty is described.

2. Experimental methods

The principle of the operation of dispensing is simple: a disposable tip, usually of

polypropylene, is attached to the piston pipette and, with the piston at the aspiration

lower limit, the tip is dipped in the liquid. When moved to the aspiration upper limit the

piston aspirates the liquid. To expel the liquid the piston is slid between the volume

defining marks. The volume delivered by the micropipette is determined using a

gravimetric method and water as the calibration liquid at a temperature of (20 ± 0,5) ºC.

The calibration of micropipettes was carried out using four different gravimetric

methods based on different ISO standards.

In all four methods a suitable mass comparator is needed for registering the mass

delivered for a given pipetted volume. The water and room temperature must be

carefully controlled during the measurements.

The first method, the “normal” method, is based on ISO 4787 [1] and ISO 8655

[2]. The micropipette is wetted five times to reach equilibrium with respect to humidity.

Then the tip is changed and wetted again. The weight of the dry beaker is determined,

and so is the volume pipetted. The beaker is weighted again and the mass obtained by
difference is a measure for the volume of the micropipette. The beaker is cleaned after

each volume addictions and this procedure is repeated ten times.

The second method is the “evaporation” method based only in ISO 8655. The

micropipette is wetted five times and then the tip is changed and wetted one time. The

weight of the beaker with a 3 mm water film is determined (m0). A stopwatch is started

and the testing volume is pipetted. The beaker is weighted again (m1), then the test

volume is pipetted again and added to the beaker (m2). The mass obtained by difference

(m1-m0, m2-m1, …, m10-m9) is a measure for the volume of the micropipette. The

stopwatch is stopped after the tenth measurement and the weighing vessel is left on the

balance for the time used for the ten measurements and its mass is recorded (m12). The

value determined due to evaporation loss is added to each measurement. To calculate

the evaporation loss the following formula is used: (m11-m12)/10.

The third method is the “without evaporation” method, is also based on the ISO

8655 but the evaporation loss is not taken in count. The micropipette is wetted five

times, and then the tip is changed and wetted once more. The weight of the beaker with

a 3 mm water film is determined and the testing volume is pipetted. The beaker is

weighted again and the mass obtained by difference is a measure for the volume of the

micropipette. This cycle is repeated ten times.

The last method is the “without evaporation using oil” method. This method is

very similar to the third and is also based in ISO 8655. First of all, the beaker must be

filled with 5 mm film of oil. Due to the fact that the oil does not evaporate the beaker

can be weighted any time before the start of the measurements. As in the others

methods, the micropipette is wetted five times and the tips are replaced as described

before. A new tip is wetted and filled with the testing volume and delivered into the
beaker, with caution in order to ensure that the water goes beneath the oil. It is

important not to deliver near the beaker walls. After delivering, the beaker is weighted

again and the mass obtained by difference is a measure for the volume of the

micropipette. The procedure is repeated ten times. Within each measurement the mass

of the beaker must be registered.

Five tests were preformed to each micropipette of 5 µl, 100 µl, 250 µl and 1000

µl in all four methods.

3. Evaluation of measurement uncertainty

The evaluation of the uncertainty of all four methods follows what is commonly

referred as “mainstream GUM [3]” and is similar to the one described in the work of E.

Batista et.al., Metrologia 2000 [4].

To determine the volume of a micropipette the following formula (1) is used:

 ρ 
× 1 − A  × [1 − γ (t − 20)]
1
V20 = ( I L − I E ) × (1)
ρW − ρ A  ρ B 

where

V20 - volume, at temperature of 20 ºC, in µl;

IL - weighting result of the recipient after the pipetting in mg;

IE - weighting result of the recipient before the pipetting, in mg;

ρW - water density, at calibration temperature t, in mg/µl;

ρA - air density, in mg/µl (0,0012 mg/µl);

ρB - mass pieces density (8,0 mg/µl);

γ - material cubic thermal expansion coefficient of the micropipette, in /ºC;

t - water temperature, in ºC
Defining 1/(ρW-ρA) as A, 1-ρA/ρB as B, 1-γ(t-20) as C, and m as IL-IE, equation (1) can be

rewritten in a simpler way as

V20 = m × A × B × C

Some of the variables in (1) depend on others. A complete overview is given in figure 1.

Figure 1- Structure of model

The expression for the standard uncertainty associated with V20 is developed

from (1), using the law of propagation of uncertainty. The resulting expression reads as:
1
2 2 2
 ∂V20 2 2  ∂V20  2  ∂V20  2  ∂V20   ∂V20 
2
 ∂V20 
2
2
u(V20) =   u (m) +   u (ρw ) +  
 u (ρ ) + 
 ∂ρ   u(ρ )2
+ 
 ∂γ   u(γ )2
+   u(t )2
 (2)
∂ ∂ρ  ∂ρ
A B
∂
 m   w  A  B    t  

One of the most important uncertainty components is the uncertainty of the

operator that is included in the standard deviation of the mean. Instead of doing all the

10 measurements with the same adjustment of the scale, the experiments were made

with scale adjusting within each measurement.

3.1 Evaluation of standard uncertainty of input variables

The uncertainty of the mass is a combination of the uncertainty of the calibration

of the comparator, the repeatability of weighting and the resolution of reading.

m = M + δmres + δmcal (3)

 The uncertainty of the temperature is essentially evaluated in the same way: the

calibration of the thermometer and its repeatability are the main uncertainty

components.

t water = t + δt cal (4)

The evaluation of the uncertainty of the density of water is more problematic.

The ISO 4787 provides a table with water densities. For the calibration uncertainty of

the thermometer (0,1 °C), the uncertainty of the water density must be obtained. This is

done by interpolating the tabled values.

For the air density, the standard uncertainty is estimated to be smaller than

0,0005 g/ml, which is obtained from a review from Spieweck and Bettin [5]. The

standard uncertainty of the density of the mass pieces is obtained from the certificate of

the comparator. The standard uncertainty of the expansion coefficient is obtained from

the last significant number in the table described in the ASTM E542 [6].

For all variables the assumed distributions are given. For the volume V20, the

normal distribution can be assumed. The results are summarized in table 1 for a

micropipette of 5 µl.

Table 1- Evaluation of uncertainty of a micropipette volume

For all variables the assumed distributions are given. For the volume V20, the normal

distribution can be assumed.

4. Discussion of results

The described work had the main purpose of finding the suitable method to calibrate

micropipettes meeting laboratory requirements and based on existing ISO standards.

 Four air displacement micropipettes were tested. Fixed volume micropipettes of

5 µl, 100 µl and 250 µl and a variable micropipette of 1000 µl. The results are shown in

figures below.

Figure 2 – Calibration of a 5 µl micropipette Figure 3 – Calibration of a 100 µl micropipette

Figure 4 – Calibration of a 250 µl micropipette Figure 5 – Calibration of a 1000 µl micropipette

As it can be seen from the figures, the best repeatable method with smallest

uncertainty for all micropipettes in almost every test is the method “without

evaporation”. This method is faster and more reliable because it does not have any

awaiting time during the procedure. Furthermore, there is no volume correction

necessary after the measurements, neither a cleaning step for the beaker with solvents to

remove the oil afterwards.

The “normal” method is the one with the lower volume results due to the

evaporation loss caused by the major evaporation surface when the liquid touches the

glass. Is also the slower method because it is necessary to clean the beaker after each

volume addition.

The volume correction in the “evaporation” method can lead to results that are

not correct, as the major part of the evaporation occurs between additions, and not

between the additions and the balance stability, so when a correction is made the

measured volume is increased in a incorrect way when compared with the real value.
The correction value is a mean value added to each of the ten measurements. This can

decrease the reliability of the method.

The use of oil was thought to prevent evaporation of water while the calibration

is undergoing, in order to have better results but after evaluating the results, it is

concluded that this method does not have any benefit over the “without evaporation”

method. In the case of the 5 µl micropipette, the results are even worse. There is a great

difficulty in delivering a small quantity of liquid inside the oil without touching the oil

or the wall of the beaker, which leads to evaporation loss and a reduction in the volume.

5. Concluding remarks

Several test were preformed in order to determine the best method for calibration of

micropipettes based on the existing ISO standards. After the evaluation of results and

uncertainty it can be concluded that the without evaporation method is the most

efficient.

The described model for the calculation of the measurement uncertainty was

described and it proved to be completely suitable for this type of gravimetric method.

The presented uncertainty analysis can also be used for other piston operating

volumetric equipment and laboratory volumetric glassware if the same gravimetric

method is applied.
References

[1] ISO 4787, 1984, Laboratory glassware - Volumetric glassware - Methods for use

and testing of capacity.

[2] ISO 8655-1/2/6, 2002, Piston-operated volumetric apparatus.

[3] BIPM, IEC, IFCC, ISO, IUPAC, IUPAP, OIML, Guide to the expression of

uncertainty in measurement (GUM), Geneva, 1995.

[4] E. Batista, A. Sabrosa, M. Ferreira, I. Castanheira., A.M.H. Van der Veen,

“Uncertainty calculation in the calibration of volumetric laboratory glassware”, in

Metrologia 2000, Brasil, 2000.

[5] Spieweck F., Bettin H., Review: Solid Liquid density determination, Tm-

Technisches Messen, 59, (1992), 237-292,.

[6] ASTM E542, Standard Practice for Calibration of Laboratory Volumetric

Apparatus, 2000.
Figure 1- Structure of model
t
A P

ρΒ
B P

H
V(20)
t

γ
C
t

rpt.

m res.

cal.
Figure 2– Calibration of a 5 µl micropipette

5 µl micropipette
5,5

5,4

5,3

5,2

5,1
Volume (µl)

5,0

4,9

4,8

4,7

4,6

4,5
1 2 3 4 5
Test
With evaporation Without evaporation
Normal With oil
Figure 3– Calibration of a 100 µl micropipette

100 µl micropipette

102,0

101,5

101,0

100,5
Volume (µ l)

100,0

99,5

99,0

98,5

98,0
1 2 3 4 5
Test
With evapo ratio n Witho ut evapo rtio n
No rmal With Oil
Figure 4– Calibration of a 250 µl micropipette

250 µl micropipette

255,0

254,0

253,0

252,0
Volume (µ l)

251,0

250,0

249,0

248,0

247,0
1 2 3 4 5
Test
With evapo ratio n Witho ut evapo ratio n
No rmal With Oil
Figure 5– Calibration of a 1000 µl micropipette

1000 µl micropipette

1011,0
1008,0
1005,0
1002,0
999,0
Volume (µ l)

996,0
993,0
990,0
987,0
984,0
1 2 3 4 5
Test
With evapo ratio n Witho ut evapo ratio n
No rmal With o il
 Table 1 - Evaluation of uncertainty of a micropipette volume
Variable Estimate Distribution Standard Sensitivity Contribution
Xi xi uncertainty coefficient ui(y)
u(xi) ci (µl)
M (µg) 5,033 Normal 0,0088 1,00 0,0088
δmres (µg) 0 Rectangular 0,001 1,00 0,001
δmcal (µg) 0 Normal (k=2) 0,005 1,00 0,005
t (°C) 19,5 Normal 0 -0,00121 0
δtcal (°C) 0 Normal (k=2) 0,05 -0,00121 -0,0000606
ρW (µg/µl) 0,9983 Rectangular 0,0000116 -5,06 -0,0000588
ρA (µg/µl) 0,0012 Rectangular 0,000000289 4,43 0,00000128
ρB (µg/µl) 7,96 Rectangular 0,0346 0,000096 0,00000332
γ (1/°C) 0,000240 Rectangular 0,00000289 -2,52 -0.00000729
V20 (µl) 5,05 U(V20), 0,02
k=2

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