Vericaton of Micropipete Calibraton

by Nathan Parsons
October 9, 2014
1 Introducton
Analytcal science ofen relies on the ability to pipete known volumes of liquids with high accuracy and
precision. It is therefore necessary to periodically verify that the systematc and random errors do not exceed
those permissible. The purpose of the current work is to verify the calibraton of Gilson P20, P100, and P1000
micropipetes and to verify the accuracy of a volumetric pipete.
2 Methods
Afer pre-wetng the tp, samples of deionised water were transferred into a beaker using Gilson Pipetman
P20, P100, and P1000 pipetes set to their maximumcapacites of 20.0 L, 100 L, and 1000 L, respectvely. A
4-gure analytcal balance was used to determine the mass of water transferred. For each pipete, 10 repeats
were made and in one additonal test, the total mass of 10 successive aliquots was measured.
Similarly, a 3-gure balance was used to determine the mass of deionised water transferred by a 5.00 mL
volumetric pipete. This was repeated 8 tmes.
In both cases, care was taken to ensure that the tp remained in contact with the side of the beaker.
3 Experimental Results
The measured masses were transformed into volumes using the Z-factor correcton shown in Equaton 1,
where Z is a correcton factor equal to the reciprocal of the density of the water and thus has the units L mg
1
.
The value of Z depends on temperature and barometric pressure and can be looked up in a table; the value
was taken to be 1.0029 L mg
1
, corresponding to standard conditons (20 C, 101.3 kPa) [1].
V = mZ (1)
Table 1: Experimentally determined errors for each pipete and corresponding permissible errors given by Gilson [1].
Pipete Selected Mean (L) Systematc Random Permissible Permissible
Volume (L) Error (L) Error (L) Sys. Error (L) Random Error (L)
P20 20.0 19.78 0.22 0.06 0.20 0.06
P100 100 100.4 -0.4 0.11 0.80 0.1
P1000 1000 997 3 0.63 8 1.5
Volumetric 5000 4950 50 39 30 -
Table 1 summarises the statstcal data for the tested pipetes. Systematc error is dened as the dierence
between the selected volume and the mean, whilst the random error is dened as the standard deviaton.
The calculated errors and maximum permissible errors are given in Table 1. Comparison of these suggests
that the P1000 is well calibrated, and the P100 is accurate, but has a slightly high randomerror, although there
is some uncertainty in this value. For the P20 pipete, the errors were of the same order as those permissible,
indicatng that the pipete is reasonably well calibrated, although again there is some uncertainty in this error.
As can be seen in Figure 1, for the ten successive aliquots, the volume per aliquot is similar to the measured
mean, although for the P1000, the value lies somewhat outside of the measured range for individual aliquots.
The mean of ten successive aliquots for P20 is slightly higher than the mean of the individual aliquots, sug-
gestng that the eect of read-out resoluton may have biased the mean of the individual aliquots.
The volumes of 10 successive aliquots were 199.4 L, 1005 L, and 9983 L for P20, P100, and P1000,
respectvely. Comparing these to the data in Table 1 suggests higher accuracy is atained by a single pipete
rather than by 10 aliquots froma pipete one-tenth the size. This can also be seen by comparing the systematc
errors shown in Table 1.
1
Figure 1: Box plots comparing the volumes delivered by Gilson and volumetric pipetes, expressed as a percentage of the
selected volume. Data points for single aliquot and 10 aliquot trials are shown as blue crosses and red pluses, respectvely.
Table 2: Measurement uncertainty estmated by the GUM method for the P1000 pipete.
Uncertainty Source Value () (L) Distributon Divisor Standard Uncertainty (L)
Room temperature (2 C) 0.4 Normal 1 0.4
Standard error on mean of data 0.20 Normal 1 0.20
Barometric pressure (10 kPa) 0.1 Normal 1 0.1
Read-out resoluton (0.1 g) 0.05 Rectangular

3 0.03
Combined Standard Uncertainty - Normal - 0.46
Expanded Uncertainty - Normal, k=2 - 0.92
Table 2 presents an estmate of uncertainty by the GUM (Guide to Uncertainty in Measurement) method.
With a 95 %condence interval, the expanded uncertainty for the P1000 pipete is 0.92 L. Similar uncertainty
analysis for the P100 and P20 pipetes gives expanded uncertaintes of 0.12 L and 0.07 L, respectvely, with
the uncertainty from the eect of room temperature and barometric pressure on the Z-factor diminishing at
lower volumes. The volumetric pipete has an expanded uncertainty of 28 L.
4 Discussion
Uncertainty can be reduced by accountng for the eect of temperature and barometric pressure on the
Z-factor. Since this eect scales linearly with mass, this reducton in uncertainty will have greatest eect on
measurements of large volumes. The Z-factor varies by 2 10
4
L mg
1
C
1
for temperatures around 20 C,
and by 1 10
5
L mg
1
kPa
1
for pressures around 100 kPa. For the P1000 pipete, measuring the temper-
ature to 0.5 C would reduce the corresponding uncertainty to 0.1 L, representng a reducton of 75 %.
As can be seen in Figure 1, the data points for P20 are split into 3 discrete values; this is due to the limit
of the read-out on the analytcal balance. The balance reads to 0.1 mg which equates to 0.5 % discriminaton
for the P20 pipete. The uncertainty of the read-out dominates for small volumes, but this can be reduced
through the use of a 5-gure balance, which would give a standard uncertainty of 0.003 L.
For the volumetric pipete, contaminaton of the internal surface at the tp led to high retenton. However,
this retenton was sporadically overcome by the contact of the tp against the side of the beaker, depending
on the exact angle of contact. Since the angle of contact had some variaton, this led to large random errors
with a bias that lowered the mean value, causing a large systematc error.
In conclusion, the P20, P100 and P1000 pipetes demonstrated reasonable calibraton, whilst the 5.00 mL
volumetric pipete was shown to be unreliable and inaccurate, likely due to contaminaton.
References
[1] Gilson, Inc., 3000 Parmenter Street, P.O. Box 620027, Middleton, WI 53562-0027, USA. Vericaton Proce-
dure for Accuracy and Precision, August 2007.
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