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 Gravimetric & Spectrophotometric Errors
Impact on Pipette Calibration Certainty[1]
John P. Clark & A. Harper Shull
Westinghouse Savannah River Company

The calibration of pipettes requires a high degree of skill and knowledge of the sources of error that can
obscure the true volume of liquid delivered from a pipette. Much has been written concerning the errors
associated with using air displacement pipettes. The same errors that contribute to the uncertainty of
volumes of liquid delivered by pipettes operated by laboratory personnel must be identified and managed
by pipette calibrators. Unless the variables are tightly controlled, it is difficult to determine if measured
volumes of liquid from the pipette being calibrated are outside of accuracy limits or due to systematic errors
in the calibration system.
The preparation of uncertainty budgets for the calibration process provides estimates of uncertainty
associated with the values generated by the measurement system. The uncertainty estimates quantify the
quality or accuracy of the calibrations. Error budgets are presented for both calibration systems. Details
are provided on developing the gravimetric error budget.

Introduction spectrophotometric pipette calibration methods.

A useful resource for those developing uncertainty
Air displacement pipettes are used to make most of the estimates is the ISO technical report ISO/TR 20461
volume measurements in chemical, environmental, Determination of uncertainty for volume measurements
medical, pharmaceutical and other laboratories. Calibration made using the gravimetric method.[4] It covers:
or verification is required to assure they are capable of
accurate and precise measurements. National, international Modeling the measurements,
and manufacturers procedures are used in testing pipettes. Standard uncertainty of a measurement according to
Currently, gravimetric and photometric methods are the the GUM [5] requirements,
most common methods used. Sensitivity coefficients to normalize each variable,
Much has been written concerning the errors associated Standard uncertainty associated with the volume
with using air displacement pipettes.[2] The same errors delivered by the pipette,
that contribute to the uncertainty of volumes of liquid Standard uncertainties of measurement,
delivered by pipettes operated by laboratory personnel Expanded uncertainties of measurement
must be identified and managed by pipette calibrators. Example for determining the uncertainty of the
Unless the variables are tightly controlled, it is difficult to measurement
determine if measured volumes of liquid from the pipette
being calibrated are outside of accuracy limits or due to In addition to providing uncertainty estimates,
systematic errors in the calibration system. traceability to national standards must be demonstrated.
Many calibration organizations are seeking national This means the uncertainty of the calibration standards
accreditation to ISO/IEC 17025 General Requirement for and instruments used in performing tests must be related
the Competence of Testing and Calibration Laboratories.[3] to a national measurement system and included in the
In it, Section 5.4.6 Estimation of Uncertainty of calculation of the measurement system uncertainty in
Measurement, provides the requirements for having and order to achieve traceability.
applying procedures for estimating the uncertainty of Balance manufactures publish performance
calibration measurements. It further specifies in section in specifications for repeatability, linearity, hysteresis, corner
5.4.6.3, When estimating the uncertainty of measurement, loading, etc., but do not provide uncertainty estimates for
all uncertainty components which are of importance in the measurements made on their products. One explanation
given situation shall be taken into account using for this could be the lack of knowledge of the contributions
appropriate methods of analysis. Pipette calibration of uncertainty from the balance user s environment,
sources of uncertainty include the measurement system, material being weighed, operator technique, etc., to the
the operator, environment and the pipettes. This paper will uncertainty of measurements. Therefore, it is incumbent
look at the sources for both the gravimetric and upon the user to develop estimates of uncertainty for the

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GRAVIMETRIC & SPECTROPHOTOMETRIC ERRORS IMPACT ON PIPETTE CALIBRATION CERTAINTY
JOHN P. CLARK & A. HARPER SHULL

conformity. Parts 1 through 6 of ISO 8655 are scheduled

to become official in mid 2002. Work is underway on Part
7 Non-gravimetric methods for the determination of
measurement error, and it should become official in late
2003. This part will cover both the photometric and
titremetric test methods.

Gravimetric Calibrations

The gravimetric pipette calibration uncertainty estimates

are listed in a table that is often called an error budget and
are based on the following model.

The pipette volume is measured using the following

model V20 =m * Z* Y
V20 = volume at 20 C
Figure 1. Gravimetric Pipette Calibration System. m = m2-m1+mE (gross weight minus the tare weight
plus the wt of evaporation)
gravimetric measurements used to calibrate or verify Z = air buoyancy correction (density of the air at the
pipette accuracy and precision. time of measurement) and density of the water
This is not the case for the Artel PCS 3 Pipette Y = coefficient of expansion for the pipette and fluid
Calibration System, which is very specific for pipette being dispensed.
calibrations. The manufacturer has a very comprehensive Each of these components has random or systematic
quality assurance program and knowledgeable scientists errors that obscure the true value of measurements.
that have invested the time and effort developing detailed The GUM requires the uncertainty sources be
measurement uncertainty estimates. This allows the users standardized, so their variances (squared standard
of their systems to evaluate the adequacy of the deviations) can be combined by adding and taking the
photometric measurements against the accuracy tolerances square root to estimate of the standard uncertainty.
of volumes they are calibrating. Their system is used
The total measurement uncertainty of the pipette
exclusively for pipette calibrations while electronic
volumes involves multiplying the standard uncertainty by
balances can be used for many applications in the typical
a k value to quantify the level of confidence for the total
laboratory. Studies were done to determine the accuracy
and precision of various adjustable pipettors at various
settings. Examples of uncertainty estimates for both
measurement systems are given below.

Pipette Calibration Systems

A new 5-place analytical balance with a pipette
calibration kit (Fig.1) and an Artel PCS 3 Pipette
Calibration System (Fig. 2) were studied to determine their
performance capabilities in calibrating pipettes. Several
tests were conducted to evaluate the sources of uncertainty
associated with both pipette calibration systems and the
accuracy and precision of each. Comparisons were made
between the two systems.
At the time of writing, the new ISO standard has not
been published. The draft copy, ISO/DIS 8665-6 Piston-
operated volumetric apparatus - Part 6 Gravimetric test
methods, states in the scope, This standard specifies the
gravimetric testing of errors of measurement of piston-
operated volumetric apparatus. The emphasis is testing
for errors which are measures of inaccuracy and
imprecision. These gravimetric test methods are the
reference test methods which shall be used as conformity
tests or type tests for declaration and certification of Figure 2. Artel PCS3 Pipette Calibration.

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 GRAVIMETRIC & SPECTROPHOTOMETRIC ERRORS IMPACT ON PIPETTE CALIBRATION CERTAINTY
JOHN P. CLARK & A. HARPER SHULL

Table 1. Uncertainty estimate of a pipette volume measurement.

(expanded) uncertainty. Guide 25 recommends reporting evaporation rates. Humidity traps are available from
uncertainties at the 95% CI, which is k=2. balance manufacturers as part of their pipette calibration
The gravimetric pipette calibration error budget, shown kits. Other techniques involve keeping the room
in Table 1, has major sections corresponding to the model humidity near ~ 60% or pipetting into capable vials.
describing the measurement system variables/errors that Studies can be done to determine the evaporation rate
make knowing the true value uncertain. These include: variations.
1. The balance which is used to measure m1 and m2, the 3. Air density parameters of the Z factor are usually
tare and gross, plus an estimate of the evaporation loss determined at time of calibration. However, some
m E . The balance sources of error include its organizations have determined the range of variation of
reproducibility, linearity and resolution (rounding up or their humidity and temperature over a year from QC
down). The manufacturer s specifications for the data and have used the range of barometric pressures
microbalance used for the gravimetric calibration can be over the year for their facility. In cases where tight
used. If studies have been done to determine to estimate controls are maintained on humidity and temperature,
the uncertainty of balance measurements, they should a constant Z factor can be used.
be used. Table 1 uncertainty estimates are based on 4. The coefficient of expansion for the pipetting device was
manufacturer s specifications and the evaluation of borrowed from the Biohit PLc. error budgets found on
balance QC Data. These estimates are not absolute and their website at www.biohit.com/pdf/app13.pdf. [6]
have uncertainty. For example, the evaluation of the QC This value is much larger than the estimate given in the
data from two different weight sets collected on 3 example of ISO/TC 20461. In correspondence with the
balances gave estimates that varied by up to 20%. Biohit Plc. authors, it was learned they chose a large value
Therefore, the average was used in the budgets. This to be conservative. Their published budgets were the first
information is shown in Figure 3 and discussed later. available on the internet.
2. Water temperature and evaporation rates contribute to A correlation between the volume delivered and the
the total uncertainty. Engineering estimates were made coefficient was determined for 1l and was multiplied
to define the interval of the uncertainty around the by the size of the volume being calibrated.This was
values. Various techniques can be used to minimize the done for the Z factor components also. An

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GRAVIMETRIC & SPECTROPHOTOMETRIC ERRORS IMPACT ON PIPETTE CALIBRATION CERTAINTY
JOHN P. CLARK & A. HARPER SHULL

engineering estimate of 2 K was picked for the precision are seen for volumes < 50 l.
heating of the pipette during calibration. This is an 6. Squaring, summing and taking the square root of these
estimate as no studies were done on this variable. two components gives a standard uncertainty. In the
Dividing these component intervals by the appropriate example above the measurement system uncertainty
number of standard deviations standardizes them. If only increases the pipetting precision from 15.97 nl to
the estimate has been derived from experimental data, 16.51 nl or by ~3.4%. This value is converted to l by
a normal distribution is assumed and should be divided by 1000 nl/l. The standard uncertainty of a
divided by the appropriate whole integer, if it has been volume measured by a pipette is usually expressed in
expanded. If not, the standard deviation is used. l or ml, not nl.
Engineering estimates or data from calibration reports, 7. The GUM recommends all uncertainties be expressed
manufacturers specifications and other sources are at the 95% confidence level. Therefore, the standard
assumed to come from rectangular distributions that uncertainty is expanded by a k factor of 2. Hence it is
have a probability of 1 and contains the true value. called the expanded uncertainty at k=2. The expanded
Therefore, the range should be divided by two and interval of 0.033 l is the best estimate of the
then be divided by the square root of 3 to obtain an uncertainty of a single volume measured by the
estimate of the standard uncertainty. operator and should contain the true volume 95% of
These standard uncertainties have different units that the time. The error budget gives documented evidence
must be normalized by converting them to the same of the quality of measurements made to calibrate/verify
unit. Multiplying them by the appropriate sensitivity a pipettes accuracy.
coefficient does this. ISO/TR 20461 reminds the reader
The estimate of uncertainty on the final calibration
that uncertainty estimates do not require exact values.
report should be the uncertainty of the average
Therefore, using the approximation of 1 microgram
volume delivered, based on the square root of the
(g) = 1 nanoliter (nl) to convert g to nl is acceptable
number of measurements made. Table 2 shows the
because the difference between them is small. After
uncertainty estimates based on average of 10
the standard uncertainties are multiplied by the
measurements as prescribed in the ISO draft
sensitivity coefficients the standard uncertainties are
standard. The uncertainty estimate provided by the
all in the same unit.
calibration organization is based on their personnel
These standard uncertainties are converted to variances and environmental conditions at time of calibration.
by squaring them. The variances are summed and the The user needs to develop estimates of volume
square root taken to give an estimate of variation for measurement uncertainty using their operators, in
the measurement system. An examination of them their environment, and on the material being
indicates the balance is the major source of uncertainty. pipetted, because they will be different than the
Therefore the choice of balance and the choice of the calibration organizations.
number of places is of utmost importance.
The precision estimate from the pipetting is reduced
5. The pipettes repeatability is usually the major source
by the square root of the 10 measurements. The
of uncertainty when considering all the variables in the
standard deviation of the mean is 5.05 nl. It is now
calibration or verification. In addition to the mechanical
only 20% larger than the calibration systems
operation and tip variation, the operator technique
combined uncertainty. Note the expanded
varies the volumes dispensed. The ISO/TR 20461
uncertainty of 0.013 l is now less than half the 0.033l
suggests the uncertainty of a volume measured by a
uncertainty of a single measurement with the pipette.
pipette can be estimated by assuming the
manufacturer s specification has a rectangular 8. The authors error budgets in Tables 1 and 2 contain
distribution with a probability of 1 in finding the volume one more piece of information. It calculates the ratio of
dispensed within this interval. The pipette precision the manufacturer s accuracy tolerance and the
value used in Table 1 was based on the average standard expanded uncertainty. This is a type of measurement
deviation of 30 calibrations based on 10 measurements system capability indices. It is desirable to have the
by different operators over several weeks. The precision measurement system uncertainty less than 1/3 of the
estimate of 15.7 nl is 4 times larger than the measurement tolerance. Another way of looking at this is to divide
system standard deviation of 4.18 nl. Both the ISO one side of the tolerance by the uncertainty to give the
Technical report and the draft ISO gravimetric number of uncertainty intervals inside the tolerance.
calibration standard report the combined uncertainty Ratios greater than 3 are very good. The ratio of the
of a gravimetric calibration system should be small manufacturer s specification over the expanded
compared to the precision of the pipetting. This has been uncertainty of the average calibration accuracy is 15.3 : 1.
confirmed by the authors experience. Smaller ratios of This calibration system uncertainty is more than
the calibration system uncertainty over the pipetting adequate for calibrating pipettes.

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JOHN P. CLARK & A. HARPER SHULL

Table 2. Uncertainty of a gravimetric calibrations average volume.

Sources of Uncertainty and Their to-day source of variation in balance measurements.[7]

Magnitude The balance standard deviation for weighing the same
weight is often two or three times the manufacturers
ISO/DIS 8665-6 specifies the use of analytical balances specification for repeatability. Better than an uncertainty
that conform to the International Organization of Legal estimate based on a calibration certificate or
metrology recommendation, OIML R 76-1. The scale manufacturers specification, is one that is based on the
graduation value of the balance used for testing should evaluation of a balance measurement control program.[8]
be chosen according to the selected volume of the A study was done to determine the uncertainty of the
apparatus under test. A table in the draft gravimetric balances based on weighing masses comparable to the
standard lists the minimum requirements for balances to volumes of water that would be weighed during a
be used for various volume calibrations. Balances have calibration. Three microbalances were evaluated daily
random and systematic errors. with two weights for both of the 7 and 6 place ranges.
In developing error budgets, manufacturer s Two different sets of weights were used during the year.
specifications are often used in estimating a balances One was the primary set, which was only used while the
uncertainty. However, it states If the standard daily set of weights was sent out for calibration. Most of
uncertainty of measurement of the balance is known (e.g. the data came from measurements of the daily set.
from the balance calibration certificate), this may be used Uncertainties were determined for the balances, based on
instead of the repeatability and linearity. The standard the weighing process variation, the uncertainty of the
uncertainty of measurement should not be more than 2 standards and half of the observed bias, based on the
to 3 times the readable graduation. apparent mass value on the calibration certificates. In all
The authors have studied the precision of several cases, the process variation was the major source of
balances and evaluated the data from various balance uncertainty. The estimates of uncertainty varied by 10 to
quality assurance programs and found there is a large day- 20%. The data are summarized in Figure 3 which gives

35 JAN FEB MAR 2003

GRAVIMETRIC & SPECTROPHOTOMETRIC ERRORS IMPACT ON PIPETTE CALIBRATION CERTAINTY
JOHN P. CLARK & A. HARPER SHULL

Figure 3. Uncertainty estimates of 3 UMT balances using 2 sets of weights.

the ISO/DIS recommended uncertainty of 0.002 mg for shown in Figure 4 and is composed of a
volumes of 10 l and below. The absolute uncertainty spectrophotometer, printer, reagent kits, calibration
increases with the size of the standards from 1 to 20 mg. standards and blanks, etc. The method offers several
The 1 and 10 mg weights were weighed on the 7-place advantages over the conventional gravimetric method.
scale and the 2 and 20 mg weights were weighed on the The major one being the time required to calibrating a
6-place scale. The calculated uncertainties are all larger pipette. Photometric calibrations routinely took less than
than the manufacturers specifications. 20% of the time required using the authors gravimetric
Evaluation of the errors associated with the balances system. Calibrating a 1l syringe from 0.1 to 1 l took
indicate the requirements recommended by the committee less than 10% of the time required doing it gravimetrically.
members that drafted ISO/DIS 8655-6 underestimate the The printouts from the actual calibration provide all of
uncertainty of electronic balance measurements. This is the basic data required for most QA systems.
especially true for small volume calibrations. The identification of the pipette, operator and
The primary value of generating error budgets is their instrument serial number, the date, time, temperature,
use as tools in determining where to make the most cost- measurements, statistics, etc. are recorded plus the date
effective process improvements. of last calibration, software version, and reagent lot
number. The documentation is satisfactory for paper
Photometric Pipette Calibrations recording keeping systems. The system can be interfaced
to a computer system to provide electronic records.
The photometric method of calibrating pipettes has The QA is comprehensive. The reagents have stated
shown significant growth and improvement in recent shelf lives. Each reagent blank will accommodate from 1
years. The authors evaluated this method of calibrating to 22 volumes of dye that are measured into it from the
pipettes to determine its capability to meet in-house pipette during calibration. The reagent cost per
accuracy and precision requirements. The system is measurement is from $0.50 and higher, depending on

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 GRAVIMETRIC & SPECTROPHOTOMETRIC ERRORS IMPACT ON PIPETTE CALIBRATION CERTAINTY
JOHN P. CLARK & A. HARPER SHULL

uncertainties that are well within customer tolerances.

Spectrophotometric Calibrations
The vendor provided information showing traceability
to the national measurement system for all of the
instruments used in calibrating the spectrophotometers,
preparing the reagents, monitoring the environmental
conditions, and validating the spectrophotometric system
against the gravimetric method. An example of the
uncertainty budget for the Artel PCS3 Pipette Calibration
System is shown in Table 3. The main components of the
system are the photometric, temperature, mixing,
reagents, stability, packaging and physics.
The instrument uncertainty addresses the errors for the
two wavelengths the vial are read and zeroed. It addresses
imprecision and non-linearity, vial imperfections, The root
Figure 4. Artel PCS 3 Pipette Calibration System.
sum square of these error sources has a relative
which of the 6 ranges of dye are being used and the uncertainty of 0.24%. The reagent uncertainty
volume being measured. The reagent costs are components contribute another 0.25% and are detailed in
progressively higher for the larger volumes. The volumes the table.
that can be calibrated range from 0.1 to 5,000 l. The manufacturer has identified the basic measurement
units of the system variables, defined the probability
Comparison of Calibration Methods distribution they came from and the range of the variables.
Next, they are converted into standard uncertainties using
Many pipettes were calibrated using both the the appropriate sensitivity coefficient to convert them to
spectrophotometric and gravimetric methods. The percents, squared them, combined the variances and took
precisions reflect the operators ability to pipette and the the square root to estimate the standard uncertainty for
variation of the calibration system. They were usually the parameters combined. The table also shows the total
comparable. About 5% of the time, the average calibrated combined and expanded uncertainties. The manufacturer
volume of a vial would be 0.5 to 0.8 % higher than the has claimed a much larger uncertainty than calculated.
other averages on different vials. This could be due to This allows for customer pipetting variation.
evaporation. However, all of the observed values on the The authors did not find any pipettes that exceeded
vial to vial comparisons fell within manufacturer s the claimed accuracy of the method that were not
specifications. confirmed by the gravimetric method.
Comparisons of measurements made at different times
were affected by many variables. A test was designed
that compared the same volume pipetted with the same
pipette under the same environmental conditions. A vial
of dye was placed on the pan of a 5 place Mettler AX205
analytical balance interfaced to a computer using Mettler
Balance Link version 2.2 to record the net weight of dye
removed by a Rainin EDP2 100 l digital pipette. The first
stable reading was recorded in the spreadsheet and the
reading from the PCS3 printer was recorded. The vial
was zeroed and another volume of dye was removed, the
first stable net weight was sent to the next cell in the
spread sheet and the dye was dispensed into the reagent
vial and read. This process was repeated 11 times.
The summary statistics of 10 calibrations are shown in
Figure 5. There was no statistically significant difference
in the average biases or standard deviations for the 10
sets of calibrations, even though there were significant
differences between individual samples in both methods. Figure 5. Comparison of gravimetric and spectrophometric
Both methods are capable of providing calibrations with average biases.

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JOHN P. CLARK & A. HARPER SHULL

Table 3. Uncertainty budget for the Artel Pipette Calibration System.

Conclusions 4. Technical Committee ISO/TC 48, ISO/TR

20461:2000(E),Determination of uncertainty for volume
The calibration of pipettes requires a reliable method measurements made using the gravimetric method,
and knowledge of the sources of error that can affect the First Edition, 2000-11-01.
quality of the measurements made during the calibration.
5. Guide to the Expression of Uncertainty in Measurement
Knowledge of the measurement system uncertainty will (GUM), BIPM, IEC, IFCC, ISO, IUPAC, IUPAP, OIML.
allow the calibrating organization to select the method First edition, 1995.
that will provide cost-effective measurements that meet
6. Riikonen, Seppo and Mannonen, Sari, Accredited
their required accuracy or error tolerances. Error budgets
Calibration and future Demands for Pipettors,
for a measurement system provide useful information in
International Biotechnology Laboratory, April 2000.
evaluating the adequacy of the measurements and to
identify the most significant areas for improvement. 7. Shull, A. H. and Clark, J.P., Balance Repeatability and
Reproducibility Effects on Measurement Uncertainty,
References MSC 2002 Proceedings, Anaheim, CA, January 25, 2002.
1. The information contained in this article was developed 8. Clark, J.P. and Shull, A.H., Methods for the Estimating
during work under Contract No. DE-AC09-96SR18500 Uncertainty of Electronic Balance Measurements, Cal
with the U.S. Department of Energy. Published as WSRC- Lab Magazine, January February March 2001, pp. 29-38.
MS-2001-00569. _________________________
2. Schiff, Leora, Difficulties in Achieving Well- John P. Clark, A. Harper Shull, Westinghouse Savannah
Characterized Accuracy and Reproducibility in River Company. Tel 803-725-3654 & 803-952-4687, johnp-
Micropipettes, Cal Lab Magazine, November-December [email protected], [email protected].
1998, pp. 24-27.
3. ISO/CASCO, Committee on Conformity Assessment, This paper was presented at the Measurement Science
ISO/IEC 17025:1999(E), First Edition. Conference, February 2002. It is reprinted here by permission.

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