Statistics in Analytical ChemistryPart 5

Calibration: Calibration design

David Coleman and Lynn Vanatta
ith many things in life,
the quality and usefulness
of the outcome depends
in large measure on the care with
which the event is planned. Calibration is no exception. While a
statistical evaluation can be made
on any set of calibration data, the
results can be meaningless if the
design of the study is flawed. This
column will discuss the steps that
should be taken to develop a calibration study. In addition, some
example designs will be presented.
It must be stressed, however, that
there is no one-formula-fits-all
answer. Every calibration situation
is different and there is no substitute for careful thinking.
The following four steps will
help ensure that a successful calibration study is conducted:
Decide the objectives of the
study
Propose a model for the calibration curve
Choose the confidence level for
the curve
Design the study carefully.
The following discussion will
look at each step in more detail.

Objectives
First and foremost, one must

decide what should be accomplished in the study. In other

words, when sample results are predicted from the calibration curve,
what topics should be addressed?
Topics to consider include:
a) Detection limits (DLs). Will the
laboratory be pushing the sensitivity
of the instrument so that detection
limits are an issue? If so, how low of
a detection level is needed? When
trace work must be done, as many
concentrations as possible should be
included in the low region. The
study should also include a blank, at
least one level that is below the
desired DL, and at least one concentration above the desired DL.
b) Data behavior. In the plot of
response versus spike concentration, is curvature in the data
expected for any of the analytes?
(The answer here is usually based
on empirical data or physical/
chemical principles.) If a nonlinear
response is expected (e.g., polynomial), concentrations should be
added in the area(s) of changing
slope. Otherwise, the wrong calibration model may be selected,
and a biased (i.e., consistently
incorrect) estimate of the calibration function could result.
c) Precision. What level of preci-

sion is needed? This answer

depends in part on the consequences of a "wrong" answer. If
someone's life depends on the
result, the data will need to be
much more precise than if only
ballpark estimates are needed.
The higher the precision (i.e., the
smaller the width of the prediction interval) desired, the greater
the number of replicates needed
in the design. (Coupled with this
subject is the confidence level
needed, as discussed below.)
d) Concentration range. What levels are expected in future samples?
Covering a wider range than necessary may widen the prediction
interval for the calibration curve
(see the previous installment of this
column).1 Doing so also runs the
risk of having too scarce a distribution of data points to define the
curve appropriately. On the other
hand, it is important to ensure that
there is at least one level slightly
above and one slightly below the
expected range; it is not wise to
extrapolate a calibration curve.
Spacing of the levels must also be
considered. As mentioned above, if
curvature is expected and/or a low
DL is desired, an adequate number
of concentrations should be included in those regions. A good
starting point is a semi-geometric
design in which each successive
level is a multiple of the previous
(e.g., twice or thrice) one. Additional concentrations can then be
added as needed.

Model postulation
At the start, it is always preferable to have some idea of the expected behavior of the data. As
mentioned above, if curvature in
the data is anticipated, the concentrations should be spaced
appropriately to allow adequate
modeling of that region(s). A common starting point is a straight

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JUNE 2003 AMERICAN LABORATORY

Table 1

Sample calibration designs

rule of thumb (but by no means a

hard-and-fast recommendation) is
to start with a 5-by-5 design (five
levels, each with five replicates)
and adapt as needed.
Some example designs are given
in Table 1. As an exercise, the
reader is invited to try evaluating
each design in light of the above
discussion and postulate why each
example was structured as it is. The
next article will discuss each layout
and will present some actual calibration designs the authors have
implemented. Happy trials!

References
1.

2.

line, with ordinary least squares

(OLS) as the fitting technique. No
matter what model is set forth, it
will be tested after the data are collected and will be revised if necessary. For a straight line, three or
more distinct concentration levels
are needed for proper testing.

fidence level. However, it can be

helpful to the design process to
have a confidence level in mind.
The higher the confidence level,
the greater the number of calibration data points that are required to
achieve any given interval width.)

Coleman D, Vanatta L. Statistics in

Analytical ChemistryPart 4, Calibration: Uncertainty intervals.
Am Lab 2003; 35(5): 602.
Coleman D, Vanatta L. Statistics in
analytical chemistry: A new American Laboratory column. Am Lab
2002; 34(18): 445.

Mr. Coleman is an Applied Statistician,

Alcoa Technical Center, MST-C, 100
Technical Dr., Alcoa Center, PA 15069,
U.S.A.; e-mail: david.coleman@alcoa.
com. Ms. Vanatta is an Analytical
Chemist, Air Liquide-Balazs Analytical Services, Box 650311, MS 301,
Dallas, TX 75265, U.S.A.; tel: 972995-7541; fax: 972-995-3204; e-mail:
lynn.vanatta@airliquide.com.

Study design
Confidence level
One can never be totally confident in an answer (the world is simply not set up that way); thus, one
must decide on a tolerable degree
of risk of being wrong. Common
choices are 95% or 99% confident
(i.e., 5% or 1% risk, respectively),
but the final selection depends on
the quality requirements of the
analysis. As stressed in the first column,2 statistics cannot make this
decision for anyone. All affected
parties should negotiate and decide
what is necessary and desirable.
People still have to think! (The alert
reader may realize that choosing a
confidence level need not precede
the calibration study design, since a
new confidence level can be chosen
at any time, even after the study
has been conducted. Only the Students t values change with the con-

After steps 113 have been evaluated and the objectives for the
calibration have been determined,
the study can be designed. The
critical decisions are the concentrations to include, and the number of replicates of each concentration. The first decision depends
primarily on the range to be covered and the areas that need
emphasis (e.g., low-DL and/or curvature regions). The second decision depends in large part on the
precision needed in the various
regions of the curve. It is not necessary that the same number of
replicates be included for each
concentration. However, replicates at multiple concentrations
are needed in order to test a critical assumption of OLS; i.e., that
the standard deviation is not
increasing with concentration. A

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