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Statistics in Analytical Chemistry:

Part 7A Review
by David Coleman and Lynn Vanatta

With the publication of the previous installment (American Laboratory, July 2003), this series of
articles has arrived at a logical
spot for reviewing what has been
presented to date. The past material can be considered introductory for exploring the main task in
calibration studies (i.e., diagnosing or analyzing the calibration
data to determine the appropriate
model and its inherent uncertainties). Before proceeding to this
next topic (which will be discussed in considerable detail), it
seems prudent to summarize the
background material as well as
clarify some confusion that was
spotted by various readers in part
3 (American Laboratory, January
2003). This article will try to
achieve both goals.
Perhaps the overall theme thus
far has been Risk and Uncertainty. Both are an inescapable
fact of life, and the two go hand
in hand. People must determine
how much risk is acceptable,
where risk is a combination of
likelihood and severity of an outcome. In statistical terms, the
likelihood part translates to
the confidence level that is

needed; typical confidence levels


chosen are 95% or 99%. Once
the confidence level has been
selected, statistics (and this column) can guide the calculation
of the uncertainty in a given set
of measurements.
After the confidence level has been
determined, the user can decide if
the results are acceptable for the
application at hand. The real-number line has been divided (somewhat artificially) into segments that
take into account the amount of
uncertainty present. The main
dividing lines are 1) the critical
level (LC), below which a measurement cannot be distinguished (statistically) from zero; 2) the detection limit (DL), below which a
measurement cannot be detected
reliably; and 3) the quantitation
limit (QL), below which a measurement value cannot be reported reliably. The reason that such low-level
measurements are not reliable is
because there is too much uncertainty associated with them.
Clearly, analysts would prefer to
make measurements in regions in
which the relative uncertainty is
minimal. As Dr. William Horwitz
once stated, In almost all cases

when dealing with a limit of detection or limit of determination, the


primary purpose of determining that
limit is to stay away from it.1
As readers of this journal are well
aware, measurements are the bread
and butter of analytical chemistry.
Thus, knowledge of associated
uncertainties is critical. With most
analytical instruments, a vital (and
ever-present) source of variation is
the calibration procedure. (In this
discussion, it is assumed that the
dominant source of uncertainty is
calibration itself; such a situation
is not always the case.) Since the
computer typically generates data
in customer-unfriendly units such
as peak area or absorbance, the
analyst must find a way to transform the raw numbers into useful
readings (typically, concentration). Calibration is the name
given to the procedure that
involves 1) preparing and analyzing a set of standards, 2) collecting
the raw-response data, and 3) finding a statistical model to fit these
data and predict (the reverse of
calibrate) unknown samples.
Before step 1 can be conducted, the
calibration study must be designed.
continued

AMERICAN LABORATORY SEPTEMBER 2003

31

STATISTICS continued

This process involves


should be used instead.
selecting 1) the actual
WLS is particularly
concentrations or levmore suitable if there is
els that will be
a systematic change in
included, and 2) the
variability with respect
number of replicates
to concentration, such
that will be analyzed
as variability that infor each standard. The
creases with increasing
levels chosen should
concentration. With
bracket the expected
WLS, weights are calworking range for the
culated and applied to
procedure (no extrapthe data. The result is
olation, please). In
that the noisy data are
addition, there should
not allowed to inflube sufficient concenence the fit as much as
trations to determine
are the better-behaved
the behavior of the
measurements.
data (i.e., deciding if a
straight line is adeAssociated with any
quate or if a higherchosen combination
Figure 1
Plot of reported concentration (obtained from the appropriate calibraorder model is needed). tion curve) versus true concentration. The reported concentrations are for samples
of model and fitting
Closely spaced levels that have been spiked with known concentrations and are then analyzed. Legend: 1)
technique is a predicsolid line45 line, representing the situation where reported concentration always
should be included in
tion interval, the
equals true concentration; 2) dashed linereported-versus-true line; 3) dotted
areas in which 1) lineuncertainty interval around the reported-versus-true line; 4) dotindividual
width of which dedetection is an issue reported-versus-true measurement for a given spiked sample.
pends on the variabil(and a low detection
ity in the data and the
study (i.e., five replicates of each of
limit is desired), 2) curvature is
confidence level desired. This
five concentrations). However,
expected, and/or 3) critical measureinterval provides a statistically
careful determination of the objecments must be made.
sound estimate of the uncertainty
tives of the study (and how to
in the next measurement (typiaccomplish the goals) is crucial to
Generally, there should be enough
cally of a sample) predicted from
the success of any calibration work.
replicates of each standard to allow
the calibration curve. Both the
for modeling of the standard deviapredicted value and its related
In step 3, the choice of a model will
tion (i.e., to determine if the variuncertainty should be reported for
depend mainly on whether there is
ability is changing with concentraany analyzed sample. Only with
curvature in one or more regions of
tion). Also, if high precision is
both pieces of information can the
the range. The choice of a fitting
desired in any or all of the concenuser decide if the value is tight
technique depends on whether or
tration range, then extra replicates
enough for his or her purposes.
not the standard deviation changes
should be added.
with concentration. If the variability
In any discipline, terminology is
is not constant over the entire range,
A good starting place (not an absodeveloped to define various conthen ordinary least squares should
lute truth, just a guide that is
cepts that are specific to that area.
not be used as the fitting technique;
adjusted as circumstances warrant)
Statistics is no exception. Six
weighted least squares (WLS)
for calibration design is a 5 5
terms were discussed in the third
32

SEPTEMBER 2003 AMERICAN LABORATORY

STATISTICS continued

article of the series (American Laboratory, January 2003). Those concepts will be presented again, this
time in conjunction with a concentration-related figure (see
Figure 1).
It should be noted that the numerical value of any of the terms may
vary as the true value being measured changes; it is generally not
the case that bias, precision, etc.,
are the same throughout the
entire working range of a measurement system. It also should be
noted that error refers to a measurement itself, while the rest of
the terms pertain to a measurement system.
Error (of a measurement)the
(usually) unknown difference
between a single reported measurement and the true value; it
can include bias and noise

34

SEPTEMBER 2003 AMERICAN LABORATORY

Bias (of a measurement system)the systematic (or average) difference between reported values and the true value

sometimes reported as plus or


minus the half-width of the
interval; accounts for noise and
(ideally) bias

Noise (of a measurement system)measurement variation


over time or space, even with no
change in the true value; sometimes called random error or
stochastic error

Resolutionthe smallest discernible difference between any


two measurements that are
reported within the working
range of the method.

Reference
Precision (of a measurement system)the consistency of measurements over time or space;
precision is the reciprocal of
noise; high precision is equivalent to low noise, and low precision is equivalent to high noise
Uncertainty (of a measurement
system)a statistical interval
within which measurement
errors are believed to occur, at
some level of confidence;

1. Detection in analytical chemistry. Importance, theory, and practice. Currie LA,


ed. Am Chem Soc, Symposium Series
361; 1988:310.

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 LiquideBalazs Analytical Services, Box 650311,
MS 301, Dallas, TX 75265, U.S.A.; tel:
972-995-7541; fax: 972-995-3204; e-mail:
lynn.vanatta@airliquide.com.

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