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Principles of analytical calibration/quantification for the separation sciences

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 Journal of Chromatography A, 1158 (2007) 33–46

Review

Principles of analytical calibration/quantification for the

separation sciences
Luis Cuadros-Rodrı́guez ∗ , M. Gracia Bagur-González, Mercedes Sánchez-Viñas,
Antonio González-Casado, Antonio M. Gómez-Sáez
Department of Analytical Chemistry, University of Granada, E-18071 Granada, Spain
Available online 16 March 2007

Abstract
Calibration is an operation whose main objective is to know the metrological status of a measurement system. Nevertheless, in analytical sciences,
calibration has special connotations since it is the basis to do the quantification of the amount of one or more components (analytes) in a sample, or
to obtain the value of one or more analytical parameters related with that quantity. Regarding this subject, the aim of analytical calibration is to find
an empiric relationship, called measurement function, which permits subsequently to calculate the values of the amount (x-variable) of a substance
in a sample, from the measured values on it of an analytical signal (y-variable). In this paper, the metrological bases of analytical calibration and
quantification are established and, the different work schemes and calibration methodologies, which can be applied depending on the characteristic
of the sample (analyte + matrix) to analyse, are distinguished and discussed. Likewise, the different terms and related names are clarified. A special
attention has been paid to those analytical methods which use separation techniques, in relation with its effect on calibration operations and later
analytical quantification.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Chemical measurement processes; Metrological and analytical calibration; Analytical quantification; Calibration schemes; Calibration methodologies

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2. Metrological fundamentals: measurement function for analytical calibration/quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3. Schemes for analytical calibration/quantification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.1. Two-standard calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.2. One-standard calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4. Methodologies for analytical calibration/quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.1. External calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.2. Matrix-matched calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3. Standard addition calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.4. Internal calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.5. Calibration by internal normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

1. Introduction

A chemical measurement process (CMP) is an analytical

method of defined structure that has been brought into a state of
∗ Corresponding author. Tel.: +34 958243296. statistical control, given the measurement conditions [1]. Cali-
E-mail address: lcuadros@ugr.es (L. Cuadros-Rodrı́guez). bration and validation are key operations in a CMP because they

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.03.030
34 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
provide traceability and comparability to the measurement, act-
ing the former as a metrological interface between measurement
standards and analytical results. Nevertheless, in analytical sci-
ences, calibration can be considered from two points of view
as metrological calibration as well as analytical calibration, not
being its requirements and aims similar necessarily. From a prac-
tical view, analytical calibration implies to verify or to state
the relationship between the measurement signal and the ana-
lyte/s quantity, whereas metrological calibration, based on the
analytical one, is just related to a “measuring system” under-
stood as a set of one or more measuring instruments and often
other devices, including any reagent and supply, assembled
and adapted to give measured quantity values within specified
intervals for quantities of specified kinds [2]. The metrological
calibration implies the daily evaluation of the metrological per-
formance of a measuring system in order to provide the quality
of the analytical results. Therefore, with a calibrated chemical
measurement system could be possible to obtain good results
although it provides erroneous indications, because these ones
can be corrected due to, in the calibration process, the error
is established. That is why, a metrological calibration leads
to the characterization of a dependence relationship between
the error committed by the measurement system and the value
of the measure, expressed in terms of parameters as devia-
tion, bias, correction, correction factor or calibration factor.
This relationship is only established for the measurement val-
ues represented by the calibration standards used, but it can be
extended interpolating into the interval covered by them through
a correction/calibration function. Specific information about the
metrological approach of the calibration in a CMP can be found
in references [3,4].
Calibration in metrology, has been recently defined in a new
revision of the International Vocabulary of Metrology (VIM)
as the operation that, under specified conditions, in a first step
establishes a relation between the quantity values with measure-
ment uncertainties provided by measurement standards and the
corresponding indications [of a measuring system]1 with asso-
ciated measurement uncertainties and, in a second step, uses this
information to establish a relation for obtaining a measurement
result from an indication [2] where a “measurement standard”
is referred to the realization of the definition of a given quantity,
with stated value and measurement uncertainty, used as a refer-
ence and it can be provided by a measuring system, a material
measure, or a certified reference material [2]. This new two-
step definition generalizes the application of the previous VIM’s
definition [5] still in effect, simplifies the wording/phrasing and
improves substantially its understanding. The main innovation
comes from the second step since as the VIM itself quoted, in
this definition the first one has been traditionally perceived as
the proper calibration. As it will be highlighted in this paper, this
second step could include all the analytical operations closely
related with calibration.
The aim of the chemical analysis of an object or a material,
is to obtain analytical information of it represented by the value
1 The text in brackets has been added by authors
of an input analytical quantity, X, through the measurement of
a physicochemical magnitude, Y, denoted as output analytical
quantity. That is why it is necessary to have a “measurement
function”, Y = f(X), which permits to link the values of the out-
put analytical quantity with those corresponding to the input
analytical quantity. This one is a function of quantities, the value
of which, when calculated using known quantity values for the
input quantities in a measurement model, is a measured quantity
value of the output quantity in the measurement model [2].
In most cases, that link, always supported in a physicochem-
ical principle, cannot be expressed in a general way by means
of a straightforward mathematical function, being necessary to
use empirical models adapted to each analytical system. At this
point, is where analytical calibration stress, due to it is the oper-
ation why an empirical mathematical function is established.
Analytical calibration is always an indirect calibration, because
is not possible to measure the quantity of a substance in a
system. There are different regulations where it can be found
some calibration definitions, that strictly speaking are defini-
tions of analytical calibration, which are consistent with the one
established in the most recent VIM’s version. Thus, a ISO stan-
dard [6] states that [analytical] calibration is a complete set of
operations which estimates under specified conditions the cal-
ibration function from observation of the response variable, y,
obtained on reference states (described by the values of vari-
able states, x). Also, an IUPAC recommendation [7], defines the
[analytical] calibration as the operation that determines the func-
tional relationship between measured values (signal intensities),
y-variable, and analytical quantities characterizing types of
analytes and their amount (content, concentration), x-variable.
It can be seen that in these definitions appear the most usual
analytical names for the y-variable (signal intensity, response),
and x-variable (type of analyte, analyte amount). This IUPAC
recommendation distinguishes two different kind of calibration
functions. The first one, for species identification and qualitative
analysis, and the second for quantitative analysis, being its main
purpose to obtain a function that allows to calculate amounts of
an analyte as a function of an instrumental signal (quantification
function). In most cases, the calibration function has to take into
account the response relations for all relevant constituents and
interferences.
These calibration functions are characteristics only for the
standards used and depend on the actual instrumental and oper-
ational conditions due to the presence of “influence quantities”,
i.e. quantities that, in a direct measurement does not affect
the quantity that is actually measured, but affects the relation
between the signal intensity and the measurement results. Small
changes in the influence quantity values could change the indi-
cation of the measurement system and that is why metrological
calibration by itself does not guarantee the comparability of the
measurements. In order to assure that the measurement system
remains in a calibration state, and that certain influence mag-
nitudes are under statistical control, some periodical controls,
named “verification” operations, are required. Verification, in
this context, is defined as the provision of objective evidence
that a given item fulfils specified requirements, taking any mea-
surement uncertainty into consideration [2]. The verification
 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
Fig. 1. Flow-chart showing up the relationships between both analytical cali-
bration, metrological calibration and analytical quantification.
is a confirmation that stated performance properties or quality
requirements of a measuring system, are achieved. So, when a
measurement system has been verified, the maintenance of its
calibration state is assured.
Thus, the main objective of calibration is to establish a
mathematical function from measurement standards which sub-
sequently is applied to infer analytical information from the
samples/materials analysed on the basis of an initial hypothesis
in which implicitly is admitted that, samples and standards are
metrologically equivalents. In order to obtain a quantification
free of errors, is necessary to satisfy two basic requirements:
(i) standards and samples composition must be as similar as
possible and (ii) standards and samples must have an identical
behaviour in the measurement system. In any case, the standard
must be representative of the sample; usually the first situation
although desirable, is not essential being enough to fulfil the
second requirement to assure representativeness. The interre-
lation between both analytical calibration and quantification is
arranged in sequence in Fig. 1.
In general, the representativeness is satisfied using standards
of the analyte in the very material to be analysed. Nevertheless,
in those separation methods in which a multi-analyte quantifi-
cation is going to be done, the calibration for each separate
analyte can be tedious or even impossible (if there are no
available standards), being necessary to take into account other
options.
In this paper, the necessary conditions to assure the represen-
tativeness and the strategies that can be adopted to minimize the
errors in the quantification when this one was not fulfilled, are
going to be established. Thus, the metrological fundamentals of
the calibration/quantification, the calibration schemes depend-
ing on the number of standards employed and the methodologies
that can be used for the calibration/quantification in separation
sciences, will be widely explained, emphasizing the practical
requirement for making measuring and discussing common mis-
conceptions and errors that might arise. Consequently, names
as external calibration, internal calibration, internal normaliza-
tion calibration, matrix-matched calibration, standard addition
calibration, and signal-ratio calibration together with terms as
external standard, surrogate or internal standard will be dis-
cussed and clarified.
35
2. Metrological fundamentals: measurement function
for analytical calibration/quantification
The simplest way to write a measurement function for each
particular analytical calibration/quantification is:
Y = Φ(x, m) · X
where X and Y represent, respectively, the analyte amount (input
analytical quantity) and the analytical signal (output analyti-
cal quantity) and Φ(x, m) is the “measurement factor” which
depends on the value x of the magnitude X, and on the type of
material (matrix) m where it was. This general expression, valid
in all circumstances, indicates that for each analyte amount and
matrix, the analytical signal is obtained by multiplying the true
analyte amount by a specific measurement factor. Nevertheless,
the simplicity of this expression is deceitful, because in practice
is impossible to characterize the Φ-parameter and to know each
one of the values it can take.
In an ideal situation, the measurement factor would just be
characteristic of the type of analyte and independent of the ana-
lyte amount and/or the matrix; in this case, the CMP could be
labelled as selective. However, in analytical sciences the mea-
suring systems show frequently low selectivity and the value
of the measures depend on different constituents in the mate-
rial subjected to the CMP and the specific “analytical system”
used defined as, the range of circumstances that contribute to
the reliability of analytical measurements, including measure-
ment system, reagents, procedures, test materials, personnel,
environment and quality assurance trials (adapted from [8]).
The “selectivity” of a measuring system is its capability, using
a specified measurement procedure, to provide measurement
results, for one or more measurands (quantities intended to be
measured), that do not depend on each other nor on any other
quantity in the system undergoing measurement [2]. Actually,
the analytical signal depends on: (i) the properties of the ana-
lyte species involved; (ii) the instrumental parameters of the
particular equipment; (iii) the experimental conditions (mostly
adjusted by the analyst); and (iv) the presence of accompany-
ing substances [9]. In order to convert the previous expression
in other one more accessible from a practical point of view, to
improve the selectivity of the CMP, minimizing the lack of selec-
tivity of the measuring system, some different strategies can be
applied. One of them, is to use a separation technique previ-
ous the measurement step in order to: (i) remove or diminish
the matrix constituents (clean-up) to do the Φ-parameter inde-
pendent from the rest of the constituents of the material, and
(ii) to separate the sample in fractions (in a continuous or in
a batch way), to obtain only an analyte in each one to assure
that the indication provided by the measuring system is only
attributed to the very analyte. This meaning of selectivity term
is coherent with IUPAC definition [10]: the extent to which the
method can be used to determine particular analytes in mix-
tures or matrices without interferences from other components
of similar behaviour.
As a consequence of this fact, the general expression of the
measurement function above described, could give rise to dif-
36 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
ferent mathematical expressions. Danzer [11] analyses a general
functional relationship adapted to the special features of a CMP
in analytical chemistry, discussing the variables that affect the
value of the analytical signal and the consequences over some
important characteristics of analytical methods.
One of the easiest way to describe the measurement function
for an analyte (not taken in consideration the matrix) is:
Y = Y0 + Yanal = Y0 + S(x) · X
which indicates that the value of the analytical signal depends
on two components: one, independent of the quantity of ana-
lyte, denoted as blank signal (Y0 ) and the other, the net signal
generated by the analyte (Yanal ). This last is expressed as a
product of two terms, where S(x) is the sensitivity of the
measurement system (not necessarily a constant because its
value can be dependent on the actual x-value), and X, the
amount/concentration of analyte. This expression belongs to the
analytical calibration function obtained in the validation step of
the CMP, when the system is fitted to a linear response. Thus if
this equation is rearranged in the way:
 
Y0
Y= + S(x) · X
X
the term in brackets will be the measurement factor:
Y0
Φ= + S(x)
X signal and sensitivity, are estimated by the independent term
The blank signals may be described by three different terms,
depending upon their origin as Currie stated [1]: (i) the
instrumental background. The null signal (which for certain
instruments may be set to zero, on the average) obtained in the
absence of any analyte- or interference-derived signal; (ii) the
(spectrum or chromatogram) baseline. It comprises the summa-
tion of the instrumental background plus signals in the analyte
(peak) region of interest due to interfering species; and (iii) the
analyte blank. This signal arises from contamination from the
reagents, sampling procedure, or sample preparation steps which
correspond to the very analyte being sought. In our opinion, the
blank signals are produced by: (i) the instrumental background;
(ii) the signal in the analyte peak region due to other accompa-
nying species which can be exogenous, e.g. from the reagents
(the so-called reagent blank), or endogenous, e.g. from interfer-
ents or matrix sample (the so-called matrix blank); and (iii) the
signal due to the presence of the very analyte in the reagents.
The combination of instrumental background and reagent blank
constitutes the method blank.
The blank signal origins a (non-analytical) net signal (it
can be considered constant for each sample in each analyti-
cal system) which is independent of those signals attributed to
matrix–analyte interactions, closely related with the existence
of a matrix effect. Ideally, when a chromatographic method is
applied, the blank signal should be zero, since the analyte must
pass through the measurement system separated from the other
components of the sample and the instrumental background also
may be set to zero.
The assessment of the blank (and its variability) is mandatory
for accurate low-level measurements, but require expert chem-
ical knowledge concerning the analytical system in question,
bearing in mind that, in a lot of cases it is very difficult to obtain
an analytical blank for the overall CMP (i.e. a sample that is iden-
tical to those being taken for analysis but containing none/not
detectable levels of the analyte(s) sought). When the absence
of a matrix effect is verified, this blank can be substituted by a
reagent or procedural blank [12] carrying out a complete anal-
ysis using the solvents and reagents only, in the absence of any
sample.
“Sensitivity”, has been defined as the quotient of the change in
the indication and the corresponding change in the value of the
quantity being measured [2]. It denotes the quantity of analytical
signal yielded by each unit of analyte amount in the measured
material and it can be constant or dependent of the very analyte
amount. It would be desirable that sensitivity could achieve the
highest value possible in every CMP, in order to obtain the best
performance characteristics for the analytical method implied.
From a calibration experiment appropriately designed in
which the analytical signal is measured from the measurement
standards, the measurement function is explicitly stated as a
representative calibration function, which can be written in a
general way as:
y = f (x)
Usually it is obtained fitting the different (x, y) pairs, by applying
a proper regression algorithm. The calibration features, blank
and the gradient, respectively, and they are identified with the
intercept and the slope when the measurement function is plotted
as a linear calibration curve.
For any standard analyte amount, the measurement factor
could be estimated by mean of a “response factor” (RF) from
the following equation:
y
RF =
x
To carry out the quantification of an analyte in a sample, the
corresponding output analytical magnitude (y-variable) is mea-
sured and a numerical value of the input analytical magnitude
(x-variable) is calculated from the inverse calibration function,
denoted as “evaluation function” [1,7] or “analytical function”,
according to the following expression:
xspl = f −1 (yspl )
where f−1 is the inverse calibration function and the subscript
“spl” indicates the sample. In a similar way, each analyte amount
in the sample could be characterized by means of a “calibration
factor” (CF), defined as:
xspl 1
CF = =
yspl RF
Thus, this feature, could be used to estimate the actual analyte
amount in a sample according to:
xspl = CF · yspl
Table 1 shows the most frequent possibilities of calibration
curves as well as the RFs and CFs associated to them, being the
 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
Table 1
More frequent calibration curves and different features for the analytical separation sciences
Calibration curve Calibration function
Linear y = bx
y = a + bx
Parabolic y = cx2
y = a + cx2
y = a + bx + cx2
linear and parabolic calibration functions the most frequently
used in analytical separation sciences.
As it can be seen, in the linear functions sensitivity is constant
and therefore independent of the amount of analyte. Never-
theless, the response factor is only constant when the blank
signal is null. In these circumstances, sensitivity and response
factor are equivalent and the calibration factor is the inverse
of each one of them. Thus, the verification of these two con-
ditions (linear response and null blank signal) is crucial in
order to avoid errors associated with the selection of certain
quantification strategies based in the use of a only measure-
ment standard for calibration purpose, as will be pointed out in
Section 4.
3. Schemes for analytical calibration/quantification
The multi-standard calibration [13] is the usual scheme for
the analytical calibration and the performance characteristic
establishment of a CMP. It is based on the measurement of a
calibration standards set, including the blank if this is manifest,
evenly spaced over the analytical method working range and
preparing replicates of each one in an independent way. Then,
applying an adequate regression algorithm (not necessarily the
Gauss least-square fitting method) to the data, a calibration
function is obtained. These days, new regression methods e.g.
robust regression, are been taken into consideration. A regres-
sion involves two steps: (i) selecting a mathematical model
(measurement function) and fitting the corresponding mathe-
matical function from the experimental data; and (ii) validation
of the model and checking of certain initial hypotheses. The
consistency of the regression results always depends on the
grade of the experimental random errors. Therefore, if these
ones are important, the conclusions of the applied statistical
tests are not reliable; for calibration purpose, this fact has spe-
cial relevance in relation to linearity or goodness-of-fit tests. It
must be taken into account that regression is only a statistical
tool useful to obtain/estimate calibration functions, being other
strategies also possible. To deal with the statistical approach
of the calibration, out of the aims of this paper, there are
available numerous guidelines [7,14–16] and technical papers
[17–21].
In order to assure the accuracy of the results in samples with
low analyte content, i.e. to avoid a leverage effect on the cali-
bration curve, it is advisable to use a homogeneous arrangement
of the calibration standards. Thus, for example, if in a 5–100
analyte amount arbitrary units, the levels chosen are 5, 25 and
100, the highest standard will have a considerable leverage effect
37
Signal blank Sensitivity Response factor
0 b [constant] b [constant]
a b [constant] a/x + b [depends on x]
0 2cx [depends on x] cx [depends on x]
a 2cx [depends on x] a/x + cx [depends on x]
a b + 2cx [depends on x] a/x + b + cx [depends on x]
on the calibration curve, being the estimation of the calibration
function strongly affected.
For multi-analyte analytical methods, a very common situ-
ation when continuous separation techniques are applied, the
calibration standards are a set of mixtures containing the differ-
ent analyte standards to be quantified in the adequate amounts.
The multi-standard is feasible when the number of analytes
allows it and analyte measurement standards are available. The
measurement analyte standards used as “calibrants” (a measure-
ment standard specifically used in calibrating, also so-called
“calibrators” [2]) in analytical sciences, are reference materials,
that can be essentially classified in two types [22]:
(1) Substance reference materials (substance RM), prepared
from single analytes. They include: (a) pure substances char-
acterized for chemical purity and/or trace purities, and (b)
standard solutions, prepared from pure substances (singles
or mixed) in the working solvents used.
(2) Matrix reference materials (matrix RM) characterized for
the composition of specified major, minor and trace chemi-
cal constituents. They may be prepared from the matrices to
be analysed or by mean of synthetic mixtures. Thus, this RM
contains the analytes of interest plus the principal chemical
compounds characterizing the matrix to be matched.
In relation to the number of calibration standards which must
be used, and the number of replicates at each calibration level,
different recommendations in recognized written standards and
guidelines can be found. So, IUPAC advises for method val-
idation purposes, the use of six or more calibration standards
that should be run, preferably triplicate on more, in a random-
ized way [23]. In the same way, the ISO 8466 states, for an
initial assessment of the calibration performance, to employ at
least five calibration standards, although it recommends 10, and
10 replicates of the lowest and highest standards [14]. Another
schemes with a non-balanced uniform-level design for calibra-
tion purposes have been recommended [24].
These severe requirements are tempered when the analyt-
ical method is running in a routine way, being enough with
three or four calibration standards, duplicated if possible; this
short-working calibration for routine analysis is termed as “con-
tinuing calibration” [13]. If the calibration curve has been well
established during method validation and a suitable calibration
control scheme is put into practice, it may be even possible to
use two alternative schemes: (i) two-standard calibration and (ii)
one-standard calibration.
38 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
3.1. Two-standard calibration
When the linearity of the calibration function has been
ensured in the validation step, a routine calibration can be
obtained from only two calibration standards, preferably mea-
sured in replicate. The standards must be selected in such way
that the sample analyte/s quantity be included in the covered
range by them. That is why the quantification can be done inter-
polating between both standards values, e.g. by applying the
next equation:
x2 (ȳspl − y1 ) − x1 (ȳspl − y2 )
xspl =
y2 − y 1
where x1 and x2 are each one of the two standards analyte
amounts, y1 and y2 the arithmetic means of the corresponding
analytical signals, and ȳspl is the arithmetic mean of the analyte
analytical signal in the sample. Also, quantification can be done
using the expression of the straight line through two points (both
calibration standards) as follows:
ȳspl − ȳ1,2 ȳ2 − ȳ1
xspl = + x̄1,2 , where b =
b x2 − x 1
being ȳ1,2 and x̄1,2 the global means of the two measured
analytical signals and analyte amounts of the two standards,
respectively, and b is the slope.
When it is known that the intercept is zero, the calibration
factors of both standards are the same and very similar to the
inverse of the slope. Then, the quantification could be easily car-
ried out calculating a pooled calibration factor, CF1,2 , according
to the expressions:
x̄1,2 1
xspl = CFpool · ȳspl , where CFpool = CF = = by evidence of acceptable linearity of the calibration function,
ȳ1,2 b the sample analytical signal should be within ±10% of the cali-
The two-standard calibration can be advisable, when the
expected range of analyte amount in the samples to be analysed
is narrow, but it could be a very laborious calibration scheme
when the samples is unknown. For pesticide residue analysis in
particular, the use of two-standard calibration has been stated
to be acceptable providing the two calibrant x-value differs in a
factor not greater than 4, and where the mean response factors,
derived from replicate determinations at each standard, does not
vary in more than a ±20%. This is indicative of an acceptable
linearity of the observable analytical signals [12].
A particular type of two-standard calibration is the one
labelled “bracketing calibration” [15,16]. The basis of this strat-
egy is to decrease, as much as possible, the interval for which the
calibration function is linear, delimiting it with two calibrants
which “bracket” the analyte amount values of the test samples
closely; both calibrants and samples, should be measured at least
twice. Assuming this fact, bracketing calibration can be applied
when there is some doubt about the linearity of the calibration
function in the range where the samples are measured and it is
specifically useful when the quantification of analyte amount in
samples is required with a high accuracy degree (in terms of
trueness and precision), e.g., in doping control analysis.
It can be also convenient when the measurement system
shows a significant drift in its detector response, being necessary
a low test samples number with the aim to minimize the total
analysis time. Whether the number of test samples is great, a
batch processing (of them) can be done.
3.2. One-standard calibration
The calibration could be performed from only one calibrant
when two conditions are fulfilled: (i) the calibration function
must be linear in the interval of analyte amount ranged from
the standard value to zero, and (ii) the blank signal must be
null in the interval previously referred to. So, it is mandatory
to check this last requirement before this calibration scheme
was applied. During the experiment, the calibrant must be repli-
cated twice at least. The quantification is carried out using the
calibration factor calculated from the pair of values, standard
analyte amount/average analytical signal, concerning the cali-
brant applying the expressions:
x1
xspl = CF · ȳspl , where CF =
ȳ1
The linearity requirement can be avoided when the analyte
amount in the selected calibration standard is very near to the
one in the sample which is possible when the features of the
sample are previously known. Then the one-standard calibra-
tion can be easy going applied (for example, for quality control
of manufactured products).
As for the preceding case, for pesticide residue analysis, it has
been stipulated that one-standard calibration may provide more
accurate results than multi-standard calibration if the detector
response is variable with time [12]. In this case, it must be tak-
ing into account that, unless further extrapolation is supported
bration standard analytical signal if the maximum residue limit
is exceeded; if it is not exceeded, the sample response should be
within ±50% of the calibration response.
4. Methodologies for analytical
calibration/quantification
In order to the calibration function, previously established
from standards, could be applied to the samples (Fig. 1), differ-
ent calibration methodologies [13] can be used, bearing in mind
factors like: (i) the availability of representative standards; (ii)
the analytes nature; (iii) the lack of precision or drift of the mea-
surement system; and (iv) the possibility of disturbance caused
in the output analytical signal by other components concomi-
tants the analyte, considered them individually (interferent) or
in a whole way (matrix effect). Obviously any of the previously
mentioned schemes: multi-, two- or one-standard calibrations
can be applied in all the methodologies.
The calibration standards are prepared from RMs containing
the analyte or a “surrogate”, that is, a pure substance (com-
pound or element) similar to analyte of interest in chemical
composition, separation and measuring which is taken to be
representative of the native analyte; this must be absent or in
a negligible initial concentration in the sample. The calibration
 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
standards can be measured in a preparation apart from the sample
(external methodology) or in one including the sample (internal
methodology). Usually, a surrogate is used in an internal method-
ology and, in this case, it is termed as “internal standard” (IS).
The IS is chemically distinct from the analytes and therefore
will not have identical chemical properties. However, it is usu-
ally selected in such way it was closely related with the analytes
and can represent their analytical behaviour to the highest degree
practicable. The most favourable way to apply IS, is to use an
isotopically labelled analyte, so the chemical properties of the
internal standard are virtually identical to the very analyte. Both,
the very analyte and the labelled one, can be measured separately
by mass spectrometry (this principle is the basis of the isotope
dilution mass spectrometry (IDMS) technique).
In calibration, the IS can be applied with two aims: (i) to
estimate a calibration feature as response factor or calibration
function (it will be the subject of a detailed comment and it will
be pointed out further on Section 4.4); and (ii) to compensate
uncontrolled analytical signal variations in the measuring sys-
tem. This strategy, in our opinion is not properly a methodology
for calibration, and should be called “signal-ratio calibration”
although the general expression to refer to it is “internal standard
method”.
The main characteristic of the signal-ratio calibration is based
on the use of a relative signal (signal-ratio), defined as the quo-
tient between the signal attributed to the analyte (analyte signal)
and to the internal standard:
ya
yR =
yIS
where the subscripts “a” and “IS” are related to the analyte and
the internal standard.
It has been widely used to correct mechanical losses of
analytes in procedures such as manual injection in gas chro-
matography. Nowadays, the use of internal standards has been
Table 2
Types and main distinctive features of different calibration methodologies
39
extended to capillary electrophoresis, in order to maintain
the injection required precision during long routine injection
sequences and/or analysis of samples in complex matrices. In
fact, many imperceptible factors affect the volume introduced
into the capillary during a pressure injection in CE. These fac-
tors, mainly related to variability in the pressure/timing of the
injection and to changes in sample solution viscosity, can be
efficiently eliminated by means of the signal-ratio calibration
[25].
The signal-ratio calibration can also be used to correct a mod-
erate proportional (no-additive) matrix effect (see Section 4.2),
since the premise is that, a matrix effect changes the signals from
analyte and the internal standard in the same degree, so that cal-
ibration on the ratio signals against analyte amount, will remain
invariant to changes in matrix (different composition between
the calibration standards and the sample analysed).
Ideally, the standard should be added exactly to the test
dissolution before the injection, in enough amount so that its ana-
lytical signal be equal or higher than the highest analyte signal
expected; thus, the signal-ratios will always be lower than 1, min-
imizing the imprecision of these signals and ensuring normally
distributed measurement errors [26].
The distinctive features of the different methodologies,
explained in next subsections, are summarized in Table 2, in
which the minimum number of preparations for a metrological
calibration/quantification are included.
4.1. External calibration
The external calibration (EC) is the most commonly
employed calibration methodology and it is named so, because
the calibration standards do not make up of the sample test
portion but they are prepared and analysed separately from the
samples. In this sense, standards and samples form part of differ-
ent analytical preparations which are measured in a sequential
40 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46

way. In bibliography, EC has been also termed as “solvent cal- ya,spl

xa,spl = CF · ya,spl = xa,std ·
ibration”, “standard calibration” or even “normal calibration”. ya,std
As external standard (ES), a solution of a RM substance in the
When the signal-ratio calibration is applied, it can be established
working solvent is used. It usually contains the analyte, although
a relative response factor (RRF) or a relative calibration factor
it is not essential, and the calibration could be carried out from a
(RCF) which are calculated from the ratio signals. The equations
surrogate. The use of a surrogate in external calibration is only
used are:
appropriate when there are no a RM containing the analyte. An
application, in which two surrogates are used as ES, can be found RF ya,std /xa,std ya,std /yIS,std xIS,std R
RRF = = = = · ystd ;
in a recent paper on the determination of polyphenols in olive RFIS yIS,std /xIS,std xa,std /xIS,std xa,std
oil by CE-UV [27]. 1
When a multi-standard calibration is performed, the linear RCF =
RRF
measurement function to estimate from data regression is:
Y = Y0 + S · X ya,spl
xa,spl = xIS,spl · RCF · = xIS,spl · RCF · yspl
R
which give rise to a calibration curve: yIS,spl

ya,std = aEC + bEC · xa,std It is habitual, but not essential, to add the same amount of inter-
nal standard to all the analytical preparations (calibrants and
where the subscript “std” are related to calibration standard, the samples). Then, due to xIS,std = xIS,spl , both terms can be elim-
intercept aEC is the method blank (Y0 ) and the slope bEC is the inated in the previous equation and a pseudo-relative response
sensitivity of the measurement system (S). The quantification factor (RRF ) or pseudo-relative calibration factor (RCF ) can
can be then performed from: be calculated according to:
ya,spl − aEC
xa,spl = ya,std /yIS,std yR 1
bEC RRF = = std ; RCF =
xa,std xa,std RRF
where the subscript “spl” are now related to sample. In cer-
tain cases, a no-linear calibration function, as polynomial, yspl
R
ya,spl
exponential, sigmoidal, etc., could be applied.When a signal- xa,spl = RCF · = RCF · yspl
R
= xa,std · R
ratio external calibration is performed, the calibration functions yIS,spl ystd
should be established from signal-ratios; e.g. for a linear func- EC is suitable for the analytical methods that could be considered
tion, the equation to be fitted is: as “matrix-effect free methods” (the so-called Type II methods
ya,std   in ISO Guide 32 [30]). But it have a main limitation that derives
= ystd
R
= aEC + bEC · xa,std
yIS,std from the assumption that, the differences between the matrices
of the standards (usually a working solvent) and the sample have
and the quantification equation is:
no effect on the calibration function, bearing in mind that if these

(ya,spl /yIS,spl ) − aEC differences are ignored, additive and/or proportional systematic
xa,spl = 
bEC matrix errors may be introduced. In general, the EC methodol-
ogy is recommended if the analyst has a good knowledge of the
To do this, it is mandatory that the sample test portion and all
experimental conditions and the contribution of interference to
the calibration standards were prepared with the same internal
the measurement is irrelevant or can be kept constant; neverthe-
standard amount.
less, in the last case, a proper correction of the matrix should be
A representative example of this procedure would be
applied [31], for example, by performing a “correction function”
the1613 EPA method for the determination of tetra- through
[32].
octa-chlorinated dioxins and furans by isotope dilution
high-resolution capillary column gas chromatography/high-
4.2. Matrix-matched calibration
resolution mass spectrometry [28]. In it, a five-standard external
calibration is used in order to estimate the linear signal-ratio
The matrix of calibrants and sample test portion never are
calibration function used for quantification purposes. A 13 C
exactly equal, although the sample has been previously subjected
isotopically labelled dioxine is used as internal standard.
to a clean-up procedure. Sometimes, this fact is irrelevant and
In routine analytical determinations, of the use of response
any errors in the determination are produced, but in other cases,
and/or calibration factors obtained from one-standard calibra-
a systematic error appears due to the matrix (matrix systematic
tion is also quite applied, being it advised too in some official
error) not being possible then to apply external calibration.
analytical method, for example, the Harmonised Methods of the
In separation techniques, this matrix effect is due to the influ-
International Honey Commission for determination of sugars in
ence on the measurement of the analyte amount, of one or
honey by HPLC or GC [29]. In this case, the equations to apply
more undetected sample components and derive from various
are:
physical and chemical processes. It tends to be variable and
xa,std 1
RF = ; CF = unpredictable in occurrence, being in some cases, difficult or
ya,std RF impossible to eliminate. The presence or absence of such effect,
 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46 41

may be demonstrated by comparing the response produced from

the analyte in a working solvent solution with that obtained from
the same amount of analyte in the presence of the sample or sam-
ple extract. Usually, the matrix produces a change in the signal
which can be: (i) constant and independent of the analyte amount
(additive or translational effect); (ii) variable and proportional
to that amount (proportional or rotational effect); or (iii) the
combination of both type of effects [31,33].
Whether matrix problems are suspected (Type III methods in
ISO Guide 32 [30]), more reliable calibration may be obtained
using as calibration standard, a matrix RM (when available)
or a pure substance RM in conjunction with free-analyte sam-
ple matrix prepared freshly (most usual). This is the so-called
“matrix-matched calibration” (MC) and may make up for matrix
effects although it does not eliminate the underlying cause. That
is why, the intensity of effect may differ from one matrix or
sample to another, and can be also affected by the “concen-
tration of matrix”. In fact, a MC is a particular type of EC in
which the calibration standards are prepared in a simulated sam-
ple that initially it does not contain analyte. Nevertheless, the
MC will not overcome chromatographic interferences caused by
overlapping/unresolved peaks from co-extracted compounds. In
addition, when matrix effect is sample dependent it is necessary
to use standard addition calibration (see next section).
Metrologically, the matrix effect origins a modification in Fig. 2. A particular example of the relationship between different types of cali-
the measurement function, due to the appearance of new terms. bration curve plots: external calibration (EC), matrix-matched calibration (MC),
Supposing a linear behaviour, it can be represented as: standard addition calibration (AC) and empirical matrix-matched calibration or
matrix-corrected calibration (MCC ). y0 , method blank signal; ym , matrix blank
Y = Y0 + Ym + Yanal = Y0 + Ym + p · S · X signal; ya,spl , signal from actual analyte in sample; xa,spl , actual analyte amount
in sample; aEC , intercept from EC-curve; aMC , intercept from MC-curve; aAC ,
where Ym indicates the matrix sample contribution to the blank, intercept from AC-curve; aYC , intercept from Youden calibration curve (or YC-
i.e. the matrix blank (this term characterizes the additive matrix curve); bEC , slope from EC-curve; bMC , slope from MC-curve; bAC , slope from
effect and can have positive or negative values) and p character- AC-curve.
izes the proportional one (It can reach values upper and down 1)
being it a feature defined in a similar way to the so-called “selec- If is necessary, the MC-function also could be performed from
tivity index” in IUPAC nomenclature [23]. It can be expressed signal-ratio calibration by using a suitable internal standard,
as: being the equations to apply:
Sm ya,std  
p= ; and so : sm = p · S = ystd
R
= aMC + bMC · xa,std
S yIS,std

where Sm is the analyte measurement system sensitivity when 

the matrix sample is present.
The presence of a significant matrix effect brings about a MC-
function different to that corresponding to EC-function which
for a linear function, can be stated as:
ya,std = aMC + bMC · xa,std
where the intercept and the slope can be expressed as:
aMC = aEC + ym = y0 + ym , and bMC = pS = pbEC . It can be
deduced that, when ym = 0 and p = 1, there is not a matrix effect
and both calibration functions are equivalents. A particular case
in which ym > 0 and p > 1 is shown in Fig. 2, as an example.
In the laboratory, the sample must be prepared in such way
the sample amount in the test portion was similar to the matrix
amount used to prepare the calibration standards.
ya,spl − aMC
xa,spl =
bMC
(ya,spl /yIS,spl ) − aMC
xa,spl = 
bMC
This methodology is particularly recommended in pro-
cedures for pesticide or drug residue analysis and other
contaminants in food and biological matrices [34,35]. Contrary
to the common belief, the reliability of quantification using
modern analytical techniques as GC–MS or even LC–MS (or
tandem MS/MS) might not be adequate. Results can be adversely
affected by the lack of selectivity caused by matrix sample effect
on ion suppression.
Owing to the variety of matrices and samples in pesticide
residue analysis, the MC-functions are prepared only in a series
of representative matrices, this is, a sample material or an extract
of a single food or feed used to represent a commodity group as
an indicator of matrix effect in the analysis of broadly similar
commodities. Matrix representativeness is usually determined
among similar biological material (or tissues) according to its
42 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
content in constituents like, water, acids, sugars, lipids, sec-
ondary plant metabolites, etc. The utility of these representative
matrices, has been demonstrated preparing MC calibrations in
cucumber extracts for the determination of 20–30 pesticides by
GC–MS [36] or LC–MS [37] in several vegetable samples.
4.3. Standard addition calibration
The establishment of a matrix-matched calibration needs a
matrix free from analyte which it is only possible when the
analytes are exogenous sample components (e.g. additives or
contaminants or additives). Nevertheless, if the analytes are
endogenous (e.g. aminoacids in biological fluids or pigments
in vegetables), and a matrix effect has been verified, it is neces-
sary to apply the method of standard addition, properly named
“standard addition calibration” (AC). There are different ways in
which the AC can be applied which have been recently reviewed
[38].
It is a particular internal calibration methodology, usu-
ally based on the analysis of several test portions made,
adding increased amounts of analyte standard to several sam-
ple aliquots. From these data, an AC-function can be obtained,
which will have the same slope as the MC-function but higher
intercept, assuming that it can be considered as a MC in which
the matrix contains analyte. Fig. 2a shows a linear AC-curve
together with the corresponding linear MC- and EC-curves.
In this case, the measurement linear function is expressed by
the equation:
Y = Y0 + Ym + Sm · (Xnative + Xadded )
= Y0 + Ym + Sm · Xnative + Sm · Xadded
where Xnative indicates the analyte amount in each test portion
which is related with the analyte originally present in the sam-
ple and Xadded represents the analyte amount added to each test
portion. The AC-curve fitted (measured analytical signal versus
added analyte amount) is given by:
ya,(spl+added) = aAC + bAC · xa,added
whose intercept (see Fig. 2a and b) is:
aAC = y0 + ym + Sm · xa,spl
where Sm is the AC-slope (bAC ), y0 is the method blank, ym is
the matrix blank, ya,spl and ya,(spl + add) are the analyte signals in
the original and added samples, respectively, and xa,add is the
added analyte amount.
From this expression is possible to quantify the analyte
amount initially present in the sample (xa,spl ):
aAC − (y0 + ym ) aAC − (y0 + ym )
xa,spl = = the results are not affected by systematic matrix errors, inde-
Sm bAC
and as it can be seen, it is necessary to correct the intercept
value (aAC ) with the total sample blank (method blank + matrix
blank).
On the other hand, in order to obtain a bias free result by
means AC, it is necessary to carry out a “Youden calibration”
(YC) to estimate the total blank of the sample. It cannot be
considered a “true” calibration, since no measurement standards
are used, but just increasing sample amounts. The YC-curve is
obtained from a plot of the measured signal against the sample
(not analyte) amount (xspl ):
ya,spl = aYC + bYC · xspl
and the corresponding intercept (aYC ) is the total sample blank
(so-called total Youden blank) [33,39]. Consequently the YC-
intercept, in agreement with the MC-intercept (see Fig. 2a and
b), is a measure of the total sample blank:
aYC = aMC = aEC + ym = y0 + ym
and the quantification equation is:
aAC − aYC
xa,spl =
Sm
When the total sample blank is null, the former equation can be
simplified to:
aAC
xa,spl =
bAC
which gives the extrapolated value of the x-intercept. Only in
this case, the analyte amount can be calculated as the quotient
between the intercept and the slope of the AC-curve. This fact
is the basis of the graphic quantification method that generally
appears in text books. Nevertheless, if the total sample blank
nullity is not fulfilled, this last approach can produce serious
errors in the quantification.
When a “one-standard addition calibration” is made, the
quantification step requires the analysis of at least two test por-
tions, one of them containing an amount of analyte standard
added. Then the sample analyte amount is obtained applying
the next equation which is a particular case of the general one
above explained:
ya,(spl+add) − ya,spl 1
RF = ; CF =
xa,add RF
ya,spl
xa,spl = CF · ya,spl = xa,add ·
ya,(spl+add) − ya,spl
being ya,spl and ya,(spl + add) the analyte signals in the original and
the added samples, respectively; and xa,add is the added standard
analyte amount.
As in the previous methodologies, the AC-calibration could
be performed from signals-ratio. The equations for calibration
and quantification are equivalent to the previous ones without
more than to substitute the analytical signals for the respective
analytical signals-ratio.
This type of calibration is the only methodology in which
pendently of the kind and magnitude of the matrix effect, so it
permits to quantify the amount of analytes in any kind of sam-
ples. That is why a “in-house” validation of analytical methods
based on the standard additions and Youden calibrations has
been proposed [40]. Its principal disadvantage lies in the fact that
a calibration for each sample is needed, which implies a lot of
work in routine analysis. To avoid this problem, a simulation of a
 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
MC-curve by means of an empirical calibration curve obtained
from the YC- and AC-curves features, has been recently pro-
posed. This new calibration function, named “matrix-corrected
calibration” (MCC ) [41], is a hypothetical matrix-matched cal-
ibration characterized by a linear curve where the x-variable is
the analyte amount (in the presence of matrix) and the y-variable
is the analytical signal (Fig. 2b).
To establish the MCC , is necessary to set up only two cali-
brations: AC and YC, which must be representative. From the
intercept (aAC ) and the slope (bAC ) of the AC and the intercept
(aYC ) of the YC, both MCC -intercept and MCC -slope can be
calculated by the equations:
aMCC = aAC − ya,spl = aYC ; bMCC = bAC
which are exclusive of each type of matrix, since each matrix
has its own AC-curve because the AC-slope, which corrects the
proportional error introduced by the matrix, may change when
different matrix amounts are used and consequently bMCC =
bAC and they are only valid provided that the amount of sample
used for analysis remains constant.
Thus, when a matrix effect exists, the analyte amount from
each measured analytical signal in any sample, can be calculated
from:
ya,spl − aMCC ya,spl − aYC
xa,spl = =
bMCC bAC
4.4. Internal calibration
There are some analytical situations in which an internal stan-
dard, employed as calibrant, is added to the sample and analysed
as a whole together with the analyte. From the same analytical
preparation, a response/calibration factor is obtained from the
IS which is applied on the analyte signal for quantification using
the following equations:
yIS,spl 1 tinguish between the signals from the internal standard(s) and
RFIS = ; CFIS =
xIS,spl RFIS
ya,spl
xa,spl = CFIS · ya,spl = xIS,spl · = xIS,spl · yspl
R
yIS,spl
Metrologically, this is the real internal calibration (IC), a one-
standard internal calibration methodology formally different to
the signal-ratio calibration; however this last is the most known
application of the use of internal standard in analytical sciences
which sometimes has derived in an inappropriate use of the term
internal calibration.
The preceding equations could be combined and the quan-
tification would be obtained from the following simplified one:
xIS,spl
xa,spl = · ya,spl = xIS,spl · yspl
R
yIS,spl
With the aim to enhance the accuracy of the results, a modifi-
cation of the methodology that uses two internal standards, has
been proposed and it has been called “double standard internal
43
method”. Initially, the quantification involved the average geo-
metrical value calculation of the two results obtained with each
standard:

xa,spl = ya,spl CFIS1 · CFIS2
This equation can be significantly simplified when: (i) two
closely related compounds, upper and lower, of the same
homologous series to which the analyte belongs, are used
as internal standards (e.g. methanol and n-propanol could be
used for ethanol determination); (ii) the analyte and inter-
nal standards calibration factors are approximately equals
(CFIS,1 ≤ CFa ≤ CFIS,2 ), being the product of the relative cali-
bration factors analyte/internal standards around 1 (CF1 CF2 = 1)
due to its differences are compensated each other [42]; and
(iii) both internal standards are added in the same amount,
xIS,1 = xIS,2 = xIS . Then, the analyte amount in the sample can
be calculated according to:

1 
xa,spl =ya,spl · xIS,spl =xIS,spl y1,spl
R · y2,spl
R
yIS1,spl · yIS2,spl
where ya,1R and yR represent the analyte/internal standard
a,2
signal-ratios 1 and 2, respectively, obtained from the sample
analysis.
The main advantage argued by the authors, arises from that
this quantification method does not need a previous calibration
(the calibration is implicit in the quantification), so its prac-
tical application is very easy. It only requires the addition of
a known and equal amount of both internal standards to each
sample. Once the sample treatment has been finished, the ana-
lyte and internal standards signals are measured. The results
obtained show a great accuracy degree, even whether during
sample treatment any analyte losing is produced. Its more impor-
tant disadvantage lies in the necessity of having two analyte
homologous (internal standards), initially absents in the sample.
To apply an IC the measurement system has to be able to dis-
the very analyte, that is why, both substances must have a differ-
ent behaviour in the measurement system but similar analytical
properties in order to be measured in a quasi-simultaneous way.
In the same portion of the test sample, the signal of the standard
and the very analyte are measured; from the first one a calibra-
tion factor is obtained, which is used to quantify the amount of
analyte from the second signal.
An exclusive feature of IC is that it is possible to carry out the
quantification of several analytes (generally from the same fam-
ily) in the same sample portion, starting from an only internal
calibration, this is, the internal standard can represent to several
analytes in a simultaneous way in the same sample. As conse-
quence, it would be possible to calculate the (percentage) mass
fraction of each analyte using only the values of the measured
analytical signals by:
xa,spl FCIS · ya,spl ya,spl
ca,spl (%) =  · 100 =  · 100 =  · 100
xi (FCIS · yi ) yi
where ca,spl indicates the percentage mass fraction of the analyte
“a”; xa,spl is the mass of this analyte in the sample and ya,spl is
44 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46

the analytical signal due to it. xi,spl represents the sum of the
masses of all the analytes and, yi,spl represents the sum of the
analytical signals attributed to these ones. This strategy is only
possible when the calibration factors are similar for all the ana-
lytes, and equal to the calibration factor of the internal standard.
The reliability of internal calibration as calibration/
quantification methodology is based on the ability of the IS to
act as surrogate. In metrological terms, this fact implies that the
response factor (or sensitivity) of the IS is constant and equal
to the calibration factors of each one of the substituted analytes,
into the interval of application of the method. That can be true
for those universal no-specific detectors which respond directly
to the substance amount (mass or concentration), as it happens
with the most classical GC detectors. However, this condition
is harder to fulfil in LC, due to the majority of common detec-
tors used, produce a dissimilar signal amount depending on the
judged analyte. Even, when MS detectors are used, and depend-
ing on the ionization system, it could be consider this premise
is satisfied when they work in “full-scan” mode (not in “SIM”
mode). In any case, and as far as possible, it is necessary to verify
that this requirement is achieved using a measurement standard
containing both analyte and internal standard.
The main advantage of internal calibration is the reduction
of analysis time, since it only needs one analytical preparation
for calibration and quantification. Moreover, this methodology
permits to make up for the losing of analyte during sample prepa-
ration and, in a moderate way, the matrix effect. That is why it is
advisable when at least one of the following circumstances are
produced: (i) the previous preparation sample process is long
and complicated; (ii) a long time for the measure is required
(e.g. chromatogram run time); and (iii) there is no or it is impos-
sible to acquire a material with analyte standard. Obviously, the
main limitation of this methodology is the availability of an ade-
quate IS. As an additional precautionary measure, the amount of
IS must be such its analytical signal (peak area or peak height)
be of the same order that those corresponding to the analyte;
otherwise, significant errors in quantification can be found, over
all if the internal standard signal is not entirely linear.
UE olive oil official analytical methods for the determina-
tion of the composition and content of waxes [43], sterols [44],
stigmastadienes [45], and aliphatic alcohols [46], by capillary-
column gas chromatography, constitute examples of common
application of IC. In those methods, an only calibration IS is
used for the quantification of some analytes of the same chemical
family.
An imaginative proposal recently reported is the “echo-peak
internal standard calibration” [47]. This is a novel calibra-
tion methodology which simulates the use of internal standard
by injecting consecutively, within a short period of time, an
unknown sample and a standard analyte solution. As a result,
the standard analyte peak elutes closely to the sample analyte
peak, thus performing a “echo peak”. For quantification pur-
poses, a calibration function is obtained from the signal-ratios
of the standard and the reference standard. The concentration of
analyte in the unknown sample is calculated from the peak area
ratio of sample and reference. As an internal standard method,
the echo-peak technique provide the possibility to compensate
the signal decreasing during the analytical sequence when the
detector response shows a significant instability with the time.
4.5. Calibration by internal normalization
When the measuring system sensitivity changes from one
analyte to another, the application of the internal standard cali-
bration requires to normalize the measured analytical signals by
determining a “normalization factor” (NF) for each analyte in
relation to a reference compound (usually, an internal standard).
This calibration methodology is called “calibration by internal
normalization” or “normalized signal calibration”.
Despite its denomination, it is properly a methodology of
double calibration external–internal, applied in two steps: (i) in
the first, a multiple external standard measurement to determine
the calibration factors of all the components (analytes and inter-
nal standard) and, the normalization factors of the analytes in
relation with the internal standard, are used; and (ii) in the sec-
ond, the calibration factor of the internal standard, previously
added to the sample, is determined and the analytes are quanti-
fied. Thus, the advantages of both methodologies are joined and
the drawbacks of each one of them when are applied separately,
are minimized.
The NF values of the analytes depend on the detector
employed, so it is necessary to estimate them for each measur-
ing system. For this purpose, in the first step, a multiple standard
calibration, which includes a known amount of each analyte and
internal standard, must be prepared and measured previously to
the sample. Then, the different normalization factors are cal-
culated as the quotient between the calibration factors of the
considered analyte (CFa ) and the internal standard (CFIS ):
CF xa,std /ya,std xa,std yIS,std xa,std 1
NF = = = · = · R
CFIS xIS,std /yIS,std xIS,std ya,std xIS,std ystd
Consequently, the NFa,std is the relative calibration factor of each
analyte in relation to a reference compound (internal standard).
It can be underlined that this NF equation is similar to the one
used to calculate the relative calibration factors (RCF) in Section
4.1.
In the second step, the calculated NF values are used to esti-
mate a normalized signal for each analyte in the sample (ya,spl N )
from the measured signals (ya,spl ):
ya,spl
N
= NF · ya,spl
These normalized signals indicate the hypothetical signals that
would be measured if the calibration factors were similar for
all of them, and equal to the calibration factor of the internal
standard. Finally, the normalized signals can be used for quan-
tification of the analytes in the sample, by applying the next
expression:
xa,spl = CFIS · ya,spl
N
= CFIS · NF · ya,spl
where it can be seen that, the calibration factor of each analyte
(CF), can be unfolded into two factors:
CF = NF · CFIS
 L. Cuadros-Rodrı́guez et al. / J. Chromatogr. A 1158 (2007) 33–46
To assure the validity of this methodology, it is necessary that
the calibration factor of internal standard keep constant.
As it has been stated in the previous section, is possible to
obtain directly the (percentage) mass fractions of each analyte
in a sample containing several analyte from the same chemical
family. Whether only the analyte mass fraction in the sample is
required, any other of the analytes in the sample can be used
as reference compound to normalize the signals, not being nec-
essary to add an internal standard neither in the measurement
standard nor in the sample.
In this case, the NF values are calculated directly from the
mass fraction (in percentage or non) of each analyte “i” in the
calibration standard (ca,std ) as:
 bration, in order to avoid misconceptions and errors for lack of
xi,std yref,std xi,std / xi,std yref,std
NF = · =  ·
xref,std yi,std xref,std / xi,std yi,std
ci,std yref,std
= ·
cref,std yi,std
where the subscript “ref” is referred to the analyte reference,

and the term xi,std is the sum of the amounts of all analyte,
including the analyte reference. Thus, the composition of one
analyte “a” in the sample (ca,spl ), expressed as mass fraction (in
percentage), is calculated as:
xa,spl CFref · ya,spl
N
ca,spl (%) =  · 100 =  · 100
xi,spl (CFref · yi,spl
N )
ya,spl
N
NFa · ya,spl
=  N · 100 =  · 100
yi,spl (NFi · yi,spl )
The calibration by internal normalization is a methodology
which has been scarcely used in analytical quantification. How-
ever, it exits a significant application example in the UE official
analytical method for the determination of methyl esters of fatty
acids by gas chromatography in olive oil [48], a representative
example in which both ones, an internal standard and an analyte,
are used as reference compound.
5. Conclusion
An overview on the problem of the analytical calibra-
tion/qualification for the separation sciences has been shown up.
The paper intends to establish a description of the metrological
fundamentals with the aim of reconciling the vocabulary and the
subjects of the measurement science with those corresponding
to the chemical analysis, when separation techniques are previ-
ously applied. We believe necessary that the chromatographers
familiarize with this terminology that nowadays is considered
as a fundamental topic of the analytical chemistry.
In addition, on the basis of the conventional multi-standard
calibration, the principles, advantages and limitations of alter-
native calibration schemes as two- and one-standard calibration
have been presented. The different calibration methodologies,
that could be applied, have been explained and their scope have
been discussed. We have tried to show how the analysts can have
a battery of possibilities that embraces from the simplest exter-
45
nal calibration with an only standard (an one-standard external
calibration) until the most tedious, for routine analysis, standard
addition calibration from several calibration standards prepared
and measured in replicate.
The selection of the appropriate methodology should be
based on a good knowledge of the metrological features of
the CMP and/or from a previous judgment on particular situ-
ations such as lack of stability or drift of the measuring system,
existence of the (additive or proportional) matrix effects, avail-
ability of proper calibration standard (from the analyte or some
surrogates), etc. The paper also contains a lot of practical
recommendations and sufficient guidance to properly apply a
chromatographical (or from other separation technique) cali-
knowledge.
Acknowledgement
We acknowledge financial support from the Spanish Min-
isterio de Educación y Ciencia (MEC), Dirección General de
Investigación (Project No. CTQ2006-15066-C02-02).
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