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Uncertainty of the result from the method of
standard addition is often underestimated due to neglect of the
covariance between the intercept and the slope. In order to
simplify the data analysis from standard addition experiments,
we propose x−y coordinate swapping in conventional linear
regression. Unlike the ratio of the intercept and slope, which is
the result of the traditional method of standard addition, the
result of the inverse standard addition is obtained directly from
the intercept of the swapped calibration line. Consequently,
the uncertainty evaluation becomes markedly simpler. The
method is also applicable to nonlinear curves, such as the
quadratic model, without incurring any additional complexity.

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pubs.acs.org/ac

Chemistry: A Simplied Path for Uncertainty Calculation in Linear

and Nonlinear Plots

Juris Meija,* Enea Pagliano, and Zoltan Mester

National Research Council Canada, 1200 Montreal Road, Ottawa, Ontario K1A 0R6, Canada

S Supporting Information

*

standard addition is often underestimated due to neglect of the

covariance between the intercept and the slope. In order to

simplify the data analysis from standard addition experiments,

we propose xy coordinate swapping in conventional linear

regression. Unlike the ratio of the intercept and slope, which is

the result of the traditional method of standard addition, the

result of the inverse standard addition is obtained directly from

the intercept of the swapped calibration line. Consequently,

the uncertainty evaluation becomes markedly simpler. The

method is also applicable to nonlinear curves, such as the

quadratic model, without incurring any additional complexity.

INTRODUCTION

It is common knowledge in analytical chemistry that the

instrumental response can be aected by the sample matrix.

This behavior leads to biased results when the analytical signal

produced from the sample is compared against the calibration

graph prepared from matrix-free standard solutions. To

overcome this problem, the method of standard addition was

introduced in the 1930s.1,2 To date, the method of standard

addition remains an essential tool in quantitation, especially

when isotope dilution cannot be applied. Like any scientic

tool, the method standard addition has witnessed new

developments, most notable of which is the concomitant use

of the internal standard.3

To wit, when more than one addition of a standard is

performed, the method of standard addition seeks the point

with zero response (assuming all signals are corrected for blank

contribution), i.e., the analytical result is as follows:4,5

x 0 = xy = 0

(1)

the covariance between these two estimates, which is not trivial

and is often neglected in practice:69

u 2(x0)

x02

a02

u 2(a1)

a12

cov(a0 , a1)

a0a1

(3)

(4)

are rather tedious; and publications on the subject are riddled

with mathematical equations.3 In addition, several recent

studies have focused on a more general problem of nonlinear

calibration curves in analytical chemistry.10,11 The metrological

aspects of nonlinear calibration curves, however, have not

witnessed proper attention, largely due to the complexity that

this problem poses.

THEORY

Variable Transformation for Standard Additions. To

the annoyance of many chemists, traditional standard addition

regression does not yield the analytical result directly. This

problem has been noted before. Notably, Tellinghuisen has

proposed an alternative approach whereby the variable-ofinterest for standard additions, x0, is cast as an explicit variable

of the tting model rather than calculated from the tting

parameters.12,13 The method involves variable transformation

(2)

the ratio of the intercept and the slope of the linear t. A

drawback of this traditional approach is that the uncertainty of

the analytical result cannot be obtained readily from the tting

parameters a0 and a1. Rather, uncertainty propagation must be

performed by combining the uncertainties of a0 and a1.

Moreover, because the intercept and the slope are correlated

Published 2014 by the American Chemical

Society

u 2 (a 0 )

where,

the observed data {x, y}, where x is the amount of the added

analyte, and y is the corresponding analytical response. For a

commonly used straight line t, y = a0 + a1x,

x0 = a0 /a1

Accepted: August 6, 2014

Published: August 6, 2014

8563

Analytical Chemistry

Article

which gives a direct estimate of the standard addition result, i.e.,

x0 = a1. Likewise, quadratic tting of y = a0 + a1x + a2x2 can be

done with a modied quadratic function, y = a0(a1 + x) + a2(a1

+ x)2, which yields x0 = a1.5 Albeit simple in its concept, the

variable change from y = a0 + a1x to y = a0(a1 + x) = a0a1 + a0x

leads to a nonlinear tting problem because of the product of

both variables, a0a1, which appears in the model equation. As a

result, no simple algebraic expressions can provide the results

and iterative minimization methods must be employed, such as

the Excel SOLVER macro.

Coordinate Swapping. Consider a typical standard

addition experiment where x is the vector of the masses of

the added standard and y is the vector of the corresponding

instrumental responses. Now, instead of tting y = a0 + a1x, one

performs tting of x = b0 + b1y, which is known as the inverse

calibration.14 Ordinary least-squares t is obtained by

minimizing the sum (a0 + a1xi yi)2 with a0 and a1 as

variables, whereas the inverse t is found by minimizing the

sum (b0 + b1yi xi)2 with b0 and b1 as variables. The advantage

of the seemingly trivial coordinate swapping becomes evident

after calculation of x0,inv = xy=0; x0,inv equals the (negative)

intercept of the inverse regression, i.e.,

x0,inv = b0

dierence between the results of direct and inverse linear

standard addition plots is irrelevant.

Nonlinear Models. Not all analytical methods provide

linear response with respect to the amount of the analyte.

When nonlinear response is observed, it is common among

chemists to employ empirical polynomials, such as the

quadratic function, in order to represent the observed curvature

while disregarding the physical basis of the nonlinearity. The

diculty of employing polynomial tting in standard addition

models, however, is in large part due to the complexity of

obtaining the analytical results. For example, the analytical

result from a quadratic standard addition t, y = a0 + a1x + a2x2,

is given as follows:16

x0 =

u 2(x0) =

x0,inv is the uncertainty of the intercept:

qcov(a0 , a1)

qd cov(a1 , a 2)

d cov(a0 , a 2)

+

2

a 2z

a2 z

2a 23z

(6)

(8)

x x 4 x2 x3

cov(a0 , a 2) = u 2(a 2)

cov(a1 , a 2) = u 2(a 2)

( x 2)2 N x 4

(9)

( x 2)2 x x 3

( x)2 N x 2

(10)

N x3 x x2

( x)2 N x 2

(11)

model y = a0 + a1x + a2x2, the uncertainty calculation in the

inverse approach, x = b0 + b1y + b2y2, remains simple:

u(x0,inv) = u(b0)

(12)

feature given the growing use of nonlinear calibration functions

in analytical chemistry (see Table 1).1618

The inverse of the linear model is also a linear model and the

same holds true for Pade[1,1] model. Unlike these two

uncertainty estimates of direct and inverse standard addition

method results. Second, the dierence between the results of

inverse and direct standard addition methods is as follows:

Approaches for the Standard Addition Method

direct calibration

approximated as follows:

u(x0,inv)

q2u 2(a1)

u 2 (a 0 )

d 2u 2(a 2)

+

+

z

4a 24z

4a 22z

regression parameter covariances are calculated as follows:

u(x0,inv)/u(x0) r 2

(7)

a21

obviates the need to evaluate the covariance between the

regression parameters which is necessary when direct regression

is employed (see eqs 3 and 4).

Equivalence between Direct and Inverse Regression.

The ordinary least-squares minimization is not symmetric with

respect to both coordinates. Consequently, many heated

discussions have ensued with regard to the numerical

dierences obtained from direct and inverse regressions.14,15

In the context of analytical chemistry, the following two

expressions are helpful to establish the metrological compatibility between the results of the direct and inverse linear

standard addition models. First, for a suciently large

coecient of determination (r2), commonly, if not universally,

encountered in analytical chemistry,

x0,inv x0

2a 2

(5)

u(x0,inv) = u(b0)

a12 4a0a 2

a1

y

y

y

y

1 r2

r2

metrologically compatible in a sense that the uncertainty of

x0 exceeds the numerical dierence between the direct and the

inverse models, x0,inv x0. Hence, it is fair to conclude that for

=

=

=

=

a0 + a1x

a0 + a1x + a2x2

(a0 + a1x)/(1 + a2x)

(a0 + a1x + a2x2)/(1 + a3x)

inverse calibration

x

x

x

x

8564

=

=

=

=

b0 + b1y

b0 + b1y + b2y2

(b0 + b1y)/(1 + b2y)

(b0 + b1y + b2y2)/(1 + b3y)

result

x0

x0

x0

x0

=

=

=

=

a0/a1

(a1 (a21 4a0a2)1/2)/2a2

a0/a1

(a1 (a21 4a0a2)1/2)/2a2

result

x0,inv

x0,inv

x0,inv

x0,inv

=

=

=

=

b0

b0

b0

b0

Analytical Chemistry

Article

type

direct

linear

quadratic

polynomial

Pade[1,1]

Pade[2,1]

function

y

y

y

y

y

y

=

=

=

=

=

=

a0x

a0 + a1x

a0 + a1x + a2x2

a0 + a1x + ... + amxm

(a0 + a1x)/(1 + a2x)

(a0 + a1x + a2x2)/(1 + a3x)

=LINEST(y,

=LINEST(y,

=LINEST(y,

=LINEST(y,

=LINEST(y,

=LINEST(y,

x, FALSE, TRUE)

x, TRUE, TRUE)c

x^{0,1,2}, FALSE, TRUE)

x^{0,...,m}, FALSE, TRUE)

x^{0,1,0}*y^{0,0,1}*x^{0,0,1}, FALSE, TRUE)

x^{0,1,2,0}*y^{0,0,0,1}*x^{0,0,0,1}, FALSE, TRUE)

a

For inverse tting x = f(y), all instances of x and y in the LINEST directive have to be interchanged. For example, the inverse linear t is performed

using LINEST(x, y, TRUE, TRUE). bSymbols x and y stand for the data input range. For example, if the values of x and y are in cells A1 to A5 and

B1 to B5, respectively, then the linear t is obtained by entering =LINEST(B1:B5, A1:A5, TRUE, TRUE). Also note that unlike many other

functions which are entered in a singe cell by pressing enter, LINEST is an array function which is entered over several cells (an array) by pressing

ctrl+shift+enter. cAlternatively, one can use =LINEST(y, x^{0,1}, FALSE, TRUE).

mathematical form of the tting model equation, however, is

irrelevant for most applications since nonlinear models are used

empirically in order to describe the observed curvature between

x and y. Consequently, the tting parameters often carry little

physical meaning. In many cases, nonlinear responses can be

made linear by using variable transformation. For example,

when the method of standard addition is used in potentiometry,

one employs linear tting function y = a0 + a1lnx, which can be

inverted to lnx = b0 + b1y.

Regressions with Excel. Calculations using Excel spreadsheets have become commonplace in science.19,20 Many tting

algorithms involve the SOLVER macro which performs

minimization of a user-dened function against the userdened argument. The use of such iterative approaches cannot

be subjected to a seamless copy-and-paste for other data, and

they do not provide the uncertainty estimates for the

parameters. On the contrary, the built-in Excel LINEST

function addresses all of the above shortcomings, and it can

be tailored for both linear and nonlinear model equations.

Table 2 summarizes useful LINEST directives in order to

perform tting on linear or polynomial models, including the

rational Pade functions, which were recently introduced for

nonlinear standard addition tting.21

(BCR-611)/Dataa

mA(AA*)/g

mA*(AA*)/g

mfinal/g

AEtBr

AEtI

2.005 99

2.008 17

2.180 35

2.001 74

2.004 35

2.009 60

1.999 61

2.005 36

2.009 01

1.999 22

2.002 76

2.006 66

1.997 26

2.004 13

2.006 93

0.000 00

0.000 00

0.000 00

0.049 49

0.050 05

0.049 89

0.099 24

0.099 49

0.099 91

0.148 14

0.148 72

0.148 83

0.197 03

0.198 03

0.200 21

2.353 82

2.358 67

2.355 67

2.350 62

2.353 49

2.358 09

2.348 73

2.354 30

2.358 00

2.346 72

2.351 28

2.354 87

2.344 29

2.352 16

2.357 14

14 610

14 633

13 422

21 863

22 161

22 155

29 786

30 806

30 427

35 088

37 794

34 243

42 221

46 070

38 640

8153

8176

7695

8190

8339

8199

8357

8652

8432

7983

8531

7731

7909

8682

7286

1925(2)k=1 ng g1.

standard additions which accounts for the (small) dierences in

the amount of sample in each aliquot, and for the (small)

dierences in the dilution factors. We also employ an internal

standard in order to attain highest precision. More details on

this precision-formalism of the standard additions can be

consulted elsewhere,3,24 and here we give only a brief summary

of the variables which are involved in constructing the standard

addition plots. When no internal standard is used, the following

variables are considered:

mA * (AA ), i

mfinal, i

* w

AEtBr, i and xi =

yi =

A*

mA(AA ), i

mA(AA ), i

(12)

*

*

When endogenous internal standard is used, there is no need to

account for the sample dilution, and the following variables are

considered:

mA * (AA ), i

AEtBr, i

* w

and xi =

yi =

A*

AEtI, i

mA(AA ), i

(13)

*

The incorporation of the mass fraction of the analyte in the

primary standard, wA*, serves to simplify the calculations. Such

practice is acceptable if the uncertainty of the wA* is negligible.

However, when the uncertainty arising from the wA* needs to

be explicitly accounted for in the combined uncertainty budget,

one can remove the wA* from eqs 12 and 13. Then, the mass

fraction of the analyte in the sample is wA = x0wA*, and the

EXPERIMENTAL SECTION

Bromide was analyzed in certied groundwater reference

material by using headspace gas chromatography mass

spectrometry (GCMS) after the aqueous derivatization with

triethyloxonium tetrauoroborate at room temperature.22

Iodide ions which are naturally present in the groundwater

were used as the internal standard for bromide since both of

these anions undergo ethylation in a similar fashion.

Experimental details are summarized in the Supporting

Information (SI) and further details can be found elsewhere.23

To demonstrate the validity of the coordinate swapping in

standard additions, and also to show the usefulness of this

approach when dealing with nonlinear standard addition

models, determination of bromide in groundwater certied

reference material was undertaken using a state-of-the-art

GCMS method.22 All relevant experimental data are given in

Table 3. Symbol mA(AA*) stands for the mass of the sample in

the mixture of the sample (A) and standard (A*), mA*(AA*) is

the mass of the standard in the corresponding mixture, mfinal is

the mass of the AA* mixtures after addition of all reagents, and

AEtBr and AEtI stand for the peak areas of EtBr and EtI.

8565

Analytical Chemistry

Article

Table 4. Determination of Bromide in BCR-611 with Standard Addition Method Using Various Direct and Inverse Calibration

Methodsa

yi = AEtBr,i/AEtI,i xi = (mA*(AA*),i/mA(AA*),i) wA*

direct calibration

y = a0 + a1x

y = a0 + a1x + a2x2

y = (a0 + a1x)/(1 + a2x)

y = (a0 + a1x + a2x2)/(1 + a3x)

inverse calibration

x = b0 + b1y

x = b0 + b1y + b2y2

x = (b0 + b1y)/(1 + b2y)

x = (b0 + b1y + b2y2)/(1 + b3y)

equations

result, w(Br)/(ng/g)

complexity of x0b

complexity of u(x0)b

eqs

eqs

eqs

eqs

24

7, 811

s1, s2s3

s4, s5s8

96.45(1.14)

93.15(3.12)

93.50(3.25)

94.93(1.17)

1

8

1

8

30

177

216

460

eqs

eqs

eqs

eqs

5,

5,

5,

5,

96.37(1.14)

93.09(3.34)

92.42(3.21)

94.94(1.12)

1

1

1

1

0

0

0

0

6

6

6

6

Mass fraction of bromide in BCR-611 as determined by the state-of-the-art quadruple isotope dilution GCMS, w(Br) = 96.28(42) ng/g.15 All

uncertainties here are combined standard uncertainties (expressed with coverage factor k = 1). bThe number of elementary arithmetic functions

(addition, multiplication, and exponentiation) needed to obtain x0 or u(x0) starting from the results given by the LINEST function (calculated for

this data set with N = 15). Although the operation count depends on variable rearrangement (e.g., three operations in a b + a b vs two

operations in 2 a b), it nevertheless provides a reasonable estimate of the mathematical complexity at-hand.

a

This way one can ensure that the uncertainty associated with

the primary standard is properly incorporated in the

uncertainty of the result. For the sake of simplicity, this was

not done in this work, also because the uncertainty in the

primary reference standard, u(wA*), is insignicant in

comparison to the uncertainty of the t for data presented in

this work.

Using data from Table 3, standard addition calculations were

performed with four modelslinear, quadratic, Pade[1,1], and

Pade[2,1] whose equations are given in Table 1. Least squares

tting was performed using LINEST function as detailed in

Table 2. For direct calibration functions, the uncertainty of x0

was obtained either from eqs 3 and 4 (linear model) or eqs

811 (quadratic model). Expressions for the Pade[1,1] and

Pade[2,1] parameter covariances are given in the SI. For all

inverse calibration functions, the uncertainty of x0 was obtained

directly from LINEST as per eq 6. The results are given in

Table 4.

In general, one can observe that the numerical results of

inverse calibration approach dier negligibly from the

corresponding results obtained from the direct tting of

analytical signal against the mass of the added analyte. In

addition, all results agree with the value established from the

state-of-the-art isotope dilution measurements.23 The calculation of the x0 values and their uncertainty estimates, however,

is signicantly simpler from the inverse regressions. Table 4

summarizes the minimum number of elementary arithmetic

functions needed to obtain x0 or u(x0) starting from the results

given by the LINEST function. In all cases, the inverse

approach provides answers without any additional calculations

whereas direct tting of y over x requires considerable amount

of additional calculations in order to obtain x0 and u(x0). In the

well-cited publication on the standard addition method, Bader

reects that once mathematical complexity sets in, it is easy to

make mistakes.7 In this vein, it is hoped that the marked

simplicity of the inverse approach will result in a wider use of

this technique and spur the much-needed evaluation of the

metrological performance of nonlinear standard addition

models. With the increased nonlinearity in the standard

addition curves, one should expect more discrepancies between

the results of the various empirical nonlinear models and also

between the direct and inverse calibration approaches. In such

chosen method of analysis or the adequacy of the measurement

model equation.

CONCLUSIONS

Although, strictly speaking, the classical and inverse regressions

are not mathematically equivalent procedures, the distinction

becomes relevant only for data sets that are very noisy or

extremely nonlinear.25 Since this is not a norm in analytical

chemistry, the inverse tting oers signicant advantages to

chemists. Given that the coordinate swapping in standard

addition experiments simplies the data analysis signicantly

over the traditional tting approach, the use of inverse

regression should be embraced in the spirit of Occams razor.

ASSOCIATED CONTENT

S Supporting Information

*

bromide and iodide sampled from the headspace of the

groundwater reference material BCR-611 with w(Br) = 93 4

ng/g and w(I) = 9.0 ng/g (Figure S1); theoretical information

and results. This material is available free of charge via the

Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: juris.meija@nrc.ca.

Notes

REFERENCES

Berlin, 1937.

(2) Kelly, W. R.; Pratt, K. W.; Guthrie, W. F.; Martin, K. R. Anal.

Bioanal. Chem. 2011, 400, 18051812.

(3) Hauswaldt, A.-L.; Rienitz, O.; Jahrling, R.; Fischer, N.; Schiel, D.;

Labarraque, G.; Magnusson, B. Accred. Qual. Assur. 2012, 17, 129

138.

(4) Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman:

New York, 1995.

(5) Tellinghuisen, J. Analyst 2005, 130, 370378.

(6) Bruce, G. R.; Gill, P. S. J. Chem. Educ. 1999, 76, 805807.

(7) Bader, M. J. Chem. Educ. 1980, 57, 703706.

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Analytical Chemistry

Article

(8) Serapinas, P.; Labarraque, G.; Charlet, P.; Ezirinskis, Z.; Juzikiene,

V. J. Anal. Atom. Spectrom. 2010, 25, 624630.

(9) Christopher, S. J.; Day, R. D.; Bryan, C. E.; Turk, G. C. J. Anal.

Atom. Spectrom. 2005, 20, 10351043.

(10) Rozet, E.; Ziemons, E.; Marini, R. D.; Hubert, Ph. Anal. Chem.

2013, 85, 63276335.

(11) Peters, F. T.; Maurer, H. H. Anal. Chem. 2007, 79, 49674976.

(12) Tellinghuisen, J. Analyst 2000, 125, 10451048.

(13) Salter, C.; de Levie, R. J. Chem. Educ. 2002, 79, 268271.

(14) Tellinghuisen, J. Fres. J. Anal. Chem. 2000, 368, 585588.

(15) Shukla, G. K. Technometrics 1972, 14, 547553.

(16) Koscielniak, P. Chemometr. Intell. Lab. 1999, 47, 275287.

(17) Kirkup, L.; Mullholland, M. J. Chromatogr. A 2004, 1029, 111.

(18) Gorazda, K.; Michalowska-Kaczmarczyk, A. M.; Asuero, A. G.;

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(19) Harris, D. C. J. Chem. Educ. 1998, 75, 119121.

(20) Kragten, J. Analyst 1994, 119, 21612165.

(21) Bartkovjak, J.; Karovicova, M. Meas. Sci. Rev. 2001, 1, 6365.

(22) DUlivo, A.; Pagliano, E.; Onor, M.; Pitzalis, E.; Zamboni, R.

Anal. Chem. 2009, 81, 63996406.

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8567

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