Journal of Applied Spectroscopy, Vol. 73, No.

2, 2006

CALIBRATION CURVES IN ATOMIC EMISSION SPECTRAL

ANALYSIS OF BIOLOGICAL SPECIMENS

K. V. Frantskevich UDC 543.423:578.085.1

An efficient algorithm and special software are described for plotting calibration curves in the form of poly-
nomials of optimal degree for atomic emission spectral analysis. The operation of the software is demon-
strated using software/hardware systems for monitoring the content of toxic elements in biological specimens.
It is shown that using optimal calibration curves leads to a decrease in the error of the spectral analysis.

Key words: atomic emission spectral analysis, calibration curve, toxic element, Chebyshev polynomials, biological
specimen.

Introduction. Currently atomic emission spectral analysis is widely used for monitoring the content of toxic
elements in samples of biological and aqueous specimens [1–3]. It is based on indirect measurements, and the value
of the absolute or relative intensity of the considered spectral line of the indicated element is taken as a measure of
the concentration (content) of the monitored element in the test sample. In this case, it is assumed that there is a one-
to-one functional relationship between the line intensity and the concentration of the corresponding element. Then
using state standard samples or calibrated reference samples with specified chemical composition, we can plot the cali-
bration curves for determination of the composition of the studied samples from the known intensity of the spectral
lines. In spectroscopy practice, the Lomakin–Scheibe equation in logarithmic form is usually considered as such a re-
lationship, which can lead to linearization of the calibration curves [4, 5]:

log I = a log C + b ,

where I is the intensity (absolute or relative) of the analytical spectral line for the given element; C is its content in
the test sample; a and b are constant coefficients. Calibration curves in such a form are convenient for photographic
recording of the spectrum with subsequent treatment of the results by hand, although they are also widely used today
for processing on personal computers [3, 6]. However, consider that the calibration curve is plotted with some error
that is part of the total error involved in making the measurement. Consequently, it is possible to reduce the error of
the determination of the content of the monitored elements in the sample by using another type of calibration charac-
teristic with less plotting error.
In the case when spectral software/hardware systems are used with computer processing of the results, it be-
comes possible to use a calibration curve passing exactly through all the experimental points. However, we need to
consider that due to the effect of random factors, a one-to-one relationship is observed only between the mathematical
expectation of the intensity of the spectral line and the elemental content known with some error. Therefore real cali-
bration curves can be represented as regression relations between the measured intensity I (or the logarithm of the in-
tensity) of the spectral line used and the corresponding content C (or the logarithm of the content) of the monitored
element. In most currently known papers devoted to the use of computers for calibration of spectral equipment [3, 7,
8], the calibration curves are represented as a dependence of the logarithm of the intensity on the logarithm of the
content. However, since the result of running the software for the software/hardware system is the value of the con-
centration for the monitored elements, calculated on the basis of the recorded intensity of the analytical spectral line,
the calibration curve is conveniently represented as a dependence of the logarithm of the content on the logarithm of
the intensity [9]. In this case, we can immediately find the parameters of the calibration curve used for operation of

Institute of Technology of Metals, National Academy of Sciences of Belarus, 11 Belynitskii-Birulya Str.,

Mogilev, 212030, Belarus; e-mail: intehmet@mogilev.unibel.by. Translated from Zhurnal Prikladnoi Spektroskopii, Vol.
73, No. 2, pp. 250–253, March–April, 2006. Original article submitted July 4, 2005.

280 0021-9037/06/7302-0280 ©2006 Springer Science+Business Media, Inc.

the software/hardware system, and we can estimate the error in determination of the content using this curve. In writ-
ing the algorithm for determining the optimal calibration curve to ensure a one-to-one correspondence between the in-
tensity of the analytical line and the concentration of the element, we also need to check the monotonicity of the
variation in the function used within the specified range.
Plotting polynomial calibration curves. When using a software/hardware system, the calibration curve can be
represented as a polynomial of degree m:

m
y = a0 + a1x + ... + am x , (1)

where y is the logarithm of the elemental content; x is the logarithm of the line intensity; the aj are the coefficients
of the calibration curves.
For plotting calibration curves of form (1), we used kits of calibrated reference samples with chemical com-
positions that simulated the average composition of the residue for a mineralized test sample of various biological
specimens. Each calibrated reference sample in the kit was subjected to k parallel tests, and based on the results of
those tests we calculated the sampling average xi:

∑
1
xi = xij , i = 1, ..., n ,
k
j=1

where xij is the logarithm of the measured intensity of the spectral line for the i-th calibrated reference sample in the
kit for the j-th parallel measurement; n is the number of calibrated reference samples in the kit.
In order to determine the coefficients ai of the calibration curve, we used the least squares method, which is
a special case of the maximum likelihood method. In this case, the best calibration coefficients for a specified degree
n
m of the polynomial is considered the one for which the sum of the squares ∆ = ∑ ωi(y~i − yi)2 of the deviations of
i=1
the estimates yi = a0 + a1xi + ... + amxm
of the calibration curve from the known values of the logarithms of the con-
i
centration ~
yi is minimum. Here the ωi are the known weights of the measurements. From the condition of an extre-
mum for the function ∆, we obtain a system of linear equations to estimate the calibration coefficients. However, when
m > 2, it is difficult to solve this system because of the loss of accuracy associated with round-off error and the close-
ness of the determinant of the system to zero. In the case when we need to increase the degree of the polynomial, all
the calculations are carried out again. So we make use of the Chebyshev orthogonalization method, which involves re-
placing expression (1) by a linear combination of orthogonal Chebyshev polynomials [7, 10]:

y (x) = b0P0 (x) + b1P1 (x) + ... + bmPm (x) .

j n
To plot the indicated polynomials Pj(x) = ∑
k
αkjx , satisfying the condition ∑ ωiPl(xi)Pj(xi) = 0 for l ) j,
k=0 i=1
we used the following convenient recursion relation:

P0 (x) = 1 , P1 (x) = x − u1 ,

Pj (x) = (x − uj) Pj−1 (x) − νj−1Pj−2 (x) , j = 2, ..., m .

n n n
Here uj = ∑ ωixiP2j−1 (xi) ⁄ Hj−1 , νj−1 = ∑ ωixiPj−1 (xi) Pj−2 (xi) ⁄ Hj−2 , Hj = ∑ ωiP2j (xi) .
i=1 i=1 i=1
The coefficients bj, βkj, and ak were determined from the formulas:

281
 n
bj = ∑ ~
yiPj (xi) ⁄ Hj , j = 0, ..., m ,
i=1

⎧β − uj βk,j−1 − νj−1 βk,j−2 for 0 ≤ k < j ; k = 0, ..., m ; j = 1, ..., m ,

⎪ k−1,j−1
βkj = ⎨1 for k = j ;
⎪
⎩0 for k > j , k < 0 ,

m
ak = ∑ bj βkj , k = 0, ..., m .
j=k

Note that for polynomials of high degree, oscillations may be present (nonmonotonic variations in the function
y(x)), which is unacceptable for making the measurements, since then the correspondence between the measured inten-
sity of the spectral line and the content of the monitored element is no longer one-to-one. So the software we devel-
oped checks the plotted polynomial for the presence of such oscillations. In the following, we used only monotonically
varying polynomials.
Then we estimated the variance of fit for the plotted calibration curve:

n
1
∑
2 2
Sr = ωi (yi − ~
yi) .
n−m−1
i=1

Using the plotted calibration curve, we find the logarithms of the contents yij in the considered calibrated ref-
erence samples, corresponding to the measured values of the logarithms of the intensities xij:

m
yij = a0 + a1xij + ... + am xij ,

and based on them, we determine the sampling variance of the measurement of yij in each sample

k
1
∑
2 2
Si = (yij − yj)
k−1
j=1

and the average variance for the measurement of the logarithm of the elemental content in all the samples used to plot
the calibration curve for the monitored element:

n
1
∑
2 2
Se = ωiSi .
kn
i=1

We checked the adequacy of fit for the plotted calibration curve for the spectral analysis data for the cali-
%
brated reference samples using the Fisher test F = S2r S2e by comparing with tabulated values for the Fisher distribu-
tion with n – m – 1 and (k – 1)n degrees of freedom for the significance level determined from experiment [11].
From among the adequate polynomials with degrees 1, ..., 5, as optimal we selected the polynomial ensuring the mini-
mum pooled variance of the logarithm of the content [8]:

2 2
2 Sr (n − m − 1) + Se (k − 1) n
S = .
kn − m − 1

The optimal degree of the polynomial was individually selected for each monitored element in each type of
test specimen (product).

282
TABLE 1 Parameters of Optimal Calibration Curves Compared with Linear Calibration Curves

Shape of Polynomial coefficients

Variance Pooled
Element calibration F-test
a0 a1 a2 a3 of fit variance
curve
Optimal –0.6865 1.0754 – – 0.0008140 0.001288 0.5583
Cd
Linear –0.6865 1.0754 – – 0.0008140 0.001288 0.5583
Optimal –4.2132 2.3675 –0.15173 – 0.002051 0.002228 0.9001
Cu
Linear –2.2846 1.2722 – – 0.004175 0.002383 2.3952
Optimal –2.4133 2.6865 –0.25971 – 0.004746 0.004492 1.0738
Zn
Linear –0.72127 1.3357 – – 0.007341 0.004951 1.7914
Optimal 28.290 –23.874 6.7234 –0.58811 0.0006706 0.001163 0.5288
Pb
Linear –3.4767 1.5440 – – 0.002392 0.001811 1.4923

Fig. 1 Optimal calibration curves for monitoring content of zinc (1), cadmium
(2), copper (3), and lead (4).

Results and discussion. The above-indicated algorithm was written in Object Pascal for the spectral soft-
ware/hardware system used in monitoring biological specimens for content of toxic elements. The software/hardware
system includes a DFS-452 diffraction spectrograph, an IVS-29 spectral excitation source, a computer system for re-
cording the spectra with linear photodetector devices and special software which lets the user control the operation of
the system in dialog mode, change various parameters for recording and processing the spectral data, display the re-
corded spectra onscreen on different scales, and measure the intensity of the spectral lines and the background. The
spectral data obtained can be written to a hard disk and read from the disk. In operating the system, the software con-
structs the calibration curves in the form of polynomials of degree 1–5 and automatically determines the optimal de-
gree of the polynomial. For this purpose, in testing the kit of calibrated reference samples, the intensity of the
analytical spectral lines was measured and in dialog mode the user entered the certificate data for each sample in the
kit (concentration and uncertainty of the preparation for each element). The data obtained in this way are recorded in
special tables for each type of monitored product individually, and are written to the hard disk so they can be used
many times in future.
Each sample in the kit of calibrated reference samples was placed in the lower graphite electrode cup; as the
upper counterelectrode, we used a graphite electrode sharpened to a cone shape with flat surface of diameter 1 mm.
Measurement conditions: a.c. arc, arc current 10 A, ignition phase 60o, pulse frequency 100 Hz, exposure time 240

283
sec, analytical interval 3 mm. Wavelengths of the analytical spectral lines used: Cd, 326.109 nm; Cu, 282.43 nm; Zn,
334.509 nm; Pb, 287.332 nm. The results obtained from testing the calibrated reference samples and the plotted opti-
mal calibration curves are shown in Fig. 1. Table 1 shows the parameters of these curves compared with linear cali-
bration curves. From the results obtained we see that for cadmium, the pooled variance depends little on the degree of
the polynomial, and takes on the minimum value for a linear curve. For copper and zinc, the pooled variance when
using the polynomial of optimal (second) degree decreases compared with the case of the linear curve, by 6.96% and
10.2% respectively. For lead, the pooled variance when using a polynomial of the optimal (third) degree decreases by
more than one and a half times compared with using a linear curve. Thus the pooled variance decreases considerably,
and polynomials of different degrees are optimal. So the software used should determine the optimal degree of the
polynomial each time when there is a change in the initial data used to plot the calibration curves.
Conclusion. Using this method ensures that the optimal calibration curves are plotted for software/hardware
systems utilized for atomic emission spectral analysis of both various biological specimens for their content of toxic
elements and also the elemental composition of metals and alloys. In this case, the calibration curve plotting error is
reduced, and consequently the error in the results of the entire analysis decreases.

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