Univariate and Multivariate Calibration for the Quantitative

Determination of Methyl-parathion in Pesticide

Formulations by FT-Raman Spectroscopy
STAVROULA G. SKOULIKA and CONSTANTINOS A. GEORGIOU *
Chemistry Laboratory, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece

A Fourier transform (FT)-Raman m ethod for the quantitative determ ination of methyl-parathion in pesticide form ulations is described. The proposed m ethod was applied to the analysis of form ulations of methyl-parathion. Univariate and m ultivariate calibration were used and com pared for quantitative analysis. Bands
observed at 634, 661, 1113, 1348, and 1527 cm 2 1 were used for
univariate calibration. Calibration curves were linear (correlation
coef cients: 0.996 0.998 and 0.994 0.999 for band intensity and
band area m easurements, respectively) in the concentration range
of 0.6 3.75 M for the 634, 661, and 1527 cm 2 1 bands; 0.05 3.75 M
for the 1113 cm 2 1 band; and 0.1 3.75 M for the 1348 cm2 1 band.
Precision ranged from 0.5 to 5.2% and 0.1 to 6.8% relative standard deviation (RSD) (n 5 4) for band intensity and band area
m easurem ents, respectively. Spectra normalization against the 802
cm 2 1 cyclohexane band improved the long-term stability of calibration, allowing the use of calibration data acquired 30 days prior to
analysis. The application of the method was extended through multivariate calibration by multiple linear regression using the 858 and
2952 cm 2 1 bands. Results obtained com pare well with those obtained by the high-perform ance liquid chromatography (HPLC)
reference m ethod. The FT-Raman method developed is rapid, simple, and safe, as toxic sam ples are analyzed ``as received without
sample pretreatment.
Index Headings: Methyl-parathion; FT-Raman; Pesticide form ulations; Multivariate calibration; Determination.

INTRO DUCTION
M eth yl-parath ion (O ,O -d im eth yl O -4 -nitro pheny l
phosphorothioate) is a nonsystem ic insecticide and acaricide with contact, stomach, and respiratory action that is
used for the control of sucking and chewing insects in a
very wide range of crops, such as cereals, fruit (including
citrus), vines, vegetables, ornamentals, cotton, and eld
crops.1 Spectrophotometric methods based on the following have been devised for the analysis of methyl-parathion formulations:
1. Alkaline hydrolysis at 55 8 C and measurement of the
reaction rate.2
2. The reaction with 3-methylbenzothiazolin-2-one hydrazone hydrochloride in the presence of Ce(IV) or
Fe(III) as oxidizing agents.3
3. The reduction by ZnHCl to the corresponding amine
and a subsequent reaction leading to a colored product.4
4. The reaction with Na 2 Fe(CN) 5 NO and hydroxylammonium chloride in alkaline medium.5
5. Derivative spectrophotometry.6
Other spectrophotometric methods that have been develReceived 18 October 1999; accepted 20 Decem ber 1999.
* Author to whom correspondence should be sent.

Volume 54, Number 5, 2000

oped for the determination of m ethyl-parathion in crops

based on acetylcholinesterase inhibition 7 and the reaction
with 4-( p-nitrobenzyl)-pyridine8 are also suitable for formulation analysis. Gas chromatographic (GC) methods 9 13
and a high-performance liquid chromatographic (HPLC)
method 1 4 have been developed and are in use for the
analysis of m ethyl-parathion formulations. Spectrophotometric m ethods require extensive m anual sample handling such as solvent evaporation, boiling, and dilutions,
and are time consuming. Chromatographic methods are
also time consuming and require sample handling using
various solvents. As methyl-parathion is a toxic cholinesterase inhibitor, alternative fast analytical methods requiring minimal sample handling are highly desirable.
The Raman spectrum of methyl-parathion has been
previously reported from the use of dispersive1 5 and a
Fourier transform (FT)-Raman spectrometer.16 The surface-enhanced Raman spectrum has also been published,17 but no application of Raman spectroscopy to the
quantitative determination of m ethyl-parathion has been
described.
In this work, an FT-Raman m ethod for methyl-parathion determination is presented. Univariate and multivariate calibration are used and compared in the analysis
of pesticide formulations. This work is part of an ongoing
project in our laboratory concerning pesticide formulation analysis. 18 ,19
EXPERIMENTAL
Sample Preparation and Chemicals. Methyl-parathion of 80% purity and xylene of technical grade were a
kind offer of Cheminova Agro A/S, Denmark. The analytical m ethyl-parathion standard of certi ed 99.5% purity was a kind offer of Bayer, Germany. Xylene of analytical reagent grade was purchased from Merck. Com mercial formulations were obtained through local m anufactur ers. A ll ch em icals w ere used w ithou t f urther
puri cation. While preparing standards and handling formulations, extreme care should be exercised to avoid
methyl-parathion contact with the skin and eyes, due to
methyl-parathion toxicity. Methyl-parathion that is slightly soluble in water at 20 8 C (55 mg/L) is toxic, with the
acute oral LD 50 for rats at 3 m g/kg, for m ale m ice at 30
mg/kg, and for rabbits at 19 m g/kg.1 Standard methylparathion solutions in the range of 0.053.75 M were
prepared by weighing the appropriate amount of m ethylparathion and dissolving in xylene.
Apparatus. FT-Raman spectra were recorded with a
Nicolet 750 FT-Raman spectrometer equipped with a
Nd:YAG laser source that emits at 1064 nm . A CaF 2

0003-7028 / 00 / 5405-0747$2.00 / 0
q 2000 Society for Applied Spectroscopy

APPLIED SPECTROSCOPY

747

TABLE I. Methyl-parathion Ram an bands (cm2 1 ).a

Ver y strong
Strong
Medium
Weak
Ver y weak
a

1348 (256)
1590 (95), 1113 (62), 2952 (49), 858 (33), 3087 (32)
1004 (28), 2852 (22), 588 (15), 786 (14)
1527 (12), 3054 (7.1), 1032 (6.9)
661 (5.4), 645 (4.7), 634 (4.5)

% Relative intensities to the 802 cm 2

in parentheses.

cyclohexan e band are shown

beamsplitter, an indium galium arsenate (InGaAs) detector, and 1808 backscattering geometry were used in the
spectrom eter. A motorized positioner focuses the laser
beam to the sample, and a manual side-to-side adjuster
allows sample adjustment for maximum optical ef ciency. To ensure that the spectrometer is ne tuned and the
detector signal m aximized, we perform ed an optical
bench alignment before each batch of m easurements.
Sample cells used were cut to 6 cm from Wilmad W G5M NMR tubes of 4.97 m m outer diameter and 0.38 mm
wall thickness. Spectra were accumulated from 100 scans
collected during 3 m in at a resolution of 4 cm 2 1 .
H igh-per for m ance liquid chrom atographic analysis
was carried out with a Jasco PU-98 Pump and a Jasco
UV-970 UV-visible detector equipped with a uPorasil Si,
10u (Waters) 4.6 3 250 m m column. The ow rate was
1.2 m L/min and detection was at 254 nm.
RESULTS AND DISCUSSION
R am a n S p ectru m of M eth yl- p arath ion . Ram an
bands of the m ethyl-parathion spectrum acquired in this
study are summ arized in Table I where the classi cation
of very strong, strong, medium, weak, and very weak
bands is based on the % relative intensities to the 802
cm 2 1 cyclohexane band. The cyclohexane external standard spectrum was measured right after the acquisition
of the methyl-parathion spectrum with the use of the

same experimental parameters (laser power, sample holder position, resolution, and number of scans).
Molecules possessing P 5 S bonds but no benzene rings
display a clearly distinguishable Raman band between
600 and 700 cm 2 1 attributed to the P5 S stretching.1 5 In
molecules possessing benzene rings, the P 5 S stretching
is overshadowed by benzene ring vibrations. 15 In the
aforementioned range, two overlapping bands at 634 and
661 cm 2 1 appear in the methyl-parathion spectrum . For
molecules containing benzene rings, additional strong
bands around 1580 cm 2 1 and a weak band around 580
cm 2 1 have been observed. 1 5,17 The 858 cm 2 1 band has
been attributed to the in-plane CH 3 rock. 20 The 1113 cm 2 1
band is characteristic of d (CCH) vibrations coupled with
CO and CN motions. 21 The 1348 and 1527 cm 2 1 bands
have been attributed to NO vibrations, 2 2 while the (NO 2 )
vibration in inorganic salts is believed to appear around
1330 cm 2 1 . 23 The intense bands appearing in the 2800
3100 cm 2 1 region are due to aliphatic and aromatic CH
stretching m odes. 15 ,2 4
A normalization procedure was undertaken in order to
compensate for variations of excitation intensity, sample
positioning, and temperature, as these factors in uence
Raman analytical signals. 25 The 802 cm 2 1 cyclohexane
band, measured right after the acquisition of each m ethylparathion spectrum , was used for the norm alization. 26 The
cyclohexane spectrum was acquired with the use of the
same experimental parameters, such as laser excitation
power, sample holder position, resolution, and num ber of
scans. During methyl-parathion quantitative analysis,
normalized % relative intensities or areas were used. Normalization results in increased stability of the calibration
between days, compensating for changes in experimental
parameters.
Q uantitative Analysis of M ethyl-parathion Form ulations. Quantitative analysis by Raman spectroscopy can

F IG . 1. FT-Raman spectra acquired during calibration: (a) 0.0500, (b) 0.300, (c) 0.600, (d ) 1.00, (e) 1.50, (f) 2.00, (g) 2.50, (h) 3.00, and (i) 3.75
M methyl-parathion standards measured in the range of (A) 1200 500, (B) 1400 1300, (C ) 1560 1480, and (D ) 3000 2900 cm 2 1 .

748

Volume 54, Number 5, 2000

TABLE II. Methyl-parathion calibration data normalized against the 802 cm2
Band
(cm 2 1 )

Concentration
range (M)

cyclohexane band.

Equation of
calibration graph

A. Band intensity calibration curve

1527
0.63.75
1348
0.13.75
1113
0.053.75
661
0.63.75
634
0.63.75
B. Band area calibration curve
1527
0.63.75
1348
0.13.75
1113
0.053.75
661
0.63.75
634
0.63.75
a

BI
BI
BI
BI
BI

(2
(2
(2
(2
(2
5
5
5
5

BA
BA
BA
BA
BA
5

0.6
9 6
2.7
0.6
0.9
(2
(2
(2
(2
(2

0.2) 1 (3.20
3) 1 (65 6 2)
6 0.8) 1 (16.5
6 0.1) 1 (1.56
6 0.1) 1 (1.40

16
14
30
4.7
5.3

6
6
6
6
6

2) 1
4) 3
8) 1
0.7)
0.7)

6
6

3
6
6

0.08) 3 C
C
0.4) 3 C
0.04) 3 C
0.05) 3 C

(53 6 1) 3 C
10 1 (74 6 2) 3 10 3
(150 6 4) 3 C
1 (10.7 6 0.3) 3 C
1 (7.9 6 0.3) 3 C

Correlation
coef cient

Detection
limita (M)

% RSD b
( n 5 4)

0.998
0.996
0.996
0.997
0.996

0.2
0.1
0.1
0.2
0.2

2.35.2
0.52.8
0.73.4
0.54.8
0.84.1

0.999
0.994
0.995
0.996
0.995

0.1
0.2
0.2
0.2
0.3

0.16.3
1.54.4
3.24.1
0.45.0
4.36.8

Calculated as three times the standard deviation of the intercept divided by the slope.
Calculated by changing sample position.

be achieved by using either univariate or m ultivariate calibration. Univariate calibration utilizes the analytical signal resulting from a single band, while m ultivariate calibration utilizes the analytical signal of m ultiple bands. 2 7
In this work both calibration techniques are used and
compared.
Univariate Calibration. For quantitative analysis of
methyl-parathion formulations the 634, 661, 1113, 1348,
and 1527 cm 2 1 bands were used. These were chosen because the solvent (xylene) and commonly used surfactants do not interfere spectrally. Furthermore, these bands
are characteristic for methyl-parathion.1 6 Band intensities
and band areas were calculated with the use of a twopoint baseline correction using the range 630 638 cm 2 1
for the 634 cm 2 1 band, 655665 cm 2 1 for the 661 cm 2 1
band, 1104 1120 cm 2 1 for the 1113 cm 2 1 band, 1336
1357 cm 2 1 for the 1348 cm 2 1 band, and 15131540 cm 2 1
for the 1527 cm 2 1 band.
Spectra acquired during calibration are shown in Fig.
1, while calibration data based on the norm alization procedure at 1.01 W excitation intensity are presented in
Table II. The calibration curves present excellent linearity
with correlation coef cients in the range of 0.9960.998
and 0.994 0.999 for band intensity and band area m easurements, respectively.
The linear range was found to be 0.63.75 M for the

634, 661, and 1527 cm 2 1 bands, 0.053.75 M for the

1113 cm 2 1 band, and 0.13.75 M for the 1348 cm 2 1 band.
Detection limits were in the range of 0.10.2 and 0.1
0.3 M for band intensity and band area m easurements,
respectively. Precision values presented in Table II were
determined by changing the position of the sample tube
between the acquisition of successive spectra. Approximately 2 m in was allowed for laser power stabilization
between subsequent measurements. 2 6 Precision ranged
from 0.55.2 and 0.16.8 % relative standard deviation
(RSD) (n 5 4) for band intensity and band area measurements, respectively.
Results of the analysis of commercial formulations are
presented in Table III. As shown by the experimental tvalues, results obtained by the proposed m ethod are identical to those acquired through the HPLC reference method 14 for four formulations. For the Folidol formulation of
Bayer Hellas, results agree with those of the reference
method when using the intensity of the 1113 and 1348
cm 2 1 bands or the area of all bands except the 634 cm 2 1
band. For the Parathion-methyl formulation of Vector, results are consistent with those of the reference method
when using the intensity of the 1348 cm 2 1 band or the
area of the 661 and 1527 cm 2 1 bands. The use of band
area is believed to give more accurate results than the use
of band intensities, 2 8 as parameters in uencing the shape

TABLE III. Results of the analysis of m ethyl-parathion formulations by univariate calibration.

Methyl-parathion concentration, M 6
Band
(cm 2 1 )
A. Band
1527
1348
1113
661
634
B. Band
1527
1348
1113
661
634

Morfos m ethyl a

Parathion-methyl
Filocropb

intensity calibration curve

1.43 6 0.03 (0.808) 2.12 6
1.40 6 0.08 (1.145) 2.10 6
1.47 6 0.01 (0.000) 2.14 6
1.6 6 0.1 (1.922)
2.2 6
1.56 6 0.01 (1.891) 2.20 6
area calibration curve
1.4 6 0.1 (1.035)
2.1 6
1.45 6 0.04 (0.391)
2.1 6
1.43 6 0.04 (0.782)
2.1 6
1.42 6 0.04 (0.978)
2.2 6
1.53 6 0.04 (1.173) 2.21 6

SD (n 5 3) found during the analysis of six formulations accompanied by the

experim ental t-value in parentheses
Parathion-methyl
Ipsilonc

0.05
0.07
0.03
0.1
0.04

(0.841)
(1.064)
(0.548)
(0.386)
(0.528)

1.57
1.51
1.55
1.58
1.53

0.1
0.1
0.1
0.1
0.07

(0.901)
(0.901)
(0.901)
(0.386)
(0.608)

1.55
1.58
1.57
1.6
1.47

Methyl-parathiond

0.05
0.01
0.03
0.06
0.01

(1.204)
(0.278)
(1.790)
(1.369)
(0.278)

1.82
1.73
1.72
1.86
1.77

0.01
0.02
0.02
0.1
0.05

(0.833)
(1.360)
(0.000)
(1.446)
(1.204)

1.77
1.71
1.68
1.78
1.77

Folidol M e

0.06
0.03
0.03
0.07
0.01

(0.843)
(0.725)
(0.907)
(1.469)
(0.000)

1.67
1.45
1.40
1.71
1.63

0.05
0.06
0.05
0.05
0.07

(0.000)
(1.011)
(1.561)
(0.173)
(0.000)

1.51
1.48
1.44
1.5
1.58

Parathion-methylf

0.07
0.04
0.03
0.05
0.07

(4.575)
(0.598)
(0.407)
(5.640)
(3.843)

3.26
3.19
3.55
3.55
4.0

0.07
0.06
0.05
0.1
0.05

(1.647)
(1.134)
(0.389)
(1.312)
(3.112)

3.15
3.58
3.61
3.00
4.0

0.08
0.08
0.01
0.05
0.1

(3.679)
(2.547)
(9.264)
(8.605)
(13.090)

0.05
0.02
0.01
0.04
0.1

(2.347)
(9.673)
(10.274)
(0.000)
(13.090)

Methyl-parathion concentration as determined through the H PLC reference m ethod:14 a 1.47 6 0.08 M (n 5 4); b 2.17 6 0.09 M (n 5 3);
c
1.52 6 0.06 M (n 5 5); d 1.77 6 0.09 M (n 5 5); e 1.42 6 0.08 M (n 5 6); and f 3.0 6 0.1 M (n 5 4). tthe or et ical values for 95% con dence
level: a 2.571; b 2.776; c 2.447; d 2.447; e 2.365; and f 2.571. Manufacturer: a Eythimiadis; b Xelafarm; c Ipsilon; d Priftis; e Bayer; and f Vector.

APPLIED SPECTROSCOPY

749

F IG . 2. Predicted methyl-parathion concentrations by the m ultivariate

regression m odel vs. true concentrations using (A) band intensity and
(B) band area measurements.

and position of Raman bands are m ore likely to affect

the intensity than the area of a band.
Relative differences between results obtained through
the FT-Raman and the HPLC reference m ethod are in the
range of (2 3.7%)(8.8%) and (2 4.8%)(5.3%) for band
intensity and band area measurements, respectively, for
the four formulations, while for the others the relative
dif feren ces r an ge b etw een ( 2 0 .01% ) (3 3.3% ) and
(0.0%)(33.3%) for band intensity and band area measurements, respectively.
It should be noted that spectra of methyl-parathion formulations were acquired nine m onths after the construction of the calibration curves. Before the normalization
procedure, results acquired through the FT-Raman m ethod were found to be inaccurate, the deviation from the
750

Volume 54, Number 5, 2000

F IG . 3. Residuals as a function of concentration for the multivariate

regression model using (A) band intensity and (B) band area m easurements.

HPLC reference method lying in the range of (7.5% )

(17%) and (5.7%)(16%) for band intensity and band
area measurements, respectively. Normalization results in
improved long-term stability of calibration, allowing the
use of previously acquired calibration data. With the normalization procedure, slopes of the normalized calibration curves could be used as an analog to the molecular
extinction coef cients of UV-visible spectrophotometry.
Multivariate Calibration. In order to extend the applicability of the proposed m ethod, a stepwise m ultiple linear regression (M L R) calib ratio n p roced ure w as
used. 2 7,29 ,30
Multiple linear regression is based on the model
R 5

CS T 1

ET

(1)

TABLE IV. Results of the analysis of m ethyl parathion form ulations by m ultivariate calibration.
FT-Raman m ethod
Band intensity
C, M 6

Formulation, manufacturer
Morfos methyl, Eythimiadis
Methyl-parathion Filocrop, Xelafarm
Methyl-parathion Ipsilon, Ipsilon
Methyl-parathion, Priftis
Folidol M, Bayer
Parathion-methyl, Vector
a

1.37
2.15
1.62
1.63
1.47
2.9

SD (n 5
6

3)

t-value

0.02
0.01
0.05
0.07
0.03
0.1
6
6
6
6
6

2.070
0.383
2.302
2.185
1.017
1.309

Band area
C, M 6
1.55
2.26
1.61
1.73
1.51
3.1

SD (n 5

0.03
0.04
0.05
0.07
0.04
0.1

3)

HPLC method
C, M 6

t-value
1.616
1.583
2.072
0.624
1.795
1.309

1.47
2.17
1.52
1.77
1.42
3.0
6

0.08
0.09
0.06
0.09
0.08
0.1

SD
(n
(n
(n
(n
(n
(n
5

tthe ore tica la

4)
3)
5)
5)
6)
4)

2.571
2.776
2.447
2.447
2.365
2.571

ttheor etical for 95% con dence level.

where R represents the spectral data m atrix, C the concentrations m atrix, S the sensitivity matrix, and E T the
residuals matrix for residuals that are due to random errors and model inadequacies. 2 7,3 0
The sensitivity matrix S that is necessary for prediction
of unknown sample concentrations is determined via calibration steps, in which a set of standards is analyzed. 3 0
To build the calibration m odel, we used the 634, 661,
750, 858, 1113, 1348, 1527, 2734, and 2952 cm 2 1 bands,
and seventeen standards in the range of 0.053.75 M. The
selection of the most signi cant bands was performed
through the for ward and backward selection procedures
by the multiple regression module of the Statistica Software from StatSoft Inc.
For band intensity (BI) measurements the calibration
model equation was
C 5

2 (0.01 6
2

0.05) 1

(0.09 6

(0.24 6

0.02) 3

BI 29 5 2

0.03) 3
(r 5

BI 85 8
0.998)

and for band area (BA) measurements was

C 5
2

2 (0.03 6
(0.008 6

0.05) 1

(0.024 6

0.002) 3

BA 29 52

0.002) 3
(r 5

BA 8 58
0.998).

Band intensities and band areas were calculated with

a two-point baseline correction using the range 850 865
cm 2 1 for the 858 cm 2 1 band and 29462958 cm 2 1 for the
2952 cm 2 1 band.
The model vs. actual scatter plots are shown in Fig. 2,
while the residuals plots along with the root m ean square
error (RMSE)3 0 are shown in Fig. 3. Form ulation analysis
is achieved without sample dilution, minimizing manual
handling of toxic samples. Results of the analysis of the
comm ercial formulations based on band intensity and
band area measurements are presented in Table IV. As
shown by the experimental t-values, results obtained by
the proposed m ethod are identical to those acquired
through the reference method 1 4 for all formulations using
either band intensity or area measurements. Relative differences between results obtained through the FT-Raman
and the reference method are in the range of (2 7.9%)
(6.6% ) and (2 2.2%)(6.3%) for band intensity and band
area measurements, respectively.
CONCLUSION
The proposed analytical method based on m ultivariate
calibration using band intensity or band area measurements is accurate and precise. The m ethod is safe, as it

eliminates sample pretreatment and keeps handling of

toxic samples to a minimum. The proposed m ethod is
also fast and simple so it can be perform ed by m inimally
trained personnel. Multivariate calibration based on stepwise linear regression was found to be superior to univariate calibration in terms of concentration prediction.
Norm alization using cyclohexane as the external standard
improves the long-term calibration stability. The FT-Raman method can be used to replace existing spectroscopic
and chromatographic m ethods that require extensive
manual sample handling and is faster. The advantages of
the FT-Raman technique can be further exploited by application to other pesticide formulations.
ACKNOWLEDGMENT
We gratefully acknowledge support, in the form of a scholarship to
S.G.S., from the Greek State Scholarship Foundation.
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