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The Resolution of Dye

Binary Mixtures by
Bivariate Calibration Using
Spectrophotometric Data
a

P. L. Lpez-de-Alba , L. Lpez-Martnez , K.
a

Wrbel-Kaczmarczyk , K. Wrbel-Zasada & J.

Amador-Hernndez

Instituto de Investigaciones Cientficas ,

Universidad de Guanajuato , 36000, Mxico
Published online: 22 Aug 2006.

To cite this article: P. L. Lpez-de-Alba , L. Lpez-Martnez , K. WrbelKaczmarczyk , K. Wrbel-Zasada & J. Amador-Hernndez (1996) The Resolution
of Dye Binary Mixtures by Bivariate Calibration Using Spectrophotometric Data,
Analytical Letters, 29:3, 487-503, DOI: 10.1080/00032719608000413
To link to this article: http://dx.doi.org/10.1080/00032719608000413

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ANALYTICAL LETTERS, 29(3), 487-503 (1996)

THE RESOLUTION OF DYE BINARY MIXTURES BY BIVARIATE

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CALIBRATION USING SPECTROPHOTOMETFUC DATA

Key Words: Bivariate Calibration, Dye Mixtures, Spectrophotometry

Ldpez-de-Alba, P.L. *, Ldpez-Martinez, L., Wrdbel-Kaczmrczyk, K.,

Wrdbel-Zasada, K., and Amador-Herndndez, J.
Instituto de Investigaciones Cientificas, Universidad de Guanajuato, 36000
Mexico

ABSTRACT

A new, simple spectrophotometric method for resolution of dye binary mixtures is

proposed in this work. A simple mathematical algorithm was designed, in which the data
are used from four linear regression calibration equations: two calibrations for each
component at two selected wavelengths. The method of Kaiser was applied for the
selection of the optimum two-wavelength sets for all mixtures under study. The recovery
experiments were carried out in ten mixtures of the following dyes: Tartrazine, Amaranth,
Erythrosin B, Sunset Yellow and Allura Red. The obtained results were compared with
the results of a commonly used derivative spectrophotometric procedure (zero-crossing
technique). The statistical evaluation of the method bias was performed and it was
concluded that the proposed methodoloa may be competitive with the derivative
procedure for the resolution of such dye binary mixtures.

* Author to whom the correspondence should be addressed.

487
Copyright @ 1996 by Marcel Dekker, Inc.

4xx

LOPEZ-DE-ALBA ET AL

INTRODUCTION
Several natural and artificial dyes are widely used as alimentary additives. For
many commercial products, two or more different dyes are added and legislation exists
to control the quality of such dyes and their content in the alimentay products'**.Thus,

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analytical procedures are needed to rapidly and reliably determine the dyes in food
products. Spectrophotometric methods are most frequently used for such purposes. For
the simultaneous spectrophotometric determination of two sample components, the choice
of an analytical procedure is strictly related to the observed resolution between the
individual absoi-ption peaks of these components. Such a determination is not
problematic, if the absoi-ption peaks are satisfactorily resolved, but if the individual
component signals are partly or totaly overlapped, then chemometric techniques are
needed. There exist several powerful, well established multivariate calibration techniques
which can be used in the modem spectrophotometry: classical least squares (CLS),
inverse least squares (ILS). pi-incipal component regression (PCR) or partial least squares
(PLS)'". These techniques have been widely used for the simultaneous determination of

the two or more components of the sample and excellent analytical results have been
reported. On the other hand, derivative spectrophotometry has also been satisfactorily
applied for such kind of analyses6-'*. However, most of the techniques mentioned above
require full-spectrum infonnation and the spectral data have to be processed using highly
specialized software. In many cases, especially for dye mixtures, there is no need to use
such sophisticated and expensive techniques.
In the present work a new, simple procedure is proposed for the resolution of
binary dye mixtures. The recovery experiments results are compared with the results
obtained by commonly used derivative speckophotometric procedure and the method bias
is evaluated.
OUTLINE OF THE BIVARIATE METHOD PROPOSED

ln the ideal (error-fiee conditions), the absoiption of t h o component mixture (A and

B) at one chosen wavelength may be described accoiding to the Lambert-Beer law, by

the equation

DYE BINARY MIXTURES

A,=

489

Eibc,+ E;bc,,

(1)

where: Am is the absorbance value of the mixture at this wavelength; E,

E, are molar

absorption coefficients of components A and B at this wavelength; c,, c, are the molar
concentrations of both components and b is the optical path length.
However, in real conditions, when the individual responses A, and A, are affected by

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the analyt~caland measurement errors, the calibration curve formulas for each component
at one selected wavelength (Ai), are:

A,

where: m,,

= mAi'C,

+ eAi

(2)

mB,are the slope values of linear regressions; C,, C, are the concentrations

of both components (for practical reasons the concentration units of mg L-' were used in

this work) and e,,

e,, are the intercept values, which reflect the differences between the

model and the real system.

If the measurements of the binary mixture are performed at two selected wavelengths
( 1 and 2), we have two equations:

where eml, e,,are

=

the sum of the intercepts of linear calibration at two wavelengths (e,,

eAi+ %,). The resolution of such equations set allows the evaluation of,C

values:

an4C

LOPEZ-DE-ALBA ET A L

400

This simple mathematic algorithm allows the resolution of the binary mixture by
measuring the absorbance of the mixture at two selected wavelengths and using the
parameters of the linear regression functions evaluated individually for each component
at these same wavelengths
In such a procedure the problem arises of which set of wavelengths should be

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selected to assure the best sensitivity and selectivity of the determination. In this work,
the method of Kaiser" was applied for the selection of the optimum wavelengths. A
series of sensitivity matrices, K, was created for each binary mixture:

where mA1,m, are the sensitivity parameters of the component A at two selected
wavelengths (1, 2) and mRI,mRZare these parameters for the component B. It was decided
to use the values of the linear regression calibration slope evaluated for one component

at h, as the sensitivity factor. The determinants of these matrices were calculated and the
obtained values were used as the optimization criterion: the wavelength set selected was
the one for which the highest matrix determinant value was obtained.
All calculation were performed using a simple GWBASIC program.
EXPERIMENTAL

Appurutus

A Spectronic 3000 Diode Array Milton Roy spectrophotometer with 0.35nm

resolution was used which was coupled to a 486 PC and User data Version 2.01 Milton
Roy Inst. Co software for spectral data acquisition, storage and manipulation. All data
treatment operations were carried out using an Hewlett Packard Vectra 486/66 VL
microcomputer equipped with the GRAMS/386 tm software package, version 3.01A
(Galactic Ind. Co., Salem, USA)

DYE BINARY MIXTURES

49 1

Reagents

All chemicals were of analybcal-reagent grade.

The dyes: Tartrazine (FD&C Yellow-5, YS), Amaranth (FD&C Red-2, R2) and
Erythrosin B (FD&C Red-3, R3) were obtained &om Aldrich Chemical Company and the

dyes: Sunset Yellow (FD&C Y6) and Allura Red (FD&C R40) were from "Quimica

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I.R.S.A. de Mexico".
The buffer solution (2molL-', pH 4.6) was prepared by mixing acetic acid and
sodium acetate in molar ratio 1:1 and adjusting pH with hydrochloric acid lmol.L-'.
Stock solutions containing 250mg-L-'of the dye were prepared. These solutions
were stable approximately one month14,the working solutions were prepared daily by
appropriate dilution.
Pure water of Milli-Q class (Labconco) was used throughout.

Procedures

A series of the dye solutions in the concentration range from 1 to 22 mg L-' were

prepared for one-component calibration.

All possible binary mixtures of the five dyes selected were studied: YS-Y6, Y5R2, YS-R3, Y5-R40, Y6-R2, Y6-R3, Y6-R40, R2-R3, R2-R40 and R3-R40. For each
mixture a series of ten solutions were prepared, which contained different concentrations

of the components (in the range 2 to 22 mg L" with the exception of R3, for which the
range of 2 to 12 mgL-' was used).
The spectra of all solutions were registered in the spectral range 350-65Om using
the buffer solution (5ml of 2M buffer solution diluted to 25mL) as the reference. The
absorbance values at selected wavelengths were obtained from these spectra.
First derivative spectra were calculated (AA=8.75nm) from the smoothed spectra
(25 experimental points) using the Savitsky-Golay procedure',.
RESULTS AND DISCUSSION

The individual absorption spectra of five dyes under study are presented in Fig.
1. As can be observed, these spectra are partly overlapped (A,

= 427.5nm,

492

LOPEZ-DE-ALBA ET AL

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0.6

0.4

0.2

0.0
350

415

480

545

610

Wavelength, nm
Figure 1.

The absoipfion spectra of 1 - Tartraztne (Y5, 14mg L 'j, 2- Amaranth (R2.

ISmgL'), 3 - Erythrosm B (R3, 7mgL'), 4 - Sunset Yellow (Y6, 14mgL'),
5 - Allura Red (R40, 141ngL') with the buffer solution as a blank

482.0nm,

522.0nn-1,Lac: = 527.5nm,

= 499.0nm)

and the resolution of

binary mixtures using conventional methods is impossible.

The analytical characteristics for one-component determination at wavelengths
corresponding to the absorption maximum were evaluated for Y5, Y6, R2, R3 and R40
and the obtained results are given in Table I . For the binaiy mixtures studies, the
concentration range for each dye was taken according to the linear range of the individual
calibration function.
The two wavelengths sets for the proposed bivariate procedure were selected using

the method of Kaiser. Nine wavelengths were chosen and the ( m , , A- component, i

wavenumber) slope values of the linear regression calibration were estimated for five

DYE BINARY MIXTURES

493

Table 1. The sensitivities evaluated for Y5 and R2 determination in one-component solutions at

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nine selected wavelengths (q-the slope value of linear regression calibration of i-component),

A.

m,,E-3

420

43.68

7.37

427

44.26

7.85

43 7

43 30

8.84

449

38 90

10.39

474

20 28

18.69

49 1

6 40

26.43

511

5.59

33.18

521

1.65

34.34

53 1

1.60

33.38

m,E-3

Table 2. Application of the method of Kaiser for the selection of the best wavelengths set: the
absolute values of determinants of sensitivity matrices (K).

a\A.
420
427

43 7
449
474
491
511
521
53 1

420

427

437

449

474

491

511

521

531

17

67

167

667

1107

1408

1488

1446

51

I54

668

1120

I425

1507

1465

106

630

1088

1387

1472

1431

516

962

1233

1319

1282

416

568

666

647

65

176

171

137

134

0.1
0

494

LOPEZ-DE-ALBA ET AL.

Table 3. The analytical characteristics for the dye determination in a one-component solution at

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Imax

Detection Limit

I inearity Range

(mgL.')

(mg.L-')

Y5

2 5E-2

2 - 22

0 9999

Y6

16E-2

2-22

0 9999

R2

3 1E-2

2 - 22

0 9997

R3

6E-3

2 - 12

0 9999

R40

2 OE-2

2 - 22

0 9999

Dye

12 (a=o 05)

Table 4. The selected two-wavelength sets for the binary mixture resolution by the bivariate
method proposed

Binary Mixture

1,(nm)

Y5 - Y6

427 5

494 1

Y5-R2

427 5

521 8

Y5 - R 3

427 5

527 5

Y5 - R40

449 3

484 7

Y6-R2

477 7

531 8

Y6 - R3

492 7

522 5

Y6 - R40

476 6

534 9

R2 - R3

516 7

527 6

R2 - R40

498 8

508 8

R3 - R40

493 8

527 5

1 2

495

DYE BINARY MIXTURES

Table 5 . Linear regression calibration formulas used for the bivariate algorithm.

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Calibration Equations
Binary Mixture

Component
J.1

J.2

Y5

A = 0.0442.C + 0.0023

A = 0.0049C + 0,0018

Y6

A = 0.0203C + 0.0002

A = 0.0434.C + 0.0049

Y5

A = 0.0442.C + 0.0023

A = 0.0020C + 0.0016

R2

A = 0 0079.C + 0.0005

A = 0.0343.C + 0.0029

Y5

A = 0.0442.C + 0.0023

A=O

R3

0.0028.C - 0.0008

A = 0.0886.C - 0.0070

Y5

0.0389.C + 0.0022

A = 0.01 1OC + 0.0017

R40

A = 0 . 0 1 7 4 C - 0.0014

A = 0 . 0 3 7 5 C +0.0013

Y6

A = 0.0448.C + 0.0030

A = 0.0105C + 0.0003

R2

A = 0.0203.C + 0.0018

A = 0.0332.C + 0.0037

Y6

A = 0.0438.C + 0.0049

A = 0.0226C + 0.0020

R3

A = 0.0292.C - 0.0039

A=0.0819C - 0.0068

Y6

A = 0 0445.C + 0.0029

A = 0.0082C + 0.0002

R40

0.0324.C - 0.0002

A = 0.0293.C + 0.0290

R2

A = 0 0340.C + 0.0026

A = 0.0340C + 0.0033

R3

A = 0.0652.C - 0.0062

A = 0.0886.C - 0.0070

R2

A = 0 0295.C + 0.0022

A = 0.0326.C + 0.0022

R40

A = 0 0408.C + 0.0102

A = 0.0384.C + 0.0193

R3

A = 0.0298.C - 0.0041

A = 0.0866.C - 0.0071

R40

A = 0.0403C + 0.0074

A = 0.03 14.C + 0.0344

Y5-Y6

Y5-R2

Y5-R3

Y5-R40

Y6-R2

Y6-R3

Y6-R40

R2-R3

R2-R40

R3-R40

LOPEZ-DE-ALBAET AI

496

Table 6. Recovery results for individual dyes in their binary mixtures obtained using the bivariate
method
Average Recovery

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Binary Mixtures

First Component

Fi SD, %
Second Component

*07

Y5

Y6

900kO5

100 2

YS

R2

982k04

978* 1 3

YS

R3

994i06

993*23

YS

R40

966i09

98Si09

Y6

K2

100 5 + 0 7

950k32

Y6

R3

101 2 * 0 5

101 4 * 1 9

Y6

R40

1 0 2 6 * 10 1

868*83

R2

R3

969*06

976i22

R2

R40

897i73

1010173

R3

R40

S82536

1040i52

dyes at these nine wavelengths (see Table 2, where the results obtained for Y5 and R2 are
presented as an example). Using the obtained data, the sensitivity matrices were created
for each mixture and the respective determinants were calculated. The sensitivity results
obtained for the mixture of Y5 and R2 are presented in Table 3, where it can clearly be
seen that the wavelengths 427 nm and 52 I nm should be used in the bivariate procedure.

The two-wavelength sets evaluated for all dye mixtures under study are given in Table
4. At these selected wavelengths the one-component calibration curves were obtained. For

the linear response range, in each case, the linear regression calibration function (9>
0.9990) was calculated and q,e, values were taken for the bivariate algorithm (Table 5).

Then, the resolution of the binary mixtures was performed. For each mixture type, the
recovery experiments were carried out in ten solutions containing the two components in
varying concentration ratios (from 1 : 1 1 up to 1 1: I , and the mixtures containing R3 from
1.6 up to 6 : l ) . In Table 6 the obtained results are presented, where each value was

calculated as the average recoveiy of one component in ten binary solutions prepared. As

DYE BINARY MIXTURES

497

0.008

0.004

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0.000

-0.004

-0.008

-0.012
350

41 5

480

545

610

Wavelength, nrn
Figure 2.

The first-derivative spectra of. 1 - Tartrazine (Y5, 14mgL"), 2- Amaranth

(R2, 18mgL-'), 3 - Erythrosin B (R3, 7mgE ), 4

- Sunset

Yellow (Y6,

14mgL-'), 5 - Allura Red (R40, 14mgL ) with the buffer solution as a

blank.

can be observed in tlus Table, satisfactory results (recoveries in the range 88 -104%) were
obtained for all binary mixtures.

In further development these same binary mixtures were resolved using the first
derivative spectra (the derivative spectra of all five dyes are given in Figure 2). The zerocrossing measurement method was applied. The selected wavelengths and the formula of
caIibration function for each component in ten mixtures studied are presented in Table
7. Using the equations obtained, those same recoveiy experiments were repeated, as in
the bivariate procedure. The obtained results are presented in Table 8. In this case, poorer

4Y8

LOPEZ-DE-ALBA ET AL

Table 7. The calibration formulas for the individual dyes in their binary mixtures obtained using

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the zero-crossing method for the derivative spectra.

Binary Mixture

Component

h(nm)

Calibration Equation

Y5

482 7

Y6

536 8

'D = -2.417E-4.C

0 9994

Y5

377 0

ID = 2.190E-4.C - 2.7E-6

0 9996

R2

572 9

'D

= -3

Y5-Y6

Y5-R2
Y5

382 7

R3

539 3

Y5-R3

'D

'D

047E-4.C + 2.88E-5

-2.266E-4.C - 8.08E-5

=2

110E-4.C - 5.7E-6

'D=-I.5416E-3.C + 1.288E-4
ID = -1 240E-4.C

+ 8.1E-6

?(a=0.05)

0 9998

0 9995
0 9996
0 9999

Y5

49s 8

R40

553 2

ID = -2.692E-4.C - 4.080E-4

0 9924

Y6

522 I

'D

- 6.66E-5

0 999s

R2

575 5

ID = -2 239E-4.C - 8.55E-5

0 9994

Y6

572 2

ID = -4.689E-4.C-6 11E-5

0 999s

R3

482 7

ID = 3.587E-4.C-4 85E-5

0 9993

Y6

49s 8

'D=-9.713E-5.C -1.96E-5

0 9980

R40

575 4

ID = - I 2 18E-4.C + 2.44E-5

0 9981

R2

576 9

ID = -2 21 1E-4.C - 4 11E-5

0 9994

R3

522 I

ID = 7.717E-4.C - 4.09E-5

0 9997

R2

498 8

ID = 1.361E-4.C - 8.8E-6

0 9991

R40

522 1

ID = - 1 2 1 OE-4.C

R3

49s 8

ID = 2.441E-4.C - 5.30E-5

0 9989

R40

527 + 572

ID = -2.709E-4.C - 3 65E-5

0 999s

Y5-R40

Y6-R2

= -5.143E-4.C

0 9987

Y6-R3

Y6-R4O

R2-R3

R2-R40

R3-R40

+ 9.09E-5

0 9967

499

DYE BINARY MIXTURES

Table 8. Recovery results for the individual dyes in their binary mixtures (n = 10) obtained using
derivative spectrophotometricmethod.
Average Recovery

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Binary Mixture

Ti SD, %

First Component

Second Component

Y5

Y6

93.4 f 1.5

104.9 f 3.1

Y5

R2

96.6 f 3.3

90.3 f 7.2

Y5

R3

99.4

f 2.3

97.3 f 1.0

Y5

R40

81.0 f 22.3

97.6 f 4.0

Y6

R2

98.9 f 0.6

96.7

Y6

R3

980+15

105 1 f 1 8

Y6

R40

104 8 f 49 6

1089f9 1

R2

R3

1 1 2 9 1 13 1

985519

R2

R40

1077* 1 6

117 0 f 20 8

R3

R40

1129f166

I04 0 f 5 9

* 8.7

average recoveries were obtained (in the range 80-1 13%) as compared with the results

of the bivariate procedure (see Table 6).

To study the observed differences in more detail, the individual recovery results
obtained in ten solutions (containing different dye ratios) were considered for each binary
mixture under study. In Figure 3 the relationship is given between the real and the found
concentrations of Y5 and R40 in their mixture. As can be seen, much better results were
obtained for each composition of the mixture using the bivariate procedure (Fig.3a) than
using common derivative procedure (Fig.3b).
The obtained results suggest that the proposed bivariate procedure could be
competitive to the derivative spectrophotometric method in applications involving the
resolution of dye binary mixtures. The evaluation of method bias was camed out using
statistical tests. The F- and T- tests were performed for the results obtained for each
component in each mixture, and the difference between the results obtained in two

Downloaded by [Michigan State University] at 16:37 04 February 2015

500

LOPEZ-DE-ALRA 6 T AL

T
I!

Figure 3.

1'2

16

The relation between the real and the found concentrations of Y5 and R40
in their mixtures: (.4)
bivariate procedure
(B) first derivative procedure.

procedures tested were considered statistically significant only in the case when for both
components the t orf value was higher than the theoretical valueI3. First the F-test was
performed, andfq

<Aeor( a=0.05) were found for the mixtures:

Y6-R2, Y6-R3, Y6-R40,

Y2-R3, Y2-R40, R3-R40, while for the mixtures: Y5-Y6, YS-W, Y5-R3, Y5-R40,

signifigant differences in precision were detected between both procedures under study.
Better precision is obtained using the bivariate procedure, (see Tables 6 and 8). For the

DYE BINARY MIXTURES

real

cY.5 , rug.L-'

12

16

50 1

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-0

12

1;1
- 12

3
9
c

4 -

- It5

real

I
12

I
16

cR,,, mg.~-'

..
#

Figure 3. Continued

former group of mixtures, the parametric T-test was applied and for the latter group the
non-paramehic test of Wilcoxon was applied. The results obtained indicate that for the
mixtures Y5-Y6 and Y6-R3 there exists statistically signifigant differences between
results obtained by the two procedures tested (texp> t,e,n). Better recoveries are obtained
by the bivariate procedure (see Tables 6 and 8).

CONCLUSIONS
In this work, a new bivariate procedure was proposed for the resolution of the twocomponent mixtures of dyes. The results of the recovery experiments carried out in ten

LOPEZ-DE-ALBA ET AL.

502

binary mixtures and the results of the statistical evaluation of method bias indicate that
this procedure may be competitive and, in some cases, even superior to commonly used

first derivative spectrophotometricprocedure as applied to the resolution of the binary

mixtures of the dyes. Simplicity is an important advantage of the presented procedure.
There is no need for full-spectrum information and no spectral data processing is

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required.
ACKNOWLEDGEMENTS

The authors gratefully acknowledge financial support from the CONACyT

(MCxico), project 3 179-E9307.
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(1980)

2. R. L. Hall, Flavor Research and Food Acceptance, Reinhold Publishing Co., New York
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3. P. M. Fredericks, J. B. Lee, P. R. Osborn and D. A. J. Swinkels, AppZ. Spectrosc., 39,303
(1985).
4. D. Haaland and E. V. Thomas, Anal. Chem., 60, 1193 (1988)
5 . D. Haaland and E. V. Thomas, Anal. Chem., 60, 1202 (1988).

6. F. Salinas, A. Espinosa-Mansilla and P. L. Lopez-de-Alba, Analius, 22, 188 (1994).

7. P. L. Lbpez-de-Alba and L. Lopez-Martinez, 1 Rudiounul. NucZ. Chem., Letters 164 (5), 309

(1992).

8. J. A. Murillo, J. Rodriguez, J. M. Lemus and A. Alaiion, Analyst, 115, 1117 (1990).

DYE BINARY MIXTURES

503

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Received: 22 August, 1995

Accepted: 20 October, 1995