ANALYTICA CHIMICA ACTA

ELSEVIER Analytica Chimica Acta 345 (1997) 87-98

Application of micellar effects to the simultaneous kinetic determination of pyridoxal and pyridoxal-5’-phosphate

Francisco Morales, Dolores Sicilia, Soledad Rubio, Dolores Pkrez-Bendito*

Department of Analytical Chemistry Faculty of Sciences, University of Cbrdoba, Cdrdoba, Spain

Received 19 September 1996; received in revised form 24 January 1997; accepted 3 February 1997

Abstract

A method for the simultaneous kinetic determination of pyridoxal (PAL) and pyridoxal-S-phosphate (PALP) based on their cyanide-catalyzed aerobic oxidation in the presence of cetyltrimethylammonium bromide (CTAB) micelles is developed. The fluorescent reaction products yielded from PAL-cyanide and PALP-cyanide reactions [4-pyridoxolactone (PL) and 4-

pyridoxic acid S-phosphate (PAP), respectively] concentrate on the CTAB micellar surface and this ‘local concentration’ effect increases their quantum yield which results in apparently increased reaction rates. Because of the different partition or ‘binding’ constants of PL and PAP to CTAB (&,=50&6 M-’ and KpAp= 13901tlO M-l), this micellar medium accelerates

PALxyanide and PALP-cyanide systems in a different extent leading to the kinetic discrimination of both vitamers. PAL and

PALP are determinated simultaneously over the concentration range 1 x 1 O-* -6x 10m7 M with relative errors less than about

5%. The role of CTAB micelles on the studied systems is discussed.

Keywords: Multicomponent kinetic methods; Micelles; Pyridoxal, Pyridoxal-5’.phosphate

1. Introduction

Multicomponent kinetic determinations have aroused increased interest in recent years [l]. Inves- tigations have been fundamentally addressed to reach

mathematical discrimination (digital computers offer

the possibility of rapid data acquisition and processing thus permitting the use of complex chemometric methods) or instrumental discrimination (multidetec- tion devices such as diode array systems allow signal- time-wavelength data to be obtained simultaneously

and this possibility has fostered the development of procedures that benefit from both kinetic and spectral

*Corresponding author. Fax: 957218606.

0003-2670/97/L% 17.00 1x1 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(97)00107-4

differences). Recently, micellar aggregates have been proposed for chemical discrimination in multicompo-

nent kinetic determinations and, although the results have so far been highly promising, there remains much to be explored in this field [2,3].

Reagent organization, and the different microenvir- onments provided by, in micellar media may modify

reaction kinetics thus facilitating multicomponent determinations. Micelles can alter the apparent rate

constant of two or more species that interact with a

common reagent by both altering their intrinsic reac-

tivity and, more generally, binding in a different extent

to analytes. Likewise micelles can control pathways

leading to specific products or the stereochemistry or

mechanism of some reactions. On the other hand.

88 F: Morales et al. /Analytica Chimica Acta 345 (1997) 87-98

microenvironment dependent physicochemical prop-

erties such as absorptivity, equilibrium constant, spec- tra profile, fluorescence quantum yield, fluorescence

depolarization, quenching effects, etc, can be drasti-

cally affected in the micellar medium compared to the bulk solvent. All of these effects can be conveniently exploited for kinetic multicomponent determinations.

To our knowledge only four simultaneous reaction rate methods have been implemented by using

micelles as a reaction medium [4-81. Discrimination has been achieved in these methods using different

micellar effects (e.g. micellar inhibition [4], differen- tial micellar catalysis [5,6], control of reaction path-

ways leading to specific products and spectral shifts [7,8]) which gives an idea of the versatility of these organic microheterogeneous systems. Since all devel- oped methods have involved inorganic analytes it

would be interesting to explore the analytical potential of micelles in organic multicomponent kinetic deter- minations where the similar reactivity of analytes

precludes frequently their resolution by using conven- tional differential kinetic methods.

This paper is the first in a series reporting a sys-

tematic study on the analytical potential of micelles in

organic multicomponent kinetic analysis. For this purpose the resolution of simultaneous kinetic pro-

cesses unsolved by conventional differential kinetic methods will be attempted. In this paper the influence

of cationic micelles of cetyltrimethylammonium bro- mide (CTAB) on the determination of the B6 vitamers pyridoxal (PAL) and pyridoxal-S-phosphate (PALP), based on their cyanide-catalyzed aerobic oxidation, is

reported. This reaction has been frequently used to derivatize PAL and PALP, analytes of great signifi- cance in clinical chemistry, since the reaction products

yielded [4-pyridoxolactone (PL) and 4-pyridoxic acid

5phosphate (PAP) from PAL and PALP, respectively] are highly fluorescent and stable [9, lo]. Both reactions

occur under a slightly basic medium (7.1-7.3), how- ever the maximal fluorescence intensity of the reaction products is obtained at very different pH values (a slightly basic and an acid medium for PL and PAP, respectively) and their maximal excitation wavelength differ about 40 nm (360 and 320 nm for PL and PAP, respectively). These characteristics of the reaction products hinder the simultaneous resolution of PAL and PALP at the levels of concentration present in serum (between about 5 and 45 ng ml-’ [ 11,121) and,

so procedures based on this reaction require two

measurements performed at a slightly basic and an acid medium, respectively [13,14]. Other methods developed for determining both analytes involve also

two sample aliquots, PALP being quantified after enzymatic conversion in PAL [ 15,161, or the use of separation techniques, fundamentally HPLC [ 171.

Many of these methods use derivatization with cya- nide in the determination step.

Because of the different micellar effects on reaction kinetics, these aggregates were used here with the aim

of determining simultaneously PAL and PALP. In order to elucidate the mechanism via which the sur- factant acted on PAL-CN- and PALP-CN- reactions several parameters were calculated, namely, the true

second-order rate constant of the reactions taking place in the micellar pseudo-phase (k,), the binding constant of reactants and reaction products to the micelles (KS) and the fluorescence quantum yield of reaction products in the micelle (4).

2. Experimental

2.1. Apparatus

Fluorimetric measurements were made on a Perkin-

Elmer fluorescence spectrophotometer, model MPF- 43A, fitted with 1 cm cells and xenon-arc source. The

spectrofluorimeter cell compartment was thermo- stated by circulating water through it. All measure- ments were made by using excitation and emission

slits of 5 and 7 nm spectral bandpass, respectively. A series of six fluorescent polymer samples purchased from Perkin-Elmer was used daily to adjust the spec-

trofluorimeter to compensate for changes in the source intensity. A classical stalagmometer was used for

surface tension measurements in order to determine the critical micelle concentration (c.m.c.) of the sur-

factant.

2.2. Reagents

All reagents used were of analytical-reagent grade and were employed as supplied. Bidistilled water was used throughout. Stock PAL and PALP solutions (1.0x lo-* M) were prepared by dissolving 0.0509 and 0.0663 g of pyridoxal hydrochloride (Aldrich)

E Morales et al. /Analytica Chimica Acta 345 (1997) 87-98 89

and PALP (Merck), respectively, in 25 ml of

I .0x 1 0m2 M hydrochloric acid. These solutions were stable at 4°C. protected from light, for at least one week. More dilute solutions were made from these

stock solutions prior to each set of experiments by dilution with bidistilled water. Aqueous solutions of

potassium cyanide (1.5 x lo-* M, Merck), CTAB (0.2 M, Serva) were also prepared. The buffer solution

used consisted of 0.9 M KH2P04 (pH 7.1). PL was obtained by acid hydrolysis from pyridoxic acid

(Sigma); a sample of pyridoxic acid (1.0~ lo-* M) in 1 M HCl was incubated at 37°C during 24 h. PAP

was synthesized by irradiation of PALP

( 1.0x 10 --* M), in HCl 1 M and in the presence of oxygen, with light of wavelengths longer than 300 nm

[181.

2.3. Procedure for the simultaneous resolution of bina? mixtures of PAL and PALP

In a 10 ml standard flask were placed, in sequence,

2.0 ml of 0.9 M phosphate buffer (pH 7.1), 0.8 ml of 0.2 M CTAB, appropriate volumes of analytes to give a final PAL and PALP concentrations between 0.01

and 0.6 uM and 0.2 ml of 1.5 x 1 O-* cyanide. Reagents were kept at 25fO. 1 ‘C to obtain maximum precision.

The stopclock was then started and the mixture was

diluted to the mark with bidistilled water. An aliquot of this reaction mixture was transferred to a cell kept at 60&O. 1 ‘C and the relative fluorescence intensity (X,,,/X,,=340/435 nm) was recorded as a function

of time. Measurements were started exactly 1 min after the addition of cyanide. Blank solutions were prepared like the samples but contained no analytes; their signals were subtracted from those obtained for

the samples. The analyte concentrations in each mixture were

calculated by measuring both the initial rate (v) and

the relative fluorescence intensity increment for the

reaction between t=O (initio of the recording of the kinetic curve) and t=l h (AZ,). The dependence of v

and AIF on the concentration of the analytes in the binary mixtures was found to conform to the following equations:

where X] and X2 are the concentrations of the analytes expressed as micromoles per liter. Parameter v was expressed as relative fluorescence intensity per min-

ute. The coefficients PO, pi, ,& @,, 0; and ,!$ were estimated by multiple linear regression (MLR) from

11 binary mixtures containing PAL and PALP in the above-mentioned ranges using a laboratory developed

FORTRAN 77 program. The expression of MLR for Eqs. (1) and (2) in matrix notation is

y =x/I+&.

where y is the measurement vector (AZ, or v), e the residual vector, /I the parameter vector, and X the

independent variable matrix. The least-squares esti- mate of j?, b, was obtained from

b = (X’X)_‘X’y,

where X’ is the transpose of X and (X/x)-’ the inverse

of X’X. Once the estimates of coefficients were obtained, unknown samples were analyzed by sub- stituting the measured parameters v and Alr into Eqs. (1) and (2) and calculating the analyte concen-

trations by using a laboratory-developed FORTRAN 77 program.

2.4. Kinetics

Kinetic data for the cyanide-catalyzed aerobic oxi-

dation of PAL and PALP in the presence of CTAB, were obtained by using spectrofluorimetric detection. In order to calculate the observed first-order rate

constants, kobs (s-l), as a function of CTAB concen-

tration for these reactions, several solutions were prepared in 10 ml volumetric flasks. To each flask

were added, in sequence, 2.0 ml of 0.9 M phosphate buffer (pH 7.1), appropriate volumes of 0.2 M CTAB

solution to give a final concentration between 0 and

1.6x lop2 M, 0.5 ml of 1.0x lo-” M PAL or PALP, 0.2 ml of 1.5x lop2 M cyanide and bidistilled water to

the mark. An aliquot of the reaction mixture was transferred into a thermostated cell at 60fO.l ‘C and the fluorescence intensity (X,,,/X,,,=340/435) recorded as a function of time. The observed first-

order rate constants (kobs) were calculated from linear plots of In (l, - It) vs. time.

The model used to analyze kinetic data was essen- tially that developed by Berezin et al. [ 191 where the

90 E Morales et al. /Analytica Chimica Acta 345 (1997) 87-98

chemical reaction is assumed to take place in two pseudo-phases, one associated with the micelle and

the other with the bulk solvent. According to this

model the experimental second-order rate constant, k,+,, of a reaction between species A and B can be expressed as

k~ = (kmPAPB + k;PA + k;PB)CV + k,( 1 - CV)

(1 + KAC)(l + KBC) ’

(3)

where k, and k, are the rate constants of the reactions

in the micellar and aqueous phase, respectively; tim the rate constant of the reaction resulting from collisions

between reactant A in the micellar phase and reactant B in the aqueous phase; and ti; that for the reaction between reactant B in the micellar phase and reactant

A in the aqueous phase. PA and PB are the partition coefficients for the reactants (e.g. PA=[AIMIIAlw); C is the surfactant concentration (molar&y) minus the

critical micelle concentration; V is the molar volume of the surfactant; factors CV and (1 -Cv) are the

volume fractions of the micellar and aqueous phase, respectively; and KA and KB are the binding constants

for the reactants, viz. KA = (PA - l)V and KB = (PB - l)V.

Since no photometric or fluorimetric spectral evi- dence of association of PAL or PALP to CTAB

micelles was obtained, kinetic data were analyzed considering that cyanide is in the micellar phase and the analytes in the aqueous phase. Assuming that the volume fraction of the micellar phase is small and

therefore CVcl and PV>V, Eq. (3) can be simplified

to

k li,

= k, + K,K~~[[CTAB] - c.m.c.1

1 + Ko[[CTAB] - c.m.c.1 ’ (4)

where KCN is the corresponding association constant

of cyanide to CTAB micelles. Eq. (4) is not in a particularly convenient form for analysis of kinetic data and it is usually rearranged to give

1 1 1 1 p= k+ - k, k:, - k, + k& - k, KcN[[CTAB] - c.m.c.1

(5)

which is in an attractive form because a plot of l/ (k*-k,) against l/([CTAB]-c.m.c.) should be linear, and allow the calculation of both Z& and KCN. Experi- mental second-order rate constant values as a function

of CTAB concentration (k,,,, 1 mol-’ s-l) were calcu- lated from k&[CN-1. The second-order rate con-

stants for the two reactions considered in the

aqueous medium (k,) were estimated from the kinetic curves obtained in the absence of DTAB.

2.5. Procedure for the determination of micellar

binding of the reaction products

The binding constants of PL and PAP to CTAB micelles (KS) were determined spectrofluorimetrically

[20-227 from the changes (hyperchromic shifts) induced by the cationic surfactant on the fluorescence

emission spectra of these reaction products. A series of solutions (10 ml total volume) containing a fixed concentration of PL or PAP (5 .O x 1 0e7 M) and various concentrations of CTAB (between 2.0x 10p4- 3.0~10-~M) at the working pH (7.1, adjusted with 0.18 M phosphate buffer) was prepared. An aliquot of each solution was transferred into a 1 cm quartz cell

thermostated at 60fO. 1 “C and the fluorescence inten- sity measured at X,,,/X,,=360/435 and 3201430 nm for PL and PAP, respectively. Binding constants were

calculated from the expression

[(k) - l]_l=[@) -I]-’

1

’ + yK,([CTAB] - c.m.c.) ’ (@ 1 where I is the fluorescence intensity of the systems at each concentration of CTAB, I0 is the fluorescence of the system in the absence of CTAB, and I,,, is the maximum fluorescence obtained; y is the quotient

between the molar extinction coefficient, at the exci- tation wavelength, in the presence and in the absence

of CTAB [22]. The regression between [(Z/la) - 11-l and

l/([CTAB] - c.m.c.) gives a straight line and the value of the binding constant (KS) can be obtained from the quotient of the intercept and the slope of this line, whenever the micelles do not modify the extinc- tion coefficient at the excitation wavelength (y=l).

2.6. Procedure for the determination of the &,J$aq

ratio

In order to determine the quotient between the quantum yield of PL and PAP in the presence (4,)

E Morales et al./Analytica Chimica Acta 345 (1997) 87-98 c)I

and in the absence (&J of CTAB, a series of solutions (10 ml total volume) containing concentrations of

CTAB ranging between 0 and 4.0x 1O-2 M, and con- centrations of PL or PAP ranging between 1 .O x 1 0m7 and 6.0~10~~ M, at the working pH (7.1, adjusted with 0. I8 M phosphate buffer) was prepared. An aliquot of each solution was transferred into a 1 cm quartz cell thermostated at 60fO.l”C and the fluor-

escence intensity measured at X,,/X,,=360/435,320/ 430 and 3401435 nm for PL, PAP and both PL and

PAP, respectively. Since the extinction coefficients of PL and PAP, at the excitation wavelengths studied,

were not modified by the presence of CTAB it was fulfilled that c&,l&,q=b,,,lb,q, where b, and b,, are the slopes obtained in the micellar and the aqueous med-

ium, respectively, for the linear plots I vs. PL or PAP concentration, at a determined CTAB concentration.

3. Results and discussion

PAL and PALP are oxidized in the presence of cyanide to form the fluorescent products PL (X,,,/

X,,=360/435 nm) and PAP (X,,,/&,=320/430 nm),

respectively. The kinetic simultaneous resolution of PAL and PALP based on their reaction with cyanide in an aqueous medium has not been proposed because of the problems of selectivity and sensitivity derived

from their similar reactivity and the special spectral characteristics of their reaction products (PL and PAP feature a different maximal excitation wavelength and show the maximal fluorescence intensity at different pH). Thus, poor sensitivity is obtained for PALP by

monitoring the PALPxyanide reaction at the pH conditions (slightly basic) under which it occurs since

maximal fluorescence intensity for its reaction product (PAP) is obtained in an acid medium. On the other hand, since the rate of change of the fluorescence

intensity with time is dependent on the quantum yield of the monitored species at the wavelength used, the contribution of PALP at the initial rate measured for a PAL-PALP mixture is lesser than that of PAL which

results in poor selectivity for the determination of PALP.

In order to solve this problem we investigated the effect of CTAB micelles on both the rate and the fluorescence intensity of these reactions. This cationic surfactant was selected because it has been known to

accelerate the PAL-cyanide reaction in a factor of about two compared with water and a method has been

developed for the determination of this Bh vitamer by using flow injection analysis [23]. No influence of PALP on this reaction was investigated. Likewise, no kinetic studies have been performed to elucidate the mechanism via which CTAB exerts its accelerating

effect on the PAL-cyanide reaction. It was checked that CTAB micelles influenced the

two investigated systems in the same way: they

increased their reaction rate and caused an hyperchro-

mic effect on their fluorescence emission spectrum, although the extent of these effects was considerably greater for the PALP-cyanide reaction. Therefore,

CTAB micelles were considered good candidates to solve the problems of sensitivity and selectivity asso-

ciated to the simultaneous determination of PAL and PALP by their reaction with cyanide.

Because of the different maximal excitation wave- length of the reaction products, a study was carried out

to select the excitation wavelength more appropriate to solve the PAL-PALP mixture. The study was performed at a CTAB concentration above its c.m.c.

Fig. 1 shows (A) the relative fluorescence intensity

increment between t=O (initio of the recording of the kinetic curve) and different fixed reaction times

(30 min, 1 h and 2 h) (AZ,) and (B) the initial rate as a function of the wavelength excitation for ( 1) PAL

and (2) PALP reactions. Like in the aqueous medium the derivatization reactions using cyanide were only completed 2 h after reagents had been mixed. From the results shown in this Fig. 1 it follows that there is an excitation wavelength interval, which is wider in

proportion as the reactions progress, in which the

contribution of PAL to the fluorescence intensity is greater than that of PALP and the opposite occurs for

the initial rate measurement. This fact permits the analytes to be solved with sufficient selectivity. The final fluorescence obtained for both reactions only

increased slightly after 1 h and therefore measure- ments were made at this fixed time. An excitation wavelength of 340 nm, which was estimated to pro-

vide the best possible sensitivity and selectivity for the simultaneous determination of PAL and PAL, was selected. Typical kinetic curves obtained at this exci- tation wavelength for the two reactions, in the absence (1,2) and presence (1’,2’) of CTAB micelles, are shown in Fig. 2. The use of this cationic surfactant

92

AIF

60

40

20

AIF

60

40

20

AI, 60

40

20

E Morales et al./Analytica Chimica Acta 345 (1997) 87-98

A3

<

1

2

A2

><

1

2

A,

x 1

2

330 335 340 345

Fig. 1. Effect of the excitation wavelength (X,,,) on the AI, at a fixed time: 30 min (A,), 1 h (AZ) and 2 h (A,) and on the initial rate (B) of the

PAL/cyanide (1) and PALPkyanide (2) systems. Emission wavelength=435 nm. [PAL]=[PALP]=l pM. For details, see Section 2.3.

altered the reaction rates of PALP and PAL to different

extents, thereby enabling their simultaneous determi- nation. Likewise, because of the increased fluores- cence intensity values of PAP resulting from the presence of CTAB, the determination of PALP was more sensitive than in an aqueous medium.

Fig. 3 shows the most representative dependences obtained by plotting the initial rate and AZ, as a function of CTAB concentration. The reactions were not affected by surfactant concentrations below the

c.m.c. (9.2x lop5 M, calculated under our reaction

conditions); therefore, micelles were necessary in the reaction medium to observe any effect. The initial rate and fluorescence intensity increment of the PAL- cyanide system (curve 1) were hardly modified by the presence of CTAB concentrations ranging between 5 x lop5 and 5 x lo-* M. However, the initial rate ratio for the PAL-PALP mixture was increased to the extent required to permit simultaneous resolution by using a conventional differential kinetic method.

E Morales et al. /Analytica Chimica Acta 345 (I 997) 87-98 93

60 120 180 240 3do

TIME (min)

Fig. 2. Kinetic curves, obtained at pH 7.1, for the PAL/cyanide (1 and 1’) and PALPkyanide (2 and 2’) systems in the absence (1 and

2) and presence (I’ and 2’) of 1.6~10~~ M CTAB.

[PAL]=[PALP]=O.6 FM.

3.1. Optimization of the reaction conditions

The PAL and PALP systems were optimized by The cyanide concentration present in the reaction

changing each experimental variable in turn while medium influenced both the accuracy and sensitivity keeping all others constant. The two measured para- of the simultaneous determination of PAL and PALP meters used to resolve binary mixtures of the analytes (Fig. 4 A and A’). The dependence of the AZr on the (initial rate and fluorescence intensity increment cyanide concentration was similar for both systems, between t=O, initio of the recording of the kinetic however, the vitamer PAL needed a cyanide concen- curve, and t=l h) were found to be additive through- tration about twenty-fold greater than that needed by out the analytes concentration ranges tested. In order PALP to reach maximal initial rate. At cyanide con-

to achieve the highest possible accuracy and sensitiv- centrations greater than about 1 .O x 1 Op3 M the initial ity in the determination, the reactant concentrations rate for the PAL-cyanide system became higher than should meet two requirements, namely, the v(PALP)I that corresponding to the PALP-cyanide system, v(PAL) and &(PAL)lAZr(PALP) ratios should be while AZ, ratio remained unchanged; so, at these maximal, and the absolute values for v(PALP) and cyanide concentrations the resolution of the binary AZr(PAL) should also be maximal. mixture was inadvisable since PALP was determined

0.0 ’ 1.0 3.0 5.0

[CTAB] (hi) x 10'

Fig. 3. Influence of the CTAB concentration on the al, (A) and on

the initial rate (B) of the PAL/cyanide (1) and PALPkyanide (7)

systems. [PAL]=[PALP]=I FM.

E Morales et al. /Analytica Chimica Acta 345 (1997) 87-98

60 '2

40 I

0.2 0.6 1.0 30 40 50 60 5 -7 9 0.05 0.25 0.45

[CN-] (M) x 10' TEMPERATURE ('C) PH [PHOSPHATE BUFFER] (M)

Fig. 4. Influence of (A, A’) cyanide concentration, (B, B’) temperature (C, C’) pH and (D, D’) phosphate buffer concentration on the AI, (A,

B, C, D) and on the initial rate (A’, B’, C’, D’) of the PAL/cyanide (I) and PALPkyanide (2) systems. [PAL]=[PALP]=l PM.

60

6.0-

4.0. '/

2.0. /c-=2

with a high degree of error. Maximum accuracy and

the best possible sensitivity was obtained for a cyanide

concentration of 3.0x lop4 M. The effect of the temperature was investigated over

the range 30-60°C (Fig. 4 B and B’). The accuracy and sensitivity of the simultaneous determination of

PAL and PALP increased as a function of this para- meter. Therefore, a temperature of 60°C was fixed to resolve the PAL-PALP mixture.

Oxidation of PAL and PALP catalyzed by cyanide

occurred preferentially at slightly basic pH values (Fig. 4 C and C’). Oxidation of PAL was almost

completely suppressed at pH values lower than 5.5.

On the other hand, similar reactivity was observed at pH about 8 for both analytes. A pH value close to neutrality (pH=7.1) was selected as optimal.

A phosphate buffer was used to adjust the pH of the reaction medium to the required value. An increase in the buffer concentration slightly increased the rate of both reactions (Fig. 4 D’), buffer concentrations higher than about 0.2 M sharply decreased the AI, corresponding to the PAL-cyanide system (Fig. 4 D, curve 1). The AI, value corresponding to the PALP- cyanide system was not affected by this parameter (Fig. 4 D, curve 2). Since buffer concentrations lower

than about 0.12 M did not adjust the working pH adequately, phosphate concentrations between 0.12 and 0.2 M were advisable to adjust the pH of the reaction medium.

The ionic strength, which was adjusted with sodium

chloride, had no influence on both systems up to an electrolyte concentration of 5.0x lo-’ M. Increasing sodium chloride contents gradually decreased the

initial rate and AZ, of the PALP-cyanide system. Thus for an electrolyte concentration of 1 M, the initial rate and AI, decreased about 50%.

3.2. Features of the proposed analytical method

The calibration graphs for PAL and PALP were

constructed by plotting the initial rates (v) and fluor- escence intensity increments at a fixed reaction time

(1 h) (AZ,) obtained from the fluorescence intensity- time curves for each analyte as a function of the analyte concentration. The calibration graphs for the

individual determinations were linear over the range 0.01-0.6 PM. For maximum precision, a different instrumental sensitivity was used for the lower PAL and PALP concentrations (0.01-0.06 FM). The absence of synergistic effects ensured that the para- meters obtained for a mixture of the two analytes were the sums of the corresponding parameters obtained for each analyte separately.

In order to calculate the concentration of PAL and PALP in the mixture, Eqs. (1) and (2) (see Sec- tion 2.4) were solved by using a straightforward laboratory-developed FORTRAN 77 program. Coeffi- cients p in the equations were estimated by multiple

Table I

E Morales et al. /Analytica Chimica Acta 345 (1997) 87-98

Quantitative performance of the proposed method for the determination of binary mixtures of PAL and PALP

Linear range (pM) Measured parameter Coefficients of Eqs. (1) and (2) rr SEE ’

&, or $$S.D. /3, or fl, Z&D. /“* or ,$&Y,.D.

0.01-0.06 “ (1*3)x10-* 8.3*0.5 18.3f0.5 0.998 2.3x 10. ’

0.610.2 2.43zkO.05 0.92f0.05 0.9992 0.21

0.05-0.60 h -(sf3)xlo-~ 1.6910.07 5.021kO.07 0.9993 5.2x10 ’

0.9f0.4 49* I 2611 0.9990 0.62

Instrumental sensitivity: a 3.5, b 10.5.

’ Correlation coeffkient (n=l I).

’ Standard error of the estimate.

linear regression from 11 observations made on the variables v and AI, on 11 samples containing different combinations of analyte concentrations for each stu- died instrumental sensitivity. The results obtained are shown in Table 1, which includes the statistical para- meters of these equations. The precision of the pro-

posed method, expressed as relative standard deviation (%), was 2.8% for PAL and 3.4% for PALP

in a 1 : 1 mixture, [PAL]=[PALP]=O.l PM. The predictive ability of indirect calibration by

MLR for the binary mixtures containing the analytes

in different ratios as unknown samples and making measurements under the same experimental condi- tions as those used for calibration. Table 2 sum- marizes the results obtained from Eqs. (1) and (2)

at the different analyte ratios tested. Relative errors less than 5% were obtained in most of the analyte determinations, which confirms the good accuracy of

the proposed method. The study of the effect of foreign species often

present in serum on the proposed method is summar-

ized in Table 3. The maximum mole ratio of foreign species to PAL+PAL tested was 10000. A given

compound was considered not to interfere with the determination if the interferent plus analytes mixture yield a signal comprising the range S,fo, where S, is the signal provided by the analyte in the absence of interferent and 0 is the standard deviation of the method. Most of the foreign species

tested did not interfere in the PAL and PALP deter- mination at concentrations much higher than that corresponding to the analytes. In the presence of calcium, addition of 5.0x 10m3 M EDTA was neces- sary in order to avoid its precipitation in the reaction medium.

Table 2

Multiple linear regression prediction for binary mixtures of PAL

and PALP

Analyte ratio Actual concentration (PM) Relative error (so)

PAL : PALP PAL PALP PAL PALP

112 0.01 0.02 -7.3 -0.4

0.02 0.04 3.5 1.8

0.05 0.10 5.5 -0.7

0.20 0.40 3.0 1.8

0.30 0.60 -3.8 PI.9

2: 1 0.02 0.01 I .o -5. I

0.04 0.02 1.5 3.9

0.10 0.05 4. I -4.8

0.40 0.20 3.2 2.9

I:3 0.02 0.06 0.0 ~ I.9

0.20 0.60 2.5 I .A 3: I 0.06 0.02 PO.3 -5.3

0.60 0.20 -1.2 0.4

1 : I 0.04 0.04 -1.5 3.3

0.20 0.20 1 .o -6.0

3.3. Some observations on the action of CTAB on the

PAL-cyanide and PALP-cyanide systems

Several experiments were carried out in order to elucidate the mechanism via which the surfactant induced the changes showed in Fig. 3 in the features of the PAL-cyanide and PALP-cyanide systems. The presence of an excess of micelles in the reaction

medium was essential for CTAB to exert its effect on these systems. This was concluded from the critical micelle concentration (c.m.c.) obtained under the experimental conditions used (viz. 8.0x 10e5 M, as calculated from surface tension measurements); this concentration was substantially different from the

96 E Morales et al. /Analytica Chimica Acta 345 (1997) 87-98

Table 3

Effect of some foreign species on the determination of binary mixtures of 0.3 pM PAL and 0.3 pM PALP

Foreign species

Alanine, cystine, aspartic acid, succinic acid, urea, uric acid, xanthine,

hypoxanthine, histamine, glucose, acetaldehyde, magnesium

Glutamic acid, calciuma

Glycine, glutamine, a-ketoglutarate

Ascorbic acid, citric acid, histidine, I-methylhistidine NAD,

sodium pyruvate

Iron(III), copper(H)

Pyridoxamine, pyridoxine, pyridoxamine-5-phosphate

’ In the presence of 5.0x 10m3 M EDTA.

Maximum level tested without

observing interference

(PM)

26000

3000

600

150

3

0.6

Selectivity factor

[foreign species]/

[PAL]+[PALP]

> 10000

5000

1000

2.50

5

1

c.m.c. obtained in distilled water (9.2x 10U4 M) and

lower than the analytical concentration used (1.6x lo-* M).

Since CTAB modified both the initial rate and the final fluorescence intensity of the studied systems, experiments were designed to elucidate if micellar

catalysis was due to (a) changes in the reactivity of the reactants on transfer from water to the micellar pseudo-phase, (b) ‘local’ concentration of reactants within the micellar pseudo-phase and/or (c) modifica-

tion of the quantum yield of the reaction products which would result in an apparently modified reaction rate since reaction development was monitored via their formation.

By calculating kM (or kE, or kh) (the ‘true’ second- order rate constant in the micellar phase, Eq. (3)) and

comparing this value with k, one can determine whether micellar catalysis arises from purely extrinsic concentration of the reactants in the micelles or whether environmental perturbations alter the intrinsic reactivity of the reactants in the micellar pseudo-

phase. This calculation requires to know whether both analyte and cyanide are concentrated on the micellar surface (where the reaction would take place) or whether only one of them approach CTAB micelles (so the reaction would take place at the micelle-bulk solution interface).

The potential binding of PAL and PALP to CTAB was investigated spectrophotometrically [24,25] and spectrofluorimetrically [20-221. No changes in the absorption or fluorescence spectra of PAL-PALP (5 x lop7 M) were found to arise in the presence of CTAB (up to about 4x lo-* M), so no interaction

could be proved at the working pH (7.1, 0.18 M phosphate buffer). Ionic reactants lacking hydropho-

bic groups, such as cyanide, are concentrated on the surface of oppositely charged surfactant aggregates (i.e. CTAB) although only in some favourable cases the ion distribution between the micellar and aqueous

pseudo-phases can be measured directly [26,27]. Spectral investigation of the cyanide-CTAB interac- tion was impossible since both cyanide and CTAB absorbed in the same spectral region. We assumed cyanide to concentrate on the Stern layer and displace

the bromide counter ion of the surfactant by an ion- exchange reaction. So, the reaction was assumed to occur at the micelle-bulk solution interface and kinetic

data were analyzed according to the model specified in Section 2.4.

Fig. 5 shows the dependence of the experimental second-order rate constant, kp, on the overall surfac- tant concentration of both PAL-cyanide (curve 1) and PALP-cyanide (curve 2) systems. The plot of l!

(k,-k,) against l/([CTAB]-c.m.c.) (Eq. (5)) was linear for the two studied systems, so, as it was assumed, the reactions took place in the micelle-bulk solution interface. From the intercepts of these linear plots, the true second-order rate constant in the micelle-bulk solution interface (kk) was found to be (3.73kO.08) and (23fl)l mol-’ s-i for PAL-cyanide and PALP-cyanide reactions, respectively. The sec- ond-order rate constant obtained for these reactions in the aqueous medium (k,) was (5.0*0.3) and (17.3*0.7)1 mol-’ ss’ para PAL and PALP, respec- tively. These data indicate that CTAB does not modify the intrinsic reactivity of the reactants since no sig-

F: Morales et al./Analytica Chimica Acta 345 (1997) 87-98 91

40

- 30 7 m

i -z E 20

ti

r’ 10

0

-Q o 1

0.5 1.0 1.5 2.0

[CTAEI] (M) x lo'

Fig. 5. Influence of the CTAB concentration on the second-order

rate constant for the PAL/cyanide (1) and PALP/cyanide (2)

systems.

nificant enhancement was observed for the ‘true’

second-order rate constant in the micellar pseudo- phase (k&) relative to an aqueous medium (k,) for any of the two systems studied. On the other hand, the observed micelle-induced rate augmentation could not

be attributed to the increased reactant concentrations in the micellar units since there was no evidence for binding of PAL and PALP to CTAB and cyanide was

added in excess to the reaction medium. The association constant of cyanide to CTAB (KCN)

was calculated from the slope of the plot l/(&-k,,,) vs. l/([CTAB]-c.m.c.). Values of 1000*200 and

700*90 M-’ were obtained from the linear plots corresponding to the PAL-cyanide and PALP-cyanide systems, respectively. These KCN values were very

similar, within experimental errors, and confirmed the above hypothesis of association of cyanide to the micellar surface.

In order to determine wether CTAB micelles mod- ified the fluorescent characteristics of the reaction

products (PL and PAP) in an enough extent to account

for the micelle induced augmentation observed, CTAB-PL and CTAB-PAP interactions were investi- gated. Evidence of these interactions was provided by the hyperchromic effect that CTAB induced in the fluorescence emission spectrum of PL and PAP. The partition or ‘binding’ constants of these compounds to

the cationic surfactant were determined spectrofluor- imetrically (Section 2.5). A plot of [(l/lo)-I]-’ against l/([CTAB]-c.m.c.) was linear in the surfac- tant concentration ranges 8.0 x 1 O-j-2.8 x lo-* M and

2.0x 1O-4A.Ox 10-s M for PL and PAP, respectively. From the quotient between the intercept and the slope

of these lines, the binding constants of PL and PAP were found to be KpL=50f6 M-’ and

Kp~p=l390* 10 M-‘. These values showed that the

binding of PAP to CTAB was considerably greater than that of PL.

The quantum yield of both PL and PAP calculated, at their maximal excitationemission wavelengths, were found to increase with increased CTAB concen- trations up to about 2.8x 10M2 and 4.0x lo-’ M,

respectively. Maximal &,/4aq ratios obtained at these wavelengths were 1.5 and 2.3 for PL and PAP, respec-

tively (see Section 2.6). The quantum yield for PAP increased about twice (&J&+=2.1) and that for PL

hardly was modified (&,/&r==l.l) at the working excitation-emission wavelengths (340/435 nm) and

at the CTAB concentration used as a reaction medium (1.6x lo-’ M). These ratios were roughly equal to the

u,/u,~ ratios for PAL and PALP, respectively, there- fore, the effect of the CTAB micellar system on these reactions can be accounted for in terms of sensitization

k CN ;

, w.1 / PLW

PLm E PL w

K PAP PAP, E PAP w

K PL

+ PALP W

ICTAB1 micelle

k

CN; _ x.2

, PAP,

Fig. 6. Oxidation of PAL and PALP at the CTAB micellar surface.

98 E Morales et al./Analytica Chimica Acta 345 (1997) 87-98

of their reaction products. Fig. 6 outlines the possible

reactions and equilibria present in the micellar solu- tion according to data derived from the study here

performed.

4. Conclusions

The results reported in this work confirm the poten-

tial of micelles for simultaneous kinetic analysis of organic compounds with similar structure and reac- tivity. The presence of micelles in the reaction med-

ium introduces a dependence of the rate constant on several parameters (binding or partition constant,

specific rate constant, etc) which act as differential elements for the reactivity of the different analytes involved. Thus, kinetic discrimination of PAL and

PALP, based on their reaction with cyanide, was

possible by concentration of the fluorescent reaction products (PL and PAP) on CTAB micellar aggregates in a different extent (&=50f6 M-’ and

KPAP=1390f10 M-l) which resulted in a different increase of their quantum yield and as consequence in a differential increased reaction rate. So, micelles open up new prospects for improving the selectivity

of kinetic determinations.

Acknowledgements

We gratefully acknowledge financial support from CICyT (Project No. PB91-0840).

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