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Available online at www.sciencedirect.com

Journal of Chromatography A, 1175 (2007) 283–288

Study on the kinetics of keto-enol tautomerism ofp-hydroxyphenylpyruvic acid using capillary electrophoresis

Lu Huang a,b, Ying Huang a,b,c, Yuwu Chi a,b, Jinming Lin a,b,Lishuang Yu a,b, Liangjun Xu d, Guonan Chen a,b,∗

a Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety (Fuzhou University),Fuzhou, Fujian 350002, China

b Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350002, Chinac College of Chemistry and Materials Science, Fujian Normal University, Fuzhou, Fujian 350007, China

d Analytical and Testing center, Fuzhou University, Fuzhou, Fujian 350002, China

Received 4 August 2007; received in revised form 18 October 2007; accepted 19 October 2007Available online 1 November 2007

bstract

The kinetics of keto-enol tautomerism of p-hydroxyphenylpyruvic acid (pHPP) as a model of �-carbonyl compounds in aqueous solution atoom temperature (25 ◦C) was first investigated by capillary electrophoresis with UV detection at 200 nm. The two tautomers could be separatednd detected within 3 min. Since the ketonization of enolic pHPP varied with the buffer composition and buffer pH, the kinetics of pHPP was

tudied under different conditions, and relevant distributing fractions of enolic pHPP, ketonization rate constants and half-life were determined. Inddition, �-CD played an important part in the separation of the two tautomers, thus, the interaction between pHPP and �-CD was also investigatedy electrochemical techniques. 2007 Elsevier B.V. All rights reserved.

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eywords: p-Hydroxyphenylpyruvic acid; Kinetics; Keto-enol tautomerism; C

. Introduction

Keto-enol tautomerism is the most common tautomeric phe-omenon. The determination of keto-enol equilibrium constantsas been at the center of physical organic studies of keto-enolautomerism for many years. A number of measurements ofquilibrium constants for the tautomerism of keto-enol systems1–11] and theoretical investigations [12–17] can be found in theiterature. Up until the present time, keto-enol tautomerism haseen studied by many methods, such as infrared spectroscopy18,19], Raman spectroscopy [20], UV absorption [21–23], X-ay [24], NMR [25–27], photoelectron spectroscopy [28], mass

pectrometry [29], spectrophotometry [30] and HPLC [31].

Capillary electrophoresis is an important separation tech-ology, offering the advantage of minimal sample volume

∗ Corresponding author at: Department of Chemistry, Fuzhou University,uzhou, Fujian 350002, China. Tel.: +86 591 87893315; fax: +86 591 83713866.

E-mail address: gnchen@fzu.edu.cn (G. Chen).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.10.078

ry electrophoresis

equirement, short analysis time and high separation efficiency.owever, the application of CE in the determination of physic-chemical constant, such as rate constant, is very poor [32]. Thehysicochemical constant is a very important property in theharmaceutical industry [33], and the rate constant is a partic-larly important physicochemical parameter in the study of theegradation of the medicament [32].

In this paper, CE was first used to measure the ketonizationate constant for keto-enol tautomerism of pHPP, which is aodel of �-carbonyl compounds. The most important function

f CE is that the change in concentration of both the enolic andetonic pHPPs can be directly monitored during the keto-enolautomerism by using �-CD as the additive to complex withHPPs for the sake of their simultaneous separation.

p-Hydroxyphenylpyruvic acid (pHPP) is an important inter-ediate in the metabolism of tyrosine [21]. Its level in blood and

rine is used to diagnose a congenital metabolic defect knowns tyrosinemia. When individuals suffer from this inborn errorf metabolism, the level of pHPP in body fluid is dramaticallyncreased due to deficiency of the p-hydroxyphenylpyruvate

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84 L. Huang et al. / J. Chrom

xidase. In addition, liver disease, avitaminosis and malig-ancy can also cause elevation of pHPP level in urine34].

There are some papers dedicated to the determination ofHPP by fluorescence quenching [35], GC [36,37], HPLC [38]r capillary electrophoresis (CE) [39]. However, few papers areedicated to the keto-enol tautomerism [21]. Using electrochem-cal techniques, pHPP is demonstrated to have two tautomers byhen and coworkers [21]: enolic form exists mainly in freshlyrepared solution and ketonic form exists mainly in equilibriumolution. Moreover, Chen et al. studied the chemical oxidationf pHPP in aqueous solution by CE with an electrochemilumi-escence detection system [40].

In this study, the two tautomers of pHPP were separatedimultaneously and determined under the optimized conditionsy CE. Based on the fact that the kinetics of keto-enol tau-omerism can be influenced by the pH and composition ofhe aqueous solution, the distributing fractions and ketoniza-ion rate constants of enolic pHPP were measured underhree different conditions: (1) running buffer was 20 mmol/La2HPO4–NaOH (pH 9.0) containing 8 mmol/L �-CD andHPP was injected by dissolving it in this buffer; (2) run-ing buffer was 20 mmol/L Na2HPO4–NaH2PO4 (pH 7.0)ontaining 8 mmol/L �-CD and pHPP was injected by dis-olving it in this buffer; (3) running buffer was 20 mmol/La2HPO4–NaH2PO4 (pH 7.0) containing 8 mmol/L �-CD andHPP was injected by dissolving it in water. Under all aboveonditions, enolic and ketonic pHPPs could be completely sep-rated within 3 min. The rate constants under three differentonditions given above were 0.0596, 0.0276 and 0.0061, respec-ively, and the half-life was 12, 25, 114 min, respectively. The

ethod based on CE is simple and fast, and can be usedo study the kinetics of keto-enol tautomerism of other com-ounds.

In addition, since �-CD played an important part in theeparation of the two tautomers of pHPP and might complexith them, electrochemical study on the interaction of pHPP

nd �-CD was also performed to demonstrate the complexationetween �-CD and pHPPs.

. Materials and methods

.1. Instrument

The CE instrument used in the experiments was a Beckman/ACE MDQ system with UV detection (Beckman, Fullerton,A, USA) and a System 32-Karat Software for instrumentontrol and data collection. Electrophoresis was performed inntreated fused silica capillaries (Ruifeng, Yongnian, Hebei,hina), 40 cm (effective length 30 cm) × 50 �m I.D. (365 �m.D.).An electrochemical analyzer (CHI440, Shanghai Chenhua,

hina) was employed for cyclic voltammetric and differential

ulse voltammetric measurements with a glassy carbon elec-rode. All potentials were measured against an Ag/AgCl (3 MCl) reference electrode. The parameters used for the mea-

urements were shown as follow: (a) linear sweep and cyclic

A 1175 (2007) 283–288

oltammetry (CV): scan rate 200 mV/s and (b) differential pulseoltammetry (DPV): pulse period 1 s, pulse amplitude 50 mV,ulse width 20 ms.

.2. Chemicals

p-Hydroxyphenylpyruvic acid and �-CD were obtained fromigma (St. Louis, MO, USA). Disodium hydrogen phosphate,odium dihydrogen phosphate and sodium hydroxide (analyticalrade) were from Shanghai Chemical (Shanghai, China). Theater was prepared using a Milli-Q Academic/A10 equipment

Millipore, Bedford, MA, USA).

.3. Electrophoretic procedure

Prior to use, all solutions were degassed by sonication. Newapillaries were conditioned with: 1 M HCl for 10 min, 0.1 MaOH for 20 min, water for 10 min, followed by acetonitrile

nd methanol each for 5 min at 20 psi. The samples were intro-uced hydrodynamically at the anodic end of the capillary by aressure of 0.5 psi for 5 s. The separations were carried out at0 kV at room temperature (25 ◦C) and the enolic and ketonicHPPs were directly detected at 200 nm on the cathodic side.etween running, the capillaries were flushed with electrophore-

is medium for 2 min (20 psi).

. Results and discussion

.1. Study on the kinetics of keto-enol tautomerism ofHPP by capillary electrophoresis

There are two tautomers of pHPP, i.e. enolic and ketonic formn the aqueous solution (see Fig. 1). As reported by Chen andoworkers [21], enolic form of pHPP remained very stable inethanol medium. However, the enolic form of pHPP would

e converted to the ketonic form in aqueous solution, which iso-called tautomerism.

.1.1. Kinetic equationsSince water is superfluous during the tautomerism, its con-

entration can be considered to be constant in the reaction.herefore, the tautomerism of pHPP is a first-order reaction and

Fig. 1. Keto-enol tautomerism of pHPP.

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q. (1) also can be expressed as,

ln Cenol = kt − ln C0 (2)

here C0 is the initial concentration of pHPP and Cenol is theoncentration of enolic pHPP at a tautomerism time t. If theinetic reaction is of first-order, a straight line can be obtainedhen ln Cenol is plotted against t. The slope of the line is therst-order rate constant k.

In our study, the optimization of experimental conditions wasainly in the interest of lessening the separating time. Since a

igh voltage was applied during the separating process, it wasetter to reduce the separating time in order to reduce the experi-ental error. For the sake of this, the true ketonization time t was

alculated by adding the equilibrium time before electrophoreticnalysis to the migration time of the enolic pHPP. Besides, Cenolould not be measured directly. It was calculated by subtractingketo from C0 in respect that pHPP was mainly in its ketonic

orm in the completely balanced solution and the concentrationf the enolic form could be neglected in our calculations.

.1.2. Optimum conditions for electrophoresisTo determine the optimum conditions for the separation of

he enolic and ketonic pHPP, the composition and pH values ofhe buffer solutions were varied to optimize the conditions forhe separation.

In this study, the two tautomers of pHPP could not be sep-rated in buffer solutions without additives. Thus, differentdditives were added to improve the resolution, and satisfac-ory separation and good sensitivity could be obtained by using

it1

ig. 2. Optimization for capillary electrophoresis. (a) Effect of �-CD concentration:mmol/L �-CD (pH 7.0); (c) effect of buffer pH: 20 mmol/L NaH2PO4–Na2HPO4

issolved in methanol as a concentration of 10−3 mol/L, and was diluted with water befonditions seen Section 2.3.

r. A 1175 (2007) 283–288 285

-cyclodextrin (�-CD) as the additive in phosphate buffer. Ashown in Fig. 2(a), with the increasing of the concentration, theigration time is decreasing. It might be related to the inclusion

omplexation of �-CD with the two tautomers. When the tau-omers were included in �-CD, their negative electric chargeseduced since �-CD is neutral so that the complexes formedy �-CD and either of the two tautomers could migrate faster.hough the two tautomers could be completely separated when

he concentration of �-CD was above 2 mmol/L, the peak shapef ketonic pHPP was not better unless the concentration of �-D was above 8 mmol/L. With a view to the analytical time andeak shape, 8 mmol/L was chosen as the optimal concentrationf �-CD.

Furthermore, the effects of concentration and pH of phos-hate buffer were also studied. As shown in Fig. 2(b), theigration time and peak height are both increased when the con-

entration of phosphate buffer ranges from 10 to 50 mmol/L.aken into account the analytical time and peak height,0 mmol/L was chosen as the concentration of the buffer. Inddition, when the pH ranges from 5.0 to 9.0, the migration times reduced while the peak height is heightened (see Fig. 2(c)).n our work, pH 7.0 and 9.0 were both utilized to measure theate constants.

.1.3. Detection linearity and repeatability

In respect that the initial concentration of pHPP utilized

n our work was 10−3 mol/L and the ketonic pHPP concen-ration during the ketonization was presumably ranged from0−5 to 10−3 mol/L, the linearity between the ketonic pHPP

20 mmol/L NaH2PO4–Na2HPO4 (pH 7.0); (b) effect of buffer concentration:with 8 mmol/L �-CD. Peak 1: enolic pHPP; peak 2: ketonic pHPP. pHPP wasore every injection as a concentration of 10−4 mol/L. For details of experimental

286 L. Huang et al. / J. Chromatogr. A 1175 (2007) 283–288

Table 1The regression equations and correlation coefficient

Conditionsa Regression equation (Y = aX + b)b Correlation coefficient Linear range (mol/L)

(1) Y = 2 × 107X + 3149.4 0.9941 10−5 to 10−3

(2) Y = 2 × 107X + 2409.8 0.9963 10−5 to 10−3

(3) Y = 2 × 107X + 885.66 0.9998 10−5 to 10−3

a (1) Running buffer was 20 mmol/L phosphate buffer (pH 9.0) containing 8 mmol/L �-CD and pHPP was dissolved in this buffer; (2) running buffer was 20 mmol/Lphosphate buffer (pH 7.0) containing 8 mmol/L �-CD and pHPP was dissolved in this buffer; (3) running buffer was 20 mmol/L phosphate buffer (pH 7.0) containing8 mmol/L �-CD and pHPP was dissolved in water.

b Where Y is the peak height (in 1 AU) and X is the ketonic concentration (in mol/L).

Table 2Migration time and peak height reproducibilities

Conditionsa Migration time (%RSD) Peak height (%RSD)

Run to run Day to day Run to run Day to day

(1) 0.66 2.2 1.4 4.3

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3p

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Fa

TK

C

(2) 0.54 1.9 1.5 3.8(3) 0.36 1.5 1.2 3.2

a Conditions are the same as in Table 1.

oncentration and its peak height was determined by ana-yzing a series of different concentrations of the completelyalanced pHPP solutions (24 h after prepared) under differentonditions ranged of 10−5 to 10−3 mol/L. The results of theegression equations and correlation coefficients are shown inable 1.

The RSD of the migration time of ketonic pHPP underifferent conditions were 0.36–0.66% (run to run, n = 5) and.5–2.2% (day to day, n = 5). And the RSD of the peak current ofetonic pHPP were 1.2–1.5% (run to run, n = 5) and 3.2–4.3%day to day, n = 5). Table 2 summarizes the peak height andigration time reproducibility achieved for ketonic pHPP under

hree different conditions. It demonstrated that this method wasrecise.

.1.4. Determination of distributing fractions of enolicHPP

The ketonization of enolic pHPP was carried out in a constantemperature water bath (25 ◦C). Fig. 3 shows the typical electro-herograms of enolic and ketonic pHPPs when the ketonizationimes were 11, 58, 98, 138, 228 and 500 min, respectively. Thehange of the peak current of enolic and ketonic pHPPs demon-trates the change of the concentrations of them during thequilibrating process. It can be seen from Fig. 3 that the peakurrent of enolic pHPP decreased gradually with the ketoniza-

ion time and the enolic pHPP migrates faster than the ketonicHPP.

As mentioned in Section 3.1.1, Cenol was calculated byubtracting Cketo from C0 while Cketo at ketonization time t

cttF

able 3etonization rate constants of pHPP under different conditions

onditionsa Regression equation Correlation coefficien

(1) –ln C = 0.0596 t + 6.8842 0.994(2) –ln C = 0.0276 t + 6.8762 0.9951(3) –ln C = 0.0061 t + 6.8862 0.9941

a Conditions are the same as in Table 1. The initial concentration of pHPP was 10−

ig. 4. The fraction of enolic pHPP at different ketonization time. Conditionsre the same as in Table 1. The initial concentration of pHPP was 10−3 mol/L.

ould be obtained from Table 1. According to this, the dis-ributing fraction of enolic pHPP, which is the ratio of Cenolo C0, was calculated under three different conditions (seeig. 4).

t Ketonization constant (10−2/min−1) Half-life (min)

5.96 122.76 250.61 114

3 mol/L.

L. Huang et al. / J. Chromatogr. A 1175 (2007) 283–288 287

Fig. 5. Cyclic voltammograms of ketonic pHPP. (a) 5 × 10−4 mol/Lketonic pHPP; (b) 5 × 10−4 mol/L ketonic pHPP + 2.5 × 10−4 mol/L�-CD; (c) 5 × 10−4 mol/L ketonic pHPP + 5.0 × 10−4 mol/L �-CD; (d)5 × 10−4 mol/L ketonic pHPP + 7.5 × 10−4 mol/L �-CD; (e) 5 × 10−4 mol/Lkpb

3k

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etonic pHPP + 1.0 × 10−3 mol/L �-CD; (f) 5 × 10−4 mol/L ketonicHPP + 1.5 × 10−3 mol/L �-CD; (g) 1.5 × 10−3 mol/L �-CD in phosphateuffer solution (pH 7.0).

.1.5. Determination of ketonization rate constants foreto-enol tautomerism of p-hydroxyphenylpyruvic acid

The correlation coefficients and ketonization rate constantsnder three different conditions are calculated and presented inable 3. It is obvious that the ketonization rate constant underondition (1) is larger than that under condition (2), and theetonization rate constant under condition (2) is larger than thatnder condition (3). This might be due to the catalysis of phos-hate buffer and bases, as reported that both phosphate buffernd bases could catalyze the keto-enol tautomerism [1,41].

In conclusion, the ketonization rate constant varied with theuffer composition and buffer pH mainly due to the catalysisf phosphate buffer and bases. Furthermore, the ketonizationate data obtained obeyed the first-order rate law well, whichalidated the initial assumption in return.

.2. Electrochemical study on the interaction betweenHPP and β-CD

As mentioned in Section 3.1.2, the electrophoretic behaviorf pHPP was strongly affected by the concentration of �-CD,hich is probably resulted from the formation of the complexesetween pHPP and �-CD. In order to demonstrate the complex-tion between pHPPs and �-CD, a series of phosphate bufferolutions (pH 7.0) containing 5 × 10−4 mol/L pHPP and dif-erent concentration of �-CD ranged from 0–1.5 mmol/L wererepared and analyzed after 12 h by cyclic voltammetry andifferential pulse voltammetry.

Fig. 5 shows the cyclic voltammograms of ketonic pHPPith different concentration of �-CD at a glassy carbon elec-

rode (GCE) over the potential range of −500 to 1500 mV. It

s observed that �-CD has no electrochemical activity, whileHPP has two oxidation peaks: one near +0.25 V is a reversibleeak and the other near +0.90 V is a irreversible peak. Withhe increasing of the concentration of �-CD, both oxidation

× 10−4 mol/L ketonic pHPP + 2.5 × 10−4 mol/L �-CD; (d) 5 × 10−4 mol/Letonic pHPP + 5.0 × 10−4 mol/L �-CD; (e) 5 × 10−4 mol/L ketonicHPP + 1.0 × 10−3 mol/L.

eaks become smaller and the peak near +0.25 V shifts posi-ively. Furthermore, the differential pulse voltammograms (seeig. 6) show that there are three oxidation peaks of pHPP (+0.35,0.70 and +0.80 V). With the increasing of the concentration of-CD, the peaks near +0.70 and +0.80 V become smaller. Espe-ially, the peaks near +0.70 V become much more smaller andhift positively.

The results obtained from the cyclic and differential pulseoltammograms suggest that the addition of �-CD to pHPP leadso the shield of the electrochemically active groups of pHPPnd the decrease of electrochemical activity of pHPP, whichemonstrates that there is complexation between pHPP and �-D in phosphate buffer solutions.

. Conclusions

This study shows the applicability of capillary electrophore-is with UV detection to determine the ketonization rateonstants of p-hydroxyphenylpyruvic acid in different aqueousolutions. The method proposed is simple, fast and can be usedor the study of rate constants for keto-enol tautomerism ofther carbonyl compounds. Moreover, pHPP and �-CD wereonfirmed to form complexes in phosphate buffer solutions bylectrochemical techniques.

cknowledgements

This project was financially supported by the National Natureciences Funding of China (20735002, 20575011), the Key Nat-ral Sciences Funding of Fujian Province, China (D0520001).

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