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0022-2313/$ - se

doi:10.1016/j.jlu

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Journal of Luminescence 126 (2007) 747–752

www.elsevier.com/locate/jlumin

Spectrofluorimetric study on the inclusion behavior of p-sulfonatedcalix [6] arene with cetyltrimethylammonium bromide

and analytical application

Chun Liu, Zhong Fu, Huapeng Yu, Hongwei Xu, Lun Wang, Yunyou Zhou�

Anhui Key Laboratory of Functional Molecular Solids, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, PR China

Received 28 July 2006; received in revised form 26 October 2006; accepted 9 November 2006

Abstract

The characteristics of host–guest complexation between p-sulfonated calix [G. Arena, S. Gentile, F. G. Gulino, D. Sciotto, C. Sgarlata,

Tetrahedron Lett. 45 (2004) 7091] arene (SC6A) and cationic surfactant cetyltrimethylammonium bromide (CTAB) were studied by

fluorescence spectrometry. A 1:1 stoichiometry for the complexation was established and the complex constant was also calculated by a

deduced equation. It was found the fluorescence of the complex could be remarkably quenched by an appropriate amount of ceftriaxone

sodium (CTRX). Based on the results, a novel spectrofluorimetric method for determination of CTRX was developed with a linear range

of 9.2� 10�7–8.5� 10�5mol L�1 and a detection of 3.5� 10�7mol L�1. The proposed method was used to determine CTRX in their

commercial preparations with satisfactory results. Moreover, the probable interaction mechanisms of the systems were also discussed.

r 2006 Elsevier B.V. All rights reserved.

Keywords: p-sulfonated calix [6] arene; CTAB; Fluorescence; Ceftriaxone sodium

1. Introduction

Water-soluble calixarenes are a versatile family ofmolecules that have attracted much attention in recentyears due to their ability to form host–guest arrangementseither in solution or the solid state [1]. For example, somewater-soluble calix[n]arenes (n ¼ 4, 6 and 8) and resorci-narenes towards quaternary ammonium ions [2,3], tri-methylammonium cations [4–6], dyes [7,8], native aminoacids [9,10], and small neutral organic molecules [11] havebeen investigated extensively. Among these water-solublecalixarenes, p-sulfonated calix[n]arenes, which have flexibleand often poorly defined cavities that tend to bindpositively charged species and even have been often viewedas promising water-soluble hosts, have attracted the keeninterest of researchers studying different area, such asconformational flexibility [5,12], electrochemical behaviors[13–15], about their direct determination [16], interactingwith drugs [17,18] and molecular recognitions [11,19].

e front matter r 2006 Elsevier B.V. All rights reserved.

min.2006.11.006

ing author. Tel.: +86553 3869303, fax: +86 553 3859303.

ess: zy161299@mail.ahnu.edu.cn (Y. Zhou).

However, there are few literatures about their naturaloptical properties in particular fluorescence pro-perties owing to their weak fluorescence in the aqueoussolution [20].Surfactant molecules, which have an ionic end group as

well as a large variable hydrophobic tail, have been usedextensively in chemical synthesis, fluorescence analysis andelectrochemical analysis [21–23]. During the last few years,a number of studies on the interaction between surfactantsand cyclodextrins were reported [24–26]. From thoseresults it was concluded that the surfactant hydrophobicchain could enter inside the cyclodextrin hydrophobiccavity to form inclusion complexes with cyclodextrins ortheir derivatives. But till now, there are only a few reportson the inclusion behavior of calixarenes and theirderivatives with surfactants [27].CTRX is one of the third-generation cephalo-

sporin antibiotics characterized by a broad antibacterialspectrum and a resistance to b-lactamase-producingorganisms in addition to its antimicrobial activity(Streptococci, Staphylococci, Pneumococci, etc. [28]).A variety of analytical procedures have been reported for

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2 6 100

200

400

600

800

1000

Re

lati

ve

flu

ore

sc

en

ce

in

ten

sty

pH

84

Fig. 1. Influence of pH value on the fluorescence intensity of SC6A–

CTAB complex; CSC6A ¼ 5� 10�6molL�1, CCTAB ¼ 3� 10�5molL�1.

C. Liu et al. / Journal of Luminescence 126 (2007) 747–752748

the determination of ceftriaxone viz. derivative spectro-photometry [29,30], colorimetry [31], cyclic voltametry [32],and HPLC [33,34]. However, most of methods are basedon the reactivity of the cleaved molecule or expensiveinstrument.

In this paper, the fluorescence behaviors of p-sulfonatedcalix [6] arene in cationic surfactant of CTAB have beenstudied. Experimental results reveal that SC6A can form1:1 complex with cationic surfactants, which lead to theenhancement of fluorescence intensity of SC6A. Whenproper amounts of CTRX were added in the system, thefluorescence intensity decreased regularly. Based on theresults, a novel fluorescence spectroscopy method wasdeveloped for determination of CTRX.

2. Experimental

2.1. Apparatus

Fluorescence spectra and relative fluorescence intensitieswere measured on a model F-2500 fluorescence spectro-photometer (Hitachi, Japan) using a conventional 1� 1 cmquartz cell. Excitation and emission bandwidths were set to5 and 2.5 nm, respectively. All measurements were carriedout at room temperature. A model pHS-3C (DazhongAnalytical Instruments Factory, Shanghai, China) pHmeter was used for accurate adjustment of pH.

2.2. Reagents

All reagents used were of analytical-reagent grade or thebest grade commercially. Doubly distilled water was usedthroughout. p-sulfonated calix [6] arene was synthesizedaccording to the literature [35] and identified by IR, 1HNMR, and element analysis. Stock solution of SC6A wasprepared as 1� 10–3molL�1. CTAB was obtained fromShanghai Chemical Reagent Co., China, and 1� 10–3molL�1

stock solution was prepared in water. CTRX of drug standardsample was purchased from Chinese National Institute for theControl of Pharmaceutical and Biological Products and usedwithout further purification. Stock standard solution of1� 10–3molL�1 was prepared by dissolving standard samplein doubly distilled water as needed. Buffer solutions wereprepared by mixing 0.2molL–1 sodium monohydrogenorthophosphate solutions with 0.2molL–1 sodium dihydrogenorthophosphate solutions and adjusted to the desired valuesusing a pH meter.

2.3. Procedure

2.3.1. Inclusion procedure

0.5mL of 1� 10–4mol L�1 SC6A solution, appropriatevolumes of 1� 10–4mol L�1 CTAB and 1mL of phosphatebuffer (pH 6.7) solution were transferred into a 10mLvolumetric flask in turn. The mixed solution was diluted tofinal volume with water and stirred thoroughly; thefluorescence intensities were determined after 25min at

proper temperature controlled by a thermostatically con-trolled water bath.

2.3.2. Determination of CTRX

Into a 10mL volumetric flask were placed in turn 0.5mL of1� 10–5molL�1 SC6A solution, 0.3mL 1� 10–3molL�1

CTAB, 1mL of phosphate buffer (pH 6.7) solution andappropriate volumes of 1� 10–4molL�1 CTRX. Afterincubation for 25min at room temperature, the fluorescenceintensities were measured.

2.3.3. Procedure for dosage forms

Constitute sterile powder in a volume of water accuratelymeasured corresponding to the volume specified in thelabeling. Dilute an accurately measured volume of injectionquantitatively to obtain a solution of 1� 10–3mol L�1 thestudied drug. Carry out the procedure under calibrationcurve and calculate the concentration from the regressionequation.

3. Results and discussion

3.1. Fluorescence spectra of systems

In order to study the fluorescence properties of systems,different pH values were adjusted using different bufferssuch as citric acid—Na2HPO4 (pH 2–5), Na2HPO4–NaH2-

PO4 (pH 6–8), Na2B4O7–NaOH (pH 9–10) (Fig. 1). It canbe seen that the relative fluorescence intensity increasesslightly when the value of pH is above 6. Kunsagi-Mateet al. [36] also reported that SC6A could provide excellentconditions for the investigation of its host propertiesbetween pH 6 and pH 8.5. In this work, all the data weremeasured in a pH 6.7 Na2HPO4–NaH2PO4 buffer medium.We also found the influence of doses of buffer on thefluorescence intensity was neglectable, so 1mL of phos-phate buffer was used in the systems.

ARTICLE IN PRESSC. Liu et al. / Journal of Luminescence 126 (2007) 747–752 749

Fig. 2 shows the fluorescence spectra of SC6A in thepresence of CTAB in aqueous solution (pH 6.7). As can beseen the fluorescence intensity of SC6A increased remark-ably on the addition of an appropriate amount of1� 10�4mol L�1 CTAB, meanwhile a slight and slow shiftwas observed (426.0-440.0 nm). According to our earlierresults [18] we suppose that the fluorescence spectrachanges described above was probably induced by theformation of inclusion complexes.

The fluorescence spectra of CTRX (1), SC6A (2),SC6A+CTAB (3), and SC6A+CTAB+CTRX (4) areshown in Fig. 3. It can be seen that SC6A and CTRX havevery weak fluorescence in independent aqueous solution.However, SC6A+CTAB (3) display strong fluorescencethat can be quenched regularly on the addition ofCTRX (4).

350 400 450 5000

100

200

300

400

500

600

700

800

900

1

12

Flu

ore

scence Inte

nsity

Wavelength (nm)

Fig. 2. Fluorescence emission spectra of 5� 10�6mol L�1 SC6A in

phosphate buffer (pH ¼ 6.7) with different concentrations of CTAB:

from 1 to 12: 0.0; 0.1; 0.25; 0.5; 0.8; 1.3; 1.5; 1.8; 2.0; 2.2; 2.5

3.0� 10�5molL�1.

350 400 450 500

0

100

200

300

400

500

600

700

800

900

4

3

2

1

Rela

tive F

luore

scence Inte

nsity

Wavelength (nm)

Fig. 3. Fluorescence emission spectra: (1) CTRX, (2) SC6A, (3) SC6A+

CTAB and (4) SC6A+CTAB+CTRX, CSC6A ¼ 5� 10�6molL�1,

CCTAB ¼ 3.0� 10�5molL�1, CCTRX ¼ 1.0� 10�5molL�1, pH ¼ 6.7.

3.2. Discussion of the fluorescence behavior

To understand the fluorescence phenomena of p-sulfonated calix [6] arene in micellar solution, on the onehand, the interaction between 4-phenolsulfonate (themonomer of SC6A) and CTAB has been investigated. Itwas found that the fluorescence spectra of 4-phenolsulfo-nate could be influenced hardly when various concentra-tions of CTAB solutions were added, although CTAB isusually used to enhance the fluorescence of some sulfuricfluorophores [37,38]. Compared 4-phenolsulfonate withSC6A, the obvious difference between them is that thelatter has a cavity. The macrocyclic structure of the ligandsobviously can play an important role for the complexformation. These indicate probably the formation ofhost–guest complexes between SC6A and CTAB wouldbe the origin of the spectral changes described above.On the other hand, other cationic surfactants such as

dodecyltrimethylammonium bromide (DTAB), cetyltri-methylammonium chloride (CTAC), and cetylpyridiniumbromide (CPB) were also selected to interaction with SC6Aunder the same conditions. It was found that thefluorescence intensity of SC6A increased more evidentlyby addition of proper amount of CTAC and DTABsolution like CTAB, compared with the CPB for whichnatural fluorescence peak (460 nm) is quite close to that ofSC6A (426 nm). That suggests that all of these long-alkyltrimethylammonium cationic surfactants can form com-plexes with SC6A and enhance its fluorescence intensity.It is well known that calixarenes and their derivatives can

form non-covalent inclusion complexes with various guestmolecules of suitable size and characteristics with the aid ofelectrostatic interaction, cation–p interaction, hydrogenbonding, van de Waals, hydrophobic interaction, and so on[1]. In aqueous solution, SC6A adopts an up-downdouble–partial cone conformation, which is suited to formbi-layer arrangement [39]. When CTAB were added, theammonium cationic ion of CTAB could bind with thenegatively charged sulfonyl groups of SC6A to form saltwith the help of electrostatic interaction and the N-methylgroup may enter (or partially enter) into the cavities ofSC6A by means of hydrophobic interaction, then stabilizedby the CH–p interaction between the methyl and aromaticring [39,40]. Thus the alkyl of CTAB can but stay outsideof the SC6A as the extended long chain of complexes. It isimaginable that the structure of the complexes is so similarto the one of surfactants that they may display micelle-likestate in aqueous solution, which probably makes thefluorescence spectral of SC6A change. Besides, theremaining of CTAB molecules, which occupy interstitialspaces among the complexes, may provide a specialmicroenvironment for SC6A. Therefore, the formationsof host–guest complexes as well as microenvironment werethought to play main roles in enhancement of fluorescenceintensity of SC6A.Moreover, the N atoms of amino and thiazole moiety in

the molecule of CTRX (its structure is shown in Fig. 4),

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0 1 2 3 4 5

0

50

100

150

200

250

300

F0-F

CCTAB(×10-5) mol L-1

Fig. 5. Influence of CTAB concentration on the fluorescence intensity of

system, CSC6A ¼ 5.0� 10�6mol L�1, CCTRX ¼ 6.0� 10�6mol L�1, pH 6.7.

Table 1

The thermodynamic parameters of complexation of SC6A with CTAB

Temperature (K) KS DG

(kJmol�1)

DH

(kJmol�1)

DS

(Jmol�1K�1)

288.15 15,379 �23.09

293.15 13,478 �23.15 �2.42 1.24

298.15 11,500 �23.18

303.15 9701 �23.14

C. Liu et al. / Journal of Luminescence 126 (2007) 747–752750

which own a lone-pair electron, can be protonated in weakacid media, so that they could also bind with the negativelycharged sulphonyl groups of SC6A to form salt with theaid of electrostatic interaction and the thiazole ring mayenter into the cavity of SC6A. Consequently, the decreaseof fluorescence intensity of SC6A–CTAB system may bedue to driving some of CTAB molecules away from thecavity of SC6A with addition of CTRX.

3.3. Stoichiometry and complex constant

In this paper, a similar equation of inclusion constantKn of the complex with one guest–multiple host wasused to calculate the inclusion constant: 1=DI f ¼ ðð1=KnaÞð1=ð½H�n0ÞÞÞ þ 1=a [41]. In which, DI f ¼ I fh�g � I fg � I fhwhere, Ifh–g, Ifg, and Ifh are the fluorescence intensity of thehost–guest complex, the guest molecule and the hostmolecule, respectively. [H]0 is the original concentration ofthe CTAB, n is the number of host molecule(s) in a complexand a is a constant. By drawing the 1/DIf vs. 1=½H�

n0 graph

with different n, the n that results a straight line can be takenas the number of host molecules, consequently, the inclusionratio can be obtained as 1:n. A similar way to decide theinclusion ratio was also used in cyclodextrin system byCervero and Mendicuti [42]. From the intercept and theslope of the straight line, the inclusion constant can beobtained. It was found that the 1/DIf vs. 1=½H�

n0 graph, which

was drawn according to the change of fluorescence spectramentioned above, showed a good linear relationship(R ¼ 0.9981) when the value of n was 1. Thus, the linearequation and the inclusion constants can be acquired as:1/DIf ¼ �4.07� 10�4+3.54� 10�8 1=½H�0 and K ¼ 1.15�104Lmol�1. The large inclusion constant indicates the stronginteraction of the host and the guest molecules.

The thermodynamic parameters DH and DS of theinclusion process were determined from the temperaturedependence of the binding constants using the Van’t Hoffequation

ln KS ¼ �DH

RTþ

DS

R.

Thus, the thermodynamic parameters DH and DS wereobtained from the slope and intercept of the plot of lnKS

vs. 1/T. The results obtained are listed in Table 1. It can beseen that the complex has a large negative DH and a smallpositive DS, which indicates the formation of complex is

321

Fig. 4. Structures of SC6A (1), 4-ph

mainly driven by the favorable enthalpy with a smallentropic loss [7].

3.4. Optimization of experimental variables

3.4.1. Influence of CTAB

The effect of CTAB concentration on the fluorescenceintensity of SC6A—CTAB–CTRX system was examinedon the basis of the experimental conditions of inclusionprocess. As can be seen from Fig. 5, the fluorescencequenching intensity (DF ¼ F0�F, where F0 and F representthe relative fluorescence intensity of system in absence andpresence of CTRX) of system reaches a maximum when theconcentration of CTAB is 3.0� 10�5mol L�1. Therefore,

enolsulfonate (2) and CTRX (3).

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Table 2

Determination of cephalosporin in pharmaceutical forms (P ¼ 0.95)

Drugsa Present method

(found+SD)bOfficial method [43]

(found+SD)bExperimental

t-valuec

CRTX 1 0.9070.15 1.0070.05 1.42

CRTX 2 1.0070.07 0.9070.16 0.95

aEach vial labeled to contain 1.0 g.bStandard deviation (average of five determinations).cThe tabulated values of t at the 95% confidence limit is 2.78.

Table 3

Determination of cephalosporin in commercial preparations

Drugs Sample

content

(mgmL�1)

Drug

added

(mgmL�1)

Drug

found

(mgmL�1)

RSD

(%)

Recovery

(%)+SD

(n ¼ 6)

Sample 1 0.50 3.00 3.48 2.15 99.471.41

Sample 1 1.00 3.00 3.99 1.10 99.770.91

Sample 2 0.5 3.00 3.52 0.70 100.570.82

Sample 2 1.00 3.00 4.06 0.60 101.570.60

C. Liu et al. / Journal of Luminescence 126 (2007) 747–752 751

CTAB concentration of 3.0� 10�5mol L�1 was used insubsequent experiments.

3.4.2. Influence of reaction time and the addition order of

reagents

The effect of reaction time was studied. The resultsshowed that the maximum fluorescence quenching wasreached when the solutions were incubated at roomtemperature for 25min and remained at least 2 h. Hence,a 25min reaction time was adopted in the experiment.

The effect sequence of adding reagents on the fluores-cence quenching were also investigated, and the order:SC6A, CTAB, buffer solution, CTRX was proved to bebest.

3.4.3. Influence of SC6A

The influence of SC6A concentration on the fluorescenceintensity of the solutions containing 6.0� 10�6mol L�1

CTRX and 3.0� 10�5mol L�1 CTAB was also studiedunder the conditions established above. The experimentalresults showed the fluorescence quenching intensityreached maximum when the concentration of SC6A is5.0� 10�6mol L�1. Thus 5.0� 10�6mol L�1 SC6A wasselected for further study.

3.5. Calibration curve

According to the proposed method, the calibration curvewas constructed under the optimal conditions. It was foundthat the reciprocal of the fluorescence quenching intensity(�1/DF) was proportional to that of concentration ofCTRX (1/C) in the range 9.2� 10�7–8.5� 10�5mol L�1.The linear regression equation is as follows: 1/DF ¼

1.54� 10�3+2.44� 10�7(1/C), with a correlation coeffi-cient of 0.9979 (n ¼ 10). The 3s limit of detection (LOD) is3.5� 10�7mol L�1 (here s represents the standard devia-tion of 11 blank measurements). We have to say it issomewhat regrettable that the LOD is higher than somecorresponding data that have been reported [30]. Furtherstudy should be encouraged.

3.6. Analysis of pharmaceutical forms

The suggested method was successfully applied to thedetermination of ceftriaxone in two pharmaceutical dosageforms (Samples 1 and 2 were obtained from XINYA(Shanghai) and SIDA (Hainan) Pharmaceutical IndustriesCo. in China) following the method described in Section 2.The results obtained were compared statistically with thoseobtained by applying the official methods as shown inTable 2. As can be seen there are no significant differencesbetween the two methods in the t-test because thecalculated t values of the two method were all less thanthe corresponding the theoretical one (95% confidence).

Moreover, to check the validity of the proposed method,the standard addition method was applied by addingCTRX to the samples. The recovery of each drug was

calculated by comparing the concentration obtained fromthe (spiked) mixtures with those of the pure drugs. Table 3shows the results of analysis of the commercial reagentsand the recovery study of examined drugs.

4. Conclusion

In summary, p-sulfonated calix [6] arene and CTABcould form host–guest with good fluorescence complex andat a 1:1 stoichiometry. According to the regular quenchingfluorescence intensity of the complex with addition of by anappropriate amount of CTRX, a new method for assay thedrug based on the supramolecular system was developedand was applied satisfactorily to determination of CTRXin pharmaceutical preparations. The interaction mechan-ism of the systems was also discussed. Undoubtedly, theresearch results provide very interesting and usefulinformation for pharmaceutical and biomedical analysisby employing water-soluble calixarenes.

Acknowledgment

The authors thank the financial supports of the NationalNatural Science Foundation of P.R. China (20375001,20575001), the education Commission Natural ScienceFoundation of Anhui Province (2005KJ017ZD).

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