Spectrochimica Acta Part A 58 (2002) 501–507

Resonance Rayleigh scattering technology as a new methodfor the determination of the inclusion constant of

�-cyclodextrin

Nianbing Li a, Hongqun Luo a, Shaopu Liu a,*, Guonan Chen b

a Institute of En�ironmental Chemistry, Southwest China Normal Uni�ersity, Chongqing 400715, People’s Republic of Chinab Department of Chemistry, Fuzhou Uni�ersity, Fuzhou, 350002, People’s Republic of China

Received 6 March 2001; received in revised form 11 June 2001; accepted 14 June 2001

Abstract

The interaction of procaine hydrochloride and �-cyclodextrin in aqueous solution was studied using resonanceRayleigh scattering technology. The molar ratio of the inclusion complex was 1:1 established by spectrophotometry.The resonance Rayleigh scattering technology was first applied in the determination of the �-cyclodextrin inclusionconstant. The inclusion constant of procaine hydrochloride–�-cyclodextrin complex Kf is 1.23×102 and 1.27×102 lmol−1 for method I and 1.15×102 and 1.21×102 l mol−1 for method II. These determination results were incorrespondence with the results of the spectrophotometric and fluorescence methods. Therefore, the resonanceRayleigh scattering method can be used as a new technology for the determination of the inclusion constant. © 2002Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Cyclodextrin; Procaine hydrochloride; Inclusion constant; Resonance Rayleigh scattering technology

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1. Introduction

Cyclodextrins (CDs) are cyclic oligosacharidesproduced by the action of the CD-trans-glycosi-dase enzyme on a medium containing starch. The�-, �- and �-CDs contain six, seven or eightglucose units, respectively. They are moleculeswith a truncated cone and a hydrophobic cavity[1]. Many hydroxy groups are situated on theouter part of the ring, which makes the CDs bothhydrophilic and soluble in water. Due to their

special molecular cavity structure, CDs can in-clude other ‘guest’ molecules as ‘hosts’ to forminclusion complexes. The formation of CD com-plexes improves the physical, chemical and bio-logical properties of the guest molecule. This leadsto wider applications of CD in pharmaceutical,cosmetic, food, chemical and several other indus-tries, and analytical chemistry, etc. [2–4]. As thequantitative description of the inclusion equi-librium between CD and guest molecule, the in-clusion constant reflects the strength of thebinding force between them. So, the inclusionconstant is an important and basic parameter tothe applications of CD. Up to the present, the

* Corresponding author. Tel.: +86-23-68252748.E-mail address: linb@swnu.edu.cn (S. Liu).

1386-1425/01/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 1386 -1425 (01 )00548 -0

N. Li et al. / Spectrochimica Acta Part A 58 (2002) 501–507502

reported determination methods for the CD inclu-sion constant are: spectroscopic method [5,6], sur-face tension method [7], nuclear magneticresonance method [8], phase-solubility technique[9], fluorometry [10,11], constant currentcoulometric titration method [12], high-pressureliquid chromatography [13] and electrochemistry[14].

Resonance Rayleigh scattering (RRS) is a spe-cial elastic scattering produced when the wave-length of Rayleigh scattering (RS) is located at orclose to its molecular absorption band. In thiscase, the frequency of the electromagnetic waveabsorbed by the electron is equal to its scatteringfrequency. Owing to the intensive absorption oflight energy of the electron, re-scattering takesplace. Therefore, the scattering intensity is en-hanced by several orders of magnitude com-pared with single RS and no longer obeys theRayleigh law of I�1/�4 [15]. RRS is not onlyrelated to forced vibration caused by the action ofthe electromagnetic field of the incident light in amolecule, but is also affected by energy-level transitions of the electrons. It thereforeshows also the characteristics of the scatteringspectrum as for that of the electronic absorptionspectrum. It provides new information con-cerning molecular structure, size, form, chargedistribution, state of combination and so on.Therefore, in recent years, this technique has beenincreasingly applied to the study and de-termination of biological macromolecules [16–20],trace amounts of inorganic ions [21–26] andcationic surfactant [27]. However, until now, therehave been no reports on determining the CDinclusion constant with resonance Rayleigh scat-tering.

Our experiment discovered that when �-CDreacted with procaine hydrochloride to form aninclusion complex, not only the absorbance ofultraviolet absorption band but also the RRSintensity was obviously enhanced. The peaks ofRRS were at 251 and 332 nm, which were close toits absorption peaks of 231 and 301 nm. Com-pared with its absorption peaks, the RRS peaksshifted 20 and 31nm, respectively. In this case,there was the following relationship between theintensity of RRS and the reaction equilibrium:

(I−I0)/(I�−I)=Kf[CD] and 1/�I=1/((K2−K1)CG)+1/((K2−K1)KfCG[CD]). From this rela-tionship, the inclusion constant of the�-CD–procaine hydrochloride complex was ob-tained. The results showed that the inclusion con-stant obtained using the RRS method was thesame as those using spectrophotometric andfluorescence methods. The method was highlysensitive, concise and easily operated, and was notaffected by the system whether it had fluorescenceor not.

2. Basic principles

The intensity of resonance Rayleigh scatteringcan be obtained using the equation [18,19,21]

I=KcbEex(�ex)Eem(�em)

where Eex is the excitation function at the fixedwavelength of excitation, Eem is the emission func-tion at the fixed wavelength of emission, K is thecharacteristic constant comprising the instrumen-tal geometry factor and related parameters, c isthe analyte concentration, and b is the thicknessof the sample cell. Therefore, when the conditionsof the instrument are fixed, we can obtain thefollowing:

I�c.

Namely, the linear relationship between the in-tensity of resonance Rayleigh scattering and theanalyte concentration can be obtained.

2.1. Method I

If a guest molecule, G, forms a 1:1 inclusioncomplex with CD, the complex formation can bedescribed by the following equation

CD+G � CD–G. (1)

The inclusion constant is defined as

Kf = [CD–G]/[CD][G]. (2)

If the complex and guest molecule form reson-ance Rayleigh scattering at a measured wave-length, the intensity of RRS for the guest solution

N. Li et al. / Spectrochimica Acta Part A 58 (2002) 501–507 503

after adding CD is given by the following equa-tion:

I=K1[G]+K2[CD–G] (3)

where K1 and K2 are the ratio coefficientsbetween the intensity of RRS and concentra-tions of the guest molecule and the complexformed.

The total concentration of the guest molecule,CG, is given by the material balance:

CG= [G]+ [CD–G]. (4)

When [CD]=0 (i.e. CD is not added),

K1=I0/CG. (5)

However, when the guest molecule, G, abso-lutely reacted with CD to form the complex CD–G, there is a following equation:

K2=I�/CG (6)

where I0 and I� are the intensity of RRS of theguest molecule without addition of CD and of thecomplex formed completely. Using Eqs. (5) and(6), Eq. (3) can be transformed into

I=I0[G]/CG+I�[CD–G]/CG. (7)

According to the expression of the inclusionconstant, Eq. (2) can be further transformed into

[CD–G]= [CD][G]Kf. (8)

Therefore, Eq. (4) can be rewritten as CG=[G]+ [CD][G]Kf, i.e.

[G]=CG/(1+ [CD]Kf). (9)

Using Eq. (9), Eq. (4) can be transformed into

[CD–G]=CG Kf [CD]/(1+ [CD] Kf). (10)

Using Eqs. (9) and (10), Eq. (7) can be furthertransformed into

(I−I0)/(I�−I)=Kf[CD]. (11)

From the expression of Eq. (11), it indicatesthat (I−I0)/(I�−I) is directly proportional to[CD]. Plotting (I−I0)/(I�−I) as a function of[CD], a straight line with a slope can be obtained,and the slope is Kf.

Under experimental conditions, because thequantity of CD was greatly excessive, [CD] can be

replaced approximately with the concentration ofCD, CCD. According to Eq. (11), the I0, I� and Iwere measured in the experiment. Therefore, thevalue of Kf can be obtained from the slope of thestraight line which is obtained by plotting (I−I0)/(I�−I) against CCD.

2.2. Method II

The complex formation equilibrium and theinclusion constant (Kf) are defined as:

CD+G � CD−G (1)

Kf= [CD–G]/[CD][G] (2)

where G and CD represent the guest molecule andcyclodextrin, respectively. Also,

CG= [G]+ [CD–G] (4)

CCD= [CD]+ [CD–G] (12)

where CG and CCD are the total concentrations ofthe guest molecule and cyclodextrin, respectively.

Using Eq. (4), Eq. (3) can be transformed into

I=K1CG+ (K2−K1)[CD–G] (13)

Substituting Eqs. (10) and (5) into Eq. (13),yields:

I−I0=(K2−K1)Kf[CD]CG

1+Kf[CD](14)

That is

�I=(K2−K1)Kf[CD]CG

1+Kf[CD](15)

Therefore, one obtains:

1/�I=1

(K2−K1)CG

+1

(K2−K1)KfCG[CD](16)

where CG represents the concentration of theguest molecule, and [CD] is the equilibrium con-centration of CD. K2−K1 is the coefficient, and�I is the change in RRS intensity of the guestmolecule caused by the addition of CD. The curveof 1/�I versus 1/[CD] at the optimum RRS wave-length will give a good linearity, and Kf can beobtained when the composition ratio of the inclu-sion complex is 1:1.

N. Li et al. / Spectrochimica Acta Part A 58 (2002) 501–507504

3. Experimental

3.1. Reagents and apparatus

Procaine hydrochloride obtained from theShanghai Biochemistry Research Institute was ofpharmaceutical purity grade. �-CD was fromAldrich. The water used in the experiments wastwice-quartz-distilled. A Hitachi F-2500 spec-trofluorophotometer (Tokyo, Japan) was used forrecording fluorescence spectra and RRS spectra,using a 1 cm path length. Slit (EX/EM): 5.0nm/5.0 nm, PMT voltage: 400 V. A UV-VIS 8500spectrophotometer (Tianmei company, China)was used for recording absorption spectra.

3.2. Procedure

One millilitre of 1.5×10−3 mol l−1 aqueousprocaine hydrochloride solution was added to aseries of 25 ml volumetric flasks, and then differ-ent concentrations of �-CD were added to eachvolumetric flask. The mixtures were diluted tovolume with twice-quartz-distilled water andmixed by inversion. After 5 min, the RRS spectraof these solutions were obtained with synchronousscanning at �ex=�em (i.e. ��=0 nm), and theRRS intensities, I, were measured at the optimumRRS wavelength. In order to confirm that theRRS technology could be used as a new methodto determine the CD inclusion constant, the UV-VIS spectrophotometry and fluorescence spec-troscopy were used to determine the inclusionconstant for the inclusion complex of procainehydrochloride with �-CD under the same experi-mental conditions. Then, the inclusion constantsobtained with the three methods were compared.Absorbance was measured at the maximum ab-sorption wavelength of 307 nm; the baseline wasestablished for each measurement using a solutionof �-CD at the same concentration. The intensityof fluorescence was measured at the emissionwavelength of 350 nm and the excitation wave-length of 275.5 nm.

Job’s method of continuous variation was usedto obtain the composition ratio. The absorbanceat 307 nm was measured.

4. Results and discussion

4.1. Formation of the inclusion complex and itsmolar ratio

Procaine hydrochloride is a kind of localanaeshesia and its structure is:

The diameter of the cavity of �-CD is estimatedat 6.8 A� , while the diameter of the benzene ring inprocaine hydrochloride is about 6.7�6.8 A� ,which matches the diameter of the cavity of �-CD. Therefore, the procaine hydrochloride canenter the cavity of �-CD and react with �-CD toform a steady inclusion complex.

The UV-spectra experimental results indicatedthat �-CD had no absorption band in the UV-re-gion, but procaine hydrochloride had two absorp-tion peaks that appeared at 221 and 290 nm. Theabsorption peak of 221 nm was brought by �-NH2 (� represents a phenyl group), while thepeak of 290 nm was the characteristic absorptionpeak of bi-replacement benzene ring [28]. Absorp-tion spectra of procaine hydrochloride solutionscontaining different concentrations of �-CD areshown in Fig. 1. After procaine hydrochloride wasincluded by �-CD, the two absorption peaksshifted to 231 and 307 nm, and the ��max was 10and 17 nm, respectively. This phenomenon alsoindicated that the size of procaine hydrochloridematched the diameter of the cavity of �-CD.When �-CD included the benzene ring, the cavityof �-CD with abundant electron cloud enhancedthe electron cloud density of the guest and in-creased the conjugative effect of � electron of thebenzene ring. Therefore, the energy of electrontransition from � to �* decreased, and the maxi-mum absorption wavelength (�max) shifted to alonger wavelength. The absorbance of procainehydrochloride was enhanced with the increase in�-CD concentration. The isosbestic points wereobserved at 243 and 290 nm, indicating an obvi-ous interaction between procaine hydrochlorideand �-CD. The molar ratio of the inclusion com-

N. Li et al. / Spectrochimica Acta Part A 58 (2002) 501–507 505

plex was performed using Job’s method of contin-uous variation. The result showed that the maxi-mum ratio of [�-CD]/([G]+ [�-CD]) was 0.5,which indicated that the complex had a 1:1stoichiometry.

4.2. Determination of the cyclodextrin inclusionconstant by resonance Rayleigh scatteringtechnology

Fig. 2 shows the RRS spectra of the aqueoussolution containing 5.9×10−5 mol l−1 procainehydrochloride and different concentrations of �-CD. The RRS spectra of procaine hydrochloridesolution showed that two peaks of RRS spectraappeared at 245.5 and 331 nm. With an increasein �-CD concentration, the RRS spectra bandswere enhanced and slightly shifted to a red wave-length. The effect of time on the intensity of RRSafter the complex formed has been investigatedand the results indicated that the intensity of RRSof the inclusion complex almost remained con-stant from 5 to 25 min. When the time exceeded25 min, the intensity of RRS increased slightly.

According to the basic principle of the method,the RRS intensity (I) of the inclusion complexwas measured. The RRS intensity without addi-tion of any �-CD was I0. When �-CD was exces-

Fig. 2. Resonance Rayleigh scattering spectra of procainehydrochloride in different concentrations of �-CD. Concentra-tions of �-CD: (a) 0 mol l−1, (b) 6×10-4 mol l−1, (c)1×10−3 mol l−1, (d) 2×10−3 mol l−1, (e) 3×10−3 moll−1. Concentration of procaine hydrochloride: 5.9×10−5 moll−1.

sive, the RRS intensity of the inclusion complexcould be regarded as I�. The data of I, I0 and I�

at the peaks of 251 and 332 nm were measured,respectively. The plot of (I−I0)/(I�−I) versusthe concentrations of �-CD (C�-CD) gave a goodlinearity, and the slopes were the inclusion con-stant, Kf (Fig. 3).

However, the inclusion constant, Kf, can bedetermined using RRS spectroscopic data at 251and 332 nm by Method II. A curve of 1/�I

Fig. 1. Absorption spectra of procaine hydrochloride in differ-ent concentrations of �-CD. Concentrations of �-CD: (a)6×10−4 mol l−1, (b) 1×10−3 mol l−1, (c) 3×10−3 moll−1, (d) 4×10−3 mol l−1. Concentration of procaine hydro-chloride: 5.9×10−5 mol l−1.

Fig. 3. Plots of (I−I0)/(I�−I) versus C�-CD for the inclusionof �-CD at the RRS wavelengths of 251 nm (�) and 332 nm(�).

N. Li et al. / Spectrochimica Acta Part A 58 (2002) 501–507506

Fig. 4. Curves of 1/�I versus 1/C�-CD for the inclusion of�-CD at the RRS wavelengths of 251 nm (�) and 332 nm(�).

esi–Hildebrand equation (Eq. (17)) from UV-VISspectroscopic data.

1/�A=1/(��CG)+1/(��KfCG[�−CD]) (17)

where CG is the concentration of procaine hydro-chloride, and [�-CD] represents the equilibriumconcentration of �-CD. �A is the change in theabsorbance of procaine hydrochloride before andafter addition of �-CD, and �� is the difference ofthe molar absorpitivities between complexed andfree procaine hydrochloride. Plotting 1/�Aagainst 1/[�-CD] (if [�-CD]� [G], [�-CD] can bereplaced with C�-CD) gives a straight line withslope equal to 1/(�� Kf [G]). The Kf was directlyobtained from the intercept/slope ratio. In thesame way, the inclusion constant Kf can be deter-mined using fluoroescence spectroscopic data bythe Benesi–Hildebrand equation (Eq. (18)).

1/�F=1/(�CG)+1/(�KfCG[�−CD]) (18)

where CG and [�-CD] represent the concentrationsof procaine hydrochloride and the equilibriumconcentrations of �-CD, respectively. �F is thefluorescence intensity difference, � is the coeffi-cient. The curve of 1/�F�1/[�-CD] (when [�-CD]� [G], [�-CD] can be replaced with C�-CD)was plotted, and Kf was the ratio of the interceptto the slope. Table 1 shows the results of the threedifferent methods. The results present clearly thatthe determination value with the resonance Ray-leigh scattering method is in correspondence withthe results of the two methods and of the report,which used the constant coulometric titrationmethod [12].

against 1/[�-CD] (if [�-CD]� [G], [�-CD] can bereplaced with C�-CD) was plotted, and then astraight line with slope equal to 1/((K2−K1)Kf

CG) was obtained (Fig. 4). Kf was the ratio of theintercept to the slope.

In order to confirm and compare the experi-mental and theoretical methodology describedabove, the UV-VIS spectroscopic method andfluorescence spectroscopic method have been usedto determine the inclusion constant for the inclu-sion complex of procaine hydrochloride with �-CD. The absorbance of the procainehydrochloride in water varied with addition of�-CD. The change of the absorbance (�A) wasobserved as a function of the concentration of�-CD added. The inclusion constant value Kf forthe inclusion complex was evaluated by the Ben-

Table 1Inclusion constants of procaine hydrochloride–�-CD complex with different determination methods

Method Resonance Rayleigh scattering method UV-VIS spectroscopic Fluoroescence spectroscopicmethodmethod

Method I Method II

350332251 307Determination 332251wavelength, � (nm)

1.27 1.151.21 1.23Kf (×102 l mol−1) 1.071.23

The number of determinations (n) is 4.

N. Li et al. / Spectrochimica Acta Part A 58 (2002) 501–507 507

5. Conclusions

In this work, resonance Rayleigh scatteringmethod is first applied to study the reaction of�-CD with procaine hydrochloride. A newmethod for determination of the �-CD inclusionconstant by RRS technology was developed. Theresults obtained by this method are satisfactory.

Acknowledgements

This project is supported by the National Natu-ral Science Foundation of China, and all authorshere express their deep thanks.

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