ELSEVIER Analytica Chimica Acta 344 (1997) 137-143

ANALYTICA CHIMICA ACTA

Formation constants of polynuclear aromatic compounds and P-cyclodextrin inclusion complexes in P-cyclodextrin modified mobile phase high performance liquid chromatography system

Jing-Jau Tang, L.J. Cline Love*

Department of Chemistry, Seton Hall University, South Orange. NJ 07079, USA

Received 9 February 1996; received in revised form 13 November 1996; accepted 14 November 1996

Abstract

The effects of molecular size and shape upon the formation of inclusion complexes of b-cyclodextrin (/-/-CD) and polynuclear aromatic compounds in P-CD modified mobile phase high performance liquid chromatography (HPLC) system were studied. A correlation was established between the molecular size and shape of the probe compounds and the formation constants of the inclusion complexes. The formation constants were then used to support the elucidation of inclusion complexes formation based on the structural properties.

Kqvwordst Liquid chromatography; Cyclodextrins; Inclusion complexes

1. Introduction

Cyclodextrins are cyclic oligosaccharides made up of glucopyranose units bonded together via a-(1,4)-

linkages. They are pictured as hollow truncated cones. The toroidal structure has a hydrophilic surface result- ing from the 2, 3 and 6 position hydroxyl, and the cavity is composed of the glucoside oxygens and

methylene hydrogens giving it an apolar character. The basic property of cyclodextrins that allows them to effect numerous chemical separations is their ability to form selective inclusion complexes with a variety of guest molecules. The formation of this inclusion

complex may be caused by either a hydrophobic effect, hydrogen bonding, or the release of high energy water or modifier during complex formation or a

*Corresponding author.

0003.2670/97/$17.00 ‘c 1997 Elsevier Science B.V. All rights reserved.

PII SOOO3-2670(96)00577-6

combination of the above factors [I]. In general, binding to the cyclodextrin is governed by the mole-

cule’s ability to closely fit the cavity of the cyclodex- trin, although the polarity of the molecule also plays an important role. This fit is depending on both size and shape of the analyte concerning the cyclodextrin cavity. If the molecule is too small or too bulky, there

may be little or no binding at all. However, binding of the larger molecules to the cyclodextrin may be possible if certain groups or side chains of the mole-

cules can penetrate the cavity effectively. Alpha- cyclodextrin ((w-CD), with six glucose units, has the

smallest cavity (i.d. 5.7 A), which often is too small for a pharmaceutical molecule. Beta-cyclodextrin (p- CD), with seven glucose units, is more convenient (i.d. 7.8 A). Gamma-cyclodextrin (y-CD), with eight glu- 0 case units and an i.d. of 9.5 A, should be the best one, but it is not in fact intensively produced and remains

138 J.-J. Tang, L.J. Cline Love/Analytics Chirnica Acta 344 (1997) 137-143

impossible to use on an industrial scale. The height of all cyclodextrins is 7.8 A [2]. For the moment, /?-CD is

the most popular used natural cyclodextrin because of its low cost and unique physical characteristics, thus is used in this study.

The primary objective of this work is to demonstrate the effects of molecular size and shape upon the formation of inclusion complexes of some polynuclear aromatic compounds and P-CD. The equations

derived from chromatographic parameters can be used to directly calculate the formation constants and con- sequently to support the elucidation of inclusion com-

plexes formation based on structural properties. Benzene, naphthalene, biphenyl, fluorene, anthracene and phenanthrene are chosen as probe compounds.

They are structurally similar and are different only in

size and shape. Therefore, the effects of size and shape on the formation of inclusion complexes can be spe- cifically demonstrated.

2. Experimental

Reagents: P-CD was purchased from Sigma (St.

Louis); acetonitrile, benzene, naphthalene, biphenyl were purchased from Fisher (Fair Lawn, NJ); anthra-

cene, fluorene and phenanthrene were purchased from Aldrich (Milwaukee, WI).

Instrumentation: The high performance liquid chro- matography (HPLC) system used for the study con- sisted of a Waters Model 616 pump (Waters Chromatography, Milford, MA), a Waters column heater (set at 3O”C), a Waters Model 486 LC Spectro-

photometer at a wavelength 254 nm and a Waters WISP 7 17 autosampler. Data acquisition and chroma- tographic integration were carried out using a Beck- man LIMS System. The mobile phase contained water

and acetonitrile at the ratio of 80 : 20 (v/v). The

appropriate weights of P-CD (5.675-22.700 g) were then dissolved in 1 1 of mobile phase and filtered through a 0.45 pm Nylon-66 membrane filter (What- man, Clifton, NJ). A flow rate of 2.0 ml min-’ was

used throughout the study, and 5 ul of a solute or 20 ~1 of a mixture of solutes was injected onto a Zorbax SB- CN column (15 cmx4.6 mm i.d., 5 pm; MAC-MOD Analytical, Chadds Ford, PA). The first peak-trough combination caused by the change in refractive index of each injection was used as the dead time, to. of the

system. The average to obtained, 0.89 min, was used

for all K calculations.

3. Results and discussion

In the HPLC system using P-CD as a mobile phase modifier, complexation of an analyte with P-CD introduces a second principal equilibrium in the chro-

matographic separation. The equilibrium expressions are presented by the following set of equations in which the species concentration in the stationary phase and in the mobile phase are denoted by sub-

scripts s and m, respectively. The concentration of all species is defined in mol l-i, and [,&CD,] is the

concentration of P-CD in the mobile phase.

E,+L, SE* L,, (1)

E, + P-CD, 2 E * P-CD,, (2)

P-CD, + L, 2 P-CD * L,, (3)

E*/3-CDm+LssE*/3-CD*L,. (4)

The formation constants corresponding to Eqs. (l)-

(4) are denoted by K,, K2, K3 and K4, respectively. Eq. (1) is a reversible equilibrium of solute in the bulk solvent mobile phase, E,, with the stationary phase

sites, L,, to form a complex, E*L,. The concentration of the solute, [E,], should be kept below the bonded- phase concentration, [L,]. If [E,]>[L,], the result is a decrease in retention time and change in band shape [3]. Eq. (2) is a reversible equilibrium of solute in the bulk solvent mobile phase, E,, with the P-CD in the mobile phase, P-CD,, to form an inclusion complex,

E*P-CD,. Eq. (3) is the adsorption of P-CD onto the stationary phase of the column, and converts it into a chiral stationary phase. Eq. (4) is the adsorption of the

inclusion complex, EtP-CD,, onto the stationary phase.

Because of the hydrophilic nature of the external faces of P-CD and the hydrophobic characteristics of the nonpolar stationary phase, the interactions between them are extremely limited [4-61. The com- petition between mobile phase and the stationary phase for the P-CD plays a very important role. When the solubility of @-CD in the mobile phase is low, the

J.-J. Tang, L.J. Cline Love/Analytics Chimica Acta 344 (1997) 137-143 139

adsorption of P-CD onto the stationary phase becomes

possible. Depending on the bonding techniques, some reversed phase bonded phases still exhibit some hydrophilicity, and can interact with hydrophilic groups, such as P-CD. The equilibria expressed in

Eqs. (3) and (4) will occur when “low-soluble for ,& CD” mobile phase and “hydrophilic” columns are used [7]. The adsorption of P-CD or solute-P-CD

inclusion complex onto the stationary phase can be evident by the change of the column characteristics - retention time, efficiency, peak shape, etc. A compar- ison of column characteristics, before and after the column is exposed to the P-CD mobile phase, shows

that the column characteristics are not changed when using a P-CD modified mobile phase containing 10%

of acetonitrile in water [8]. In this study, a mobile phase containing 20% of acetonitrile in water is used.

The solubility of B-CD is better in this mobile phase [9]; therefore, the adsorption of P-CD or solute-P-CD inclusion complex onto the stationary phase, as expressed in Eqs. (3) and (4) will not happen. There is no evidence of the E,*,!-I-CD complex binding to the stationary phase in these studies; hence, the retention mechanism is actually a two equilibria process and

controlled by Eqs. (1) and (2). The capacity factor of solute, k’. is defined in the usual way as:

4[E * L] k’ = ([E,] + [E * ~-CD,]) ’

where 4 is the phase ratio, i.e., the ratio of the volume

of the stationary phase, Vs, to the volume of the mobile

phase, V,,, in the column. The combination of

Eqs. (l),(2) and (5) yields the following expression for the capacity factor in terms of Ki and K2:

[E * L] K’ = [~ml[L] ’

K* = P * ~-wlll [EmI [P-C&,1 ’

(7)

&,]KI k’ = (1 + Kz[,O-CD,]) ’

(8)

Taking the reciprocal of both sides, one obtains

J_ [Y-CD&, 1 k’ - dL]K, + d&]K, ’

(6)

(9)

By plotting l/K vs. [P-CD,] in mol l-‘, one should

obtain

(10)

1 Intercept = d,L,lK, ,

Slope K -=

Intercept 2

(11)

(12)

The value K2 is given by the ratio of slope/intercept

from the plot. Fig. 1 shows the chromatograms of the mixtures of aromatic solutes with different concentra-

tions of /?-CD added to the mobile phase as a modifier at the column temperature of 30°C. The elution orders

of all analytes except that of anthracene and phenan- threne stay unchanged when P-CD is added as a

mobile phase modifier. In the reversed phase HPLC

without P-CD added to the mobile phase, anthracene is more retained on the stationary phase. As the P-CD is added to the mobile phase, anthracene forms a more stable inclusion complex with the P-CD. The inter- action with the stationary phase decreases and anthra- cene is eluted earlier than phenanthrene; thus, the elution order of these two compounds is reversed.

The retention decreases as the [P-CD,] increases for all solutes, which is expected and predicted by

Eq. (8). In the system without P-CD added to the mobile phase, Eq. (8) becomes K=$[L,]Ki, with K directly proportional to K,, and the elution order is

according to the strength of hydrophobicity of the solutes: benzene<naphthalene<biphenyl<fluorene< phenanthrenecanthracene. Chmielowiec and Sawatzky

have obtained the same results by using a Cl8 column [lo]. When P-CD is present in the mobile phase, the value of the denominator in Eq. (8) increases as [p- CD,] is increased, and K decreases.

The dependence of l/K upon concentration of P-CD at 30°C is graphically illustrated in Figs. 2 and 3.

According to Eq. (9), linearities are obtained from the plots of l/K vs. [P-CD,], resulting with regres- sion coefficients as 0.991 or higher. The formation

constants, K2, for all compounds at column tempera- ture 30°C are listed in Table 1. Solubilities of solutes

in water at 25°C are also listed [ll], there is no correlation between the solubilities and formation constants.

The first important consideration to form a stable inclusion complex is proper fit of the molecule to the

140 L-J. Tang, L.J. Cline Love/Analytics Chimica Acta 344 (1997) 137-143

E

= A C I ^o Z B F

D

1- ,,, A, j i, ’ ,A, [l3-CD]=O.OZOM

I I,, A, /\, [BCD]=O.OlSM

r ‘A ’

A= Benzene B= Naphthalene C= Biphenyl D= Fluorene E= Anthracene F= Phenanthrene

I

[f3-CD]=O.OIOM /\, i \, /\, ____

E F

Q ID. ::-,r II A, /I

[o-CD]=O.OOSM

L o- E

d F bN_ E,. I I, n,

[l!-CD]=0 M A A

; -_ I ” ” I ” ” I ” ” I 1 ” I ". , ” ” I " I

10 20 30 40 50 60 70 Elution Time Minutes

Fig. 1. Chromatograms of the analytes using various concentrations of P-CD as a mobile phase modifier at the column temperature, 30°C.

[O-CD], M

Fig. 2. The dependence of the Inverse Capacity Factor upon the

concentration of B-CD for analytes at the room temperature of

30°C (0 fluorene, 0 anthracene, x phenanthrene).

cyclodextrin cavity. This fit is a function of both size and shape of the analyte relative to the cyclodextrin cavity. As a general rule substituted phenyl, naphthyl and biphenyl rings can be included in the cavity of the P-CD, while smaller molecules can be included in the a-CD. The enantiomers of an analyte like Norgestrel

0 ’ 0.004 ’ 0.008 ’ 0.012 ’ 0.016 ’ 0.02

[O-CD], M

Fig. 3. The dependence of the Inverse Capacity Factor upon the

concentration of P-CD for analytes at the column temperature of

30°C (0 benzene, 0 napthalene, x biphenyl).

(a S-ring steroid structure) are better separated on a y- CD [ 121. Armstrong et al. have obtained the computer projections of the inclusion complexes of d-propra- nolo1 and I-propranolol in P-CD from X-ray crystal- lographic data and indicated that both rings of the naphthyl group fit into the cavity for optimal com-

Table 1

J.-J. Tang, L.J. Cline Love/Analytics Chimica Acta 344 (1997) 137-143 141

Formation constants, K2, for solute/b-CD inclusion complexes

Compound Correlation Slope

coefficient

r

Intercept Formation constant Solubihty

in water

iG 1 ’

Benzene

0

Naphthalene

1.000 19.148

0.991 7.590

Biphenyl m 1.000 4.842 0.073 0.02668 0.00089 181.48 3.61 7000

Fluorene 0.998 1.908 0.073 0.02170 0.00089 87.93 8.25 1980

Anthracene m 0.999 1.758 0.047 0.01228 0.00057 143.16 5.39 73

Phenanthrene 0.994 0.996 0.061 0.01384 0.00075 71.97 8.19 129

0.268 0.30284 0.00328 63.23 1.73 18800

0.591 0.05546 0.00724 136.86 4.81 31700

plexation [ 131. Stefansson et al. also reported that NMR and fluorescence measurements supported the

proposed complex between 5-aminonaphthalene-2- sulfonate-n-galactose and P-CD: the naphthyl groups positioned deep in the CD cavity [14].

The benzene molecule is small and contains only one aromatic ring, can enter and exit the cavity easily, and forms a loosely fit inclusion complex with P-CD.

Phenanthrene, on the other hand, contains three aro- matic ring and is bulky; the molecule is too big to fit into the /?-CD cavity effectively. Therefore, benzene

and phenanthrene have low formation constants as expected, 63 and 72, respectively. Naphthalene in which will form inclusion complex with the whole

molecule included inside the /?-CD cavity, has a formation constant of 137. For the fluorene molecule, the existence of the methylene group between the two rings causes an interaction with the secondary hydro- xyls of the P-CD and limits the insertion of the fluorene molecule so that only half of the molecule

can get into the cavity [15]; this supposition is con- firmed by the low formation constant, 88. Armstrong

et al. have demonstrated this interaction in a study using compounds that differ only in the type or pre- sence of a heteroatom between the two aromatic rings, and resolved them on a P-CD stationary phase column

[16]. Biphenyl has two aromatic rings joined by a C-C single bond that allows the two aromatic rings to rotate freely. When interacting with P-CD, the biphenyl molecule is included inside the P-CD cavity. The

two aromatic rings would rotate to come to a position

that has the optimal fit and maximum interaction with the P-CD cavity. The resulting inclusion complex is the most stable among the compounds studied, and has

a formation constant of 18 1. Anthracene molecule contains three aromatic rings in a row. By using bond lengths, Blyshak et al. have estimated the molecular dimension for anthracene, 5.0x9.2 A [ 171. This indi- cates that the linear molecule can penetrate into the ;I- CD cavity most of the way, with a fraction of the

142 J.-J. Tang, L.J. Cline Love/Analytics Chimica Acta 344 (1997) 137-143

molecule left outside; therefore, anthracene molecule has comparable contact (interaction) with the P-CD

cavity like the naphthalene molecule. This is con- firmed by the resulting comparable formation con- stant, 143, for the anthracene*P-CD inclusion complex.

Seeman et al. have used P-CD column with a gradient going from 40% to 70% methanol in 25 min to obtain the following elution order: benzene<

naphthalene<fluorene<phenanthrene<biphenyl [ 181. This order also represents the stability of the formed

inclusion complexes between the aromatic analytes and the P-CD on the stationary phase of the HPLC

column. Analytes that formed more-stable inclusion complexes would be retained longer and be eluted later, and those formed less-stable complexes would be eluted earlier. Biphenyl formed the most stable inclusion complex, and benzene formed the least stable complex. Anthracene was not studied in that experiment. Compared to the results obtained here

using chiral mobile phase approach with P-CD as a modifier: benzene<phenanthrene<fluorene<naphtha- lene<anthracene<biphenyl. The stabilities of phenan-

threne and naphthalene are opposite in these two approaches. Armstrong et al. have reported that the retention order of the enantiomers is reversed when

using a P-CD stationary phase as opposed to using p- CD as a mobile phase additive for enantiomeric resolution of racemic nicotine and nicotine analogues [18,19].

4. Conclusion

The results presented in this study have correlated equilibrium characteristics and structural properties.

The equilibrium data can be calculated for the trans-

fer of the solute to either the stationary phase or the ,0- CD from the bulk aqueous phase. The effects of molecular size and shape upon the formation of inclusion complexes can be assessed in term of for- mation constants. The formation behavior of inclusion complexes described for formation constants and structural properties are in good agreement. This approach has its limitations when the solubility of P-CD in mobile phase is low. The adsorption of /?-CD onto the stationary phase becomes evident [7], Eqs. (3) and (4) can not be ignored, and more com-

plicate equations will have to be developred. To achieve efficient chiral separations of enantiomers through formation of inclusion complexes, there are other factors that influence the formation of the inclu- sion complexes of P-CD [20,21]. The types, sizes, numbers and positions of the substituents on the aromatic ring and the hydrophilic interaction between the hydroxyl groups outside the cavity of P-CD and

guest molecules are all important determining factors. Studies involving some of these factors using anti-

histamine drugs and mandelic acid derivatives have been reported [8].

Acknowledgements

The authors are grateful to Hoffmann-LaRoche Pharmaceutical, Inc., for their support and permission of using instruments for the entire study.

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