Anal. Chem. 1986, 58,2668-2674
2668
ACKNOWLEDGMENT We thank A* Ahmad and B. Tahiruddin for some of the measurements.
(13) (14) (15) (16)
de Clerk, K.; Buys, T. S. J . Chromatogr. 1971, 6 3 , 193. Buys, T. S.; de Clerk, K. J. Chromatogr. Sci. 1972, 70, 722. de Clerk, K.; Buys, T. S. J . Chromatogr. 1973, 8 4 , 1. Muth, J. P.; Wilson, D. J.; Overholser, K. A. J . Chromatogr. 1973, 8 7 , 1.
LITERATURE CITED AI-Thamir. W. K.; Purnell, J. H.; Laub, R. J. J . Chromatogr. 1980, 188, 79. Conder, J. R.; Young, C. L. Physicochemical Measurement by Gas chromatography: Wiley: Chichester, 1979. Conder, J. R. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1982, 5 , 341, 397. Dondi, F.: Beni, A.; Blo, G.; Bighi, C. Anal. Chem. 1981, 5 3 , 496. Excoffir, J . L.; Jaulmes, A.; VMal-Madjar, C.; Guiochon. G. Anal. Chem. 1982, 54. 1941. Jaulmes, A.; VMaCMadjar. C.; Ladurelli, A.; Guiochon, G. J . Phys , Chem. 1984, 88,5379. Conder, J. R. Anal. Chem. 1971, 43, 367. Barber, W. E.; Carr, P. W. Anal. Chem. 1981, 53, 1939. Conder, J. R.; Young, C. L. Physicochemical Measurement by Gas Chromatography;Wiley: Chichester, 1979; pp 75, 287ff. Rouchon, P.; Schoenauer, M.; Valentin, P.; Vidal-Madjar, C.: Guiochon, G. J . Phys. Chem. 1985, 8 9 , 2076. Houghton, G. J . Phys. Chem. 1963, 6 7 , 84. Haarhoff, P. C.; van der Linde, H. J. Anal. Chem. 1988, 3 8 , 573.
(18) Pollard, F. H:; Hardy, C. J. I n Vapour Phase Chromatography (First International Symposium on Gas Chromatography, London, 7956); Desty. D. H., Ed.; Butterworths: London, 1957; p 115. (19) Conder, J. R.; Rees, G. J.; McHale, Sonia, J . Chromatogr. 1983, 258, 1. (20) Littlewood, A. 6.; Phillips, C. S. G.; Price, D. T. J . Chem. SOC.1955, 1480. (21) Conder, J. R.; Young, C. L. Physicochemical Measurement by Gas Chromatography; Wiley: Chichester, 1979; pp 83, 87. (22) Scott, C. G. In Gas Chromatography 7962: van Swaay, M., Ed.; Butterworths: London, 1962; pp 36, 46. (23) Conder, J. R. J . Chromatogr. 1982, 2 4 8 , 1. (24) Hicks, C. P. Ph.D. Thesis, University of Bristol, 1970.
RECEIVED for review March 10, 1986. Resubmitted June 4, 1986. Accepted June 16, 1986. Work supported, in part, by the Science and Engineering Research Council.
Reversed-Phase Retention Behavior of Aromatic Compounds Involving P-Cyclodextrin Inclusion Complex Formation in the Mobile Phase Kazumi Fujimura,* Teruhisa Ueda, Masashi Kitagawa, Hiroaki Takayanagi, and Teiichi Ando
Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Sakyo-ku, Kyoto 606, Japan
The effect of the Inclusion complex formation of solutes with P-cyclodextrln added In the mobile phase on their retention and selectlvtty has been investlgated. A simple equatlon has been derlved that reveals the hyperbolic dependence of the capactty factor, k', on the total concentratkn of cyckdextrin, [CDh,In the mobile phase. A plot of the reciprocal of the capactty factor against [CDhgives a straight line, from the slope of which the dissociation constant, KOl of the Inclusion complex can be calculated. The KO values thus obtained In aqueous methanol were somewhat larger than those observed In 100% water. I t was possible to edlmate the K D values in 100% water from a Hnear plot of pKD vs. water content In the solutbn by extrapolation. The separatlon factor, a,of two substances has been found to be affected not only by the [CDhbut also by their K, values, indicating that three cases exist; Le., a values increase wHh a convex shape or decrease wtth a concave shape wtth an Increase ot [CDh In the moMle phase or cannot be changed by the change of [CDh.
In recent years there has been considerable interest in the utilization of cyclodextrins (CDs) as a stationary or a mobile phase in chromatography owing to their ability to form incluson complexes, i.e., host-guest compounds, with a variety of organic molecules or ions both in the solid state and in aqueous solution (1, 2). Since the host-guest interaction depends on the fitness of the structural features of the guest molecule to the cavity of CD, the introduction of CDs in the chromatographic system can be expected to vary the retention
value and hence to improve the selectivity in separating homologues and, in particular, structural or geometrical isomers. In practice, numerous studies dealing with the use of an cy- or p-CD and/or its derivative as a mobile phase additive in liquid chromatography have demonstrated that the retention time of a solute that is less hindered for inclusion becomes usually shorter reflecting that the interaction between the solute and the stationary phase is weakened by complex formation (3-21). Since the change of the retention value caused by the formation of the inclusion complex is closely related to the stability of the inclusion complex, some attempts have been made to estimate the binding or dissociation constant of the complex from the relationship between the retention value and the concentration of CD in the mobile phase (4, 9, 25, 18, 21). However, the equation used in such approaches for describing the dependence of capacity factor on the concentration of CD in the mobile phase is much too complicated; e.g., in the reversed-phase system, it does not take into account that very probably the retention of included species formed in the mobile phase onto the hydrophobic stationary phase can be neglected. Furthermore, the binding constant of the inclusion complex obtained by the chromatographic method, especially by reversed-phase liquid chromatography (RPLC) using an aqueous-organic mobile phase, is not independent of the mobile phase compositions being used, because the retention value of the solute is influenced not only by the concentration of CD but also by the type and the content of organic solvent in the mobile phase. An increase in the selectivity due to formation of the inclusion complex has also been observed in many cases, but not necessarily in all cases. Because of the lack of quantitative
0003-2700/86/0358-2668$01.50/0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
treatment of the dependence of the separation factor on the binding or dissociation constant of the complex, on the concentration of CD, and on the.type and the content of organic solvent in the mobile phase, the optimization of the eluting conditions is still very difficult. The purpose of the present work is to examine the retention mechanism of some aromatic compounds in a reversed-phase system involving p-CD inclusion complex formation in the mobile phase. The effect of the concentration of CD and the organic solvent in the mobile phase on the capacity factor and the separation factor will be discussed. An attempt to estimate the dissociation constants of CD inclusion complexes from the relationship between capacity factor and CD concentration in the mobile phase will also be described.
EXPERIMENTAL SECTION Apparatus. The liquid chromatographic system consisted of a Waters Model 6000A pump (Waters Associates, Milford, MA) connected to a Rheodyne Model 7120 sample injector with a 20-pL sample loop (Rheodyne, Inc., Berkeley, CA) and a Jasco Model UVIDEC-100-11 variable wavelength UV absorbance detector (Jasco, Tokyo, Japan) or a Shodex Model RI SE-11 differential refractometer (Showa Denko K. K., Tokyo, Japan). The columns (4 mm i.d. X 20 cm) were home packed by the balanced viscosity method using a Chemco Model 124 slurry packing apparatus (Chemco Scientific Co., Ltd., Osaka, Japan) at ca. 450 kg/cm2. Reagents and Procedure. All sample compounds were of the highest quality available and were purchased from various suppliers. a- and 0-CDs and D-glucose were also of the highest quality available and were obtained from Nakarai Chemicals, Ltd. (Kyoto, Japan). These reagents were used without further purification. Methanol, acetonitrile, and tetrahydrofuran (THF) used as an organic modifier of aqueous mobile phase were special preparations for HPLC and were purchased from Nakarai Chemicals, Ltd. Water was processed with a Milli-Q water purification system (Millipore Corp., Bedford, MA). The column packing materials were LiChrosorb RP-8 of 5 pm average particle size (E. Merck, Darmstadt, GFR). The slurry solvent for column packing was a mixture of methanol/2-propanol/carbontetrachloride/cyclohexanol/toluene (5:5:18:10.8:1.2 by volume) and the pumping solvent was methanol. The mobile phase, i.e., an aqueous-organic solvent containing CD in various concentrations, was prepared by dissolving weighed amounts of CD in purified water by ultrasonic vibration and then mixing it with an organic solvent. The CD used was vacuum dried under ca. 5 torr (133.3 Pa) at 80 "C for 8 h before use. The solvent thus obtained was filtered through a membrane filter of 0.7 pm pore size and was degassed prior to use by a vacuum-ultrasonic method. Sample solutions were prepared so as to give a concentration of 1mg/L for each solute using a solvent identical with the mobile phase in its composition except for the CD. The amount of the sample injected was usually 0.5 pL. All chromatograms were obtained at 23 "C. The flow rate was 0.7 mL/min, and the UV detector was operated at 254 nm. The dead time, to, was measured with potassium nitrite. RESULTS AND DISCUSSION Effect of Mobile Phase Additives on Retention. In reversed-phase liquid chromatography, it is known that the hydrophobic interaction, i.e., dispersion forces operating between the bonded alkyl moiety of the stationary phase and the nonpolar part of the sample molecule, plays an important role in determining the retention value of the sample solute. Since the hydrophobic interaction is affected by various factors such as the chain length and the amount of bonded alkyl moieties in the stationary phase, as well as the type and the content of the organic solvent and the additives in the mobile phase, the addition of CD in the mobile phase is expected to cause a change of the retention value of the sample solute owing to the formation of inclusion complex. The effects of additives in the mobile phase on the retention of solutes on a column of LiChrosorb RP-8 are shown in Table I. The capacity factor, k', of all solutes examined decreased to a greater extent in the presence of @-CDthan in the
2669
Table I. Effect of Additives in Mobile Phase on Capacity Factors" k' ( A k ' ) b 6 mM 0-CD
no addition
6 mM a-CD
o-cresol m-cresol p-cresol
9.2 8.8 8.9
8.9 (-0.03)
8.6 (-0.07) 9.2 (*O.OO)
rn-nitrophenol p-nitrophenol l-naphthylamine Z-naphthylamine
8.2
8.5 (-0.03) 8.5 (-0.04) 7.7 (-0.06)
7.8 (-0.11) 7.1 (-0.20) 7.4 (-0.10)
substance
36 mM D-glucose
8.8 (iO.00) 8.9 (iO.00)
7.3 15.9
8.2 (*O.OO) 5.6 (-0.23) 7.3 (fO.OO) 15.0 (-0.06) 13.1 (-0.18) 15.9 (*O.OO)
18.4
17.2 (-0.07) 10.2 (-0.45) 18.4 (AO.00)
6.2 (-0.15)
" Column, LiChrosorb RP-8 (4 mm i.d. X 20 cm); mobile phase, methanol/water (40/60); flow rate, 0.7 mL/min. Ak' indicates the variation of k' value defined as: Ak' = (k\dditive - k'&,d,)/ k LA .
10
8
4
E mM
Concentration of p-CD.
6
Figure 1. Variation of capacity factors of 1-naphthylamine(1) and 2-naphthylamine (2) in 50 % MeOH (A), 40 % CH,CN (B), and 40 % THF (C) in the presence of @-CD: column, LiChrosorb RP-8, 4 mm i.d. X 20 cm; flow rate, 0.7 mL/min; detection, UV 254 nm.
presence of a-CD at their concentration of 6 mM, whereas the addition of D-glucose, which is a constituent of the CD molecule but not capable of forming an inclusion complex, could not alter the k'values despite the concentration of D-glucose being the same as that of a-CD in the number of glucose units. These results clearly indicate that (1)the decrease in k'values caused by the addition of CDs in the mobile phase is based on the formation of an inclusion complex, resulting in weakening of the hydrophobic interaction between solutes and the stationary phase, and (2) the ability of p-CD to form an inclusion complex is larger than that of a-CD. Thus, it was considered suitable to use @-CDfor the present investigation. Effect of Organic Solvent in the Mobile Phase on Retention. Since the formation of CD inclusion complexes in the liquid phase proceeds more easily in an aqueous solution, the use of an aqueous-organic solvent as a mobile phase is essential for the present system. In the selection of an organic solvent, in a reversed-phase system the retention value and the resolution of the sample solute and the binding constant of inclusion complexes of the solute are dependent on the type of organic solvent and its content in the mobile phase. Figure 1 shows the effect of the type of organic solvent in the mobile phase on the retention and the resolution of 1and 2-naphthylamine isomers, where the content of each organic solvent tested is adjusted to 50% for methanol, 40% for acetonitrile, and 40% for T H F so that the similar k'value may be obtained for each compound when p-CD is absent in the mobile phase. Under such mobile phase conditions, the eluting power of the mobile phase in a reversed-phase system becomes identical. It is clear from Figure 1that the inclusion effect of 6-CD is strongest in aqueous methanol, whereas only
2670
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
stationary phases, respectively. The equilibrium constants are given as follows: dissociation constant of CD-S
1.5
z
p:::4 3
cn
2
1.0
5
0.5
distribution constant of S
4
distribution constant of CD-S
(3) / 6
0
50
70
60
Water content, % Figwe 2. Effect of water content in mobile phase on capacity factors of 1-naphthylamine (I), 2-naphthylamine (2), o-cresol(3), pcresoi(4), rn-nitroaniline (51,and p-nitroaniline (6): mobile phase, MeOH/H,O = (100 X ) / X , containing 6 mM @-CD. Other chromatographic conditions are the same as in Figure 1.
-
a poor effect is observed in aqueous THF. This result may be ascribed to the difference in the magnitude of the interaction between @-CDand the molecule of each organic solvent; i.e., the interaction between /3-CD and methanol molecules is so weak that the complexation of the sample molecule with @-CD is little retarded by methanol. This assumption is supported by the facts that the k‘ values of these organic solvent molecules measured on a stationary phase of @-CD bonded to silica surface using water as a mobile phase were 0.67 for methanol, 0.75 for acetonitrile, and 0.78 for THF. Actually, the formation constant of the inclusion complex of methanol with @-CDwas found to be as small as 0.32 (22). Furthermore, the solubility of @-CDin methanol is largest among these three organic solvents. I t may be concluded, therefore, that aqueous methanol is the best as a mobile phase for the present chromatographic system. The content of the organic solvent in the mobile phase also influences k ’values of the solutes. As shown in Figure 2, k‘ values increase with a decrease of the content of organic solvent in the mobile phase, and a linear relationship exists between the logarithm of k’and the content of water in the mobile phase, as generally observed in a reversed-phase system even in the presence of @-CD. This implies that the retention of the solute depends only on the hydrophobic interaction between the unincluded species and hydrophobic stationary phase, while the included species are hydrophilic enough not to be retained any longer, as will be discussed later. It is noteworthy that an increase of the water content in the mobile phase brings about an increase of the selectivity of isomers. Retention Mechanism-Theoretical Treatment. When a sample solute, S, is introduced into the column in the presence of 8-CD in the mobile phase, the following equilibria will be established
k0 (S)S
1t.l
(CD-S) s
In these equilibria, subscripts m and s denote the mobile and
In the above schemes, the dissociation and/or the protonation of sample solutes is not taken into account, since the solutes examined are nonelectrolytes or very weak electrolytes and the equilibration is treated in a neutral mobile phase. Furthermore, the distribution equilibrium of CD itself between the hydrophobic stationary phase and the hydrophilic mobile phase is assumed to be negligible because of the hydrophilic nature of the external faces of CD; this view is supported by the fact that the retention time of @-CDwas nearly the same as that of potassium nitrite used as a marker for measuring the column dead volume. For the same reason, the distribution equilibrium of the included species, (CD-S), onto the stationary phase expressed by eq 3 may also be neglected. The capacity factor, k’, of the sample solute can therefore be written as (4) where 4 denotes the phase ratio of the column. As the total concentration of CD, [CD],, added in the mobile phase is given by [CDIT = [(CD),] + [(CD-S),], eq 4 is expressed as
Since the concentration of the sample solute added is very low compared with [CD], in the mobile phase, it can be assumed that [CD], - [(CD-s),] = [CD]T. Furthermore, KO+ is equal to the capacity factor, k{, obtained in the absence of CD; therefore, eq 5 reduces to
It is clear from eq 6 that k’shows a hyperbolic dependence on [CD]T and a plot of l / k ’ vs. [CD]T gives a straight line whose slope is equal to 1/KDkd. Thus, eq 6, which is exactly the same as that proposed recently by Cline Love and Arunyanart for describing the retention characteristics of a solute in an aqueous organic mobile phase containing (3-CD on a stationary phase based on the three-phase equilibrium model in micellar chromatography (22), seems to be more useful than the one reported earlier for measuring KDvalues chromatographically (23). Although a similar equation representing the relationship between capacity factor and binding constants of CD inclusion complex in RPLC system has been proposed by Zukowski et al. ( I @ , eq 6 is much simpler and is considered to be more suitable for a RPLC system dealing with relatively small organic guest molecules. It should be noted, however, that if the sample solutes are electrolytes, the pH of the mobile phase must be adjusted so that only one
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
2671
Table 11. Effect of p-CD .Concentration in Mobile Phase on Capacity Factors"
substance
0.00 mM
2.19 mM
o-fluorophenol rn-fluorophenol p-fluorophenol o-iodophenol rn-iodophenol p-iodophenol o-nitrophenol rn-nitrophenol p-nitrophenol o-chlorophenol rn-chlorophenol p-chlorophenol o-cresol rn-cresol p-cresol o-fluoroaniline rn-fluoroaniline p-fluoroaniline o-iodoaniline rn-iodoaniline p-iodoaniline o-nitroaniline rn-nitroaniline p-nitroaniline rn-chloroaniline p-chloroaniline o-nitrotoluene rn-nitrotoluene
3.16 3.98 3.39 8.55 11.76 11.76 5.46 4.72 4.29 5.18 7.58 7.30 5.46 5.05 4.98 2.90 2.98 3.86 8.33 8.85 9.17 4.72 3.29 2.99 5.71 5.99 10.53 12.17
2.95 3.70 3.16 7.35 9.26 8.85 4.65 3.97 3.36 4.48 6.67 6.17 4.63 4.29 4.22
3
2
0.3
. -
1
Y
.
0.2
2.89 3.61 3.12 7.04 8.77 8.33 4.48 3.82 3.15 4.35 6.41 5.92 4.41 4.12 4.03 2.75 2.81 3.11 7.00 6.80 6.41 4.20 2.91 2.41 4.85 4.54
X
2.69 2.75 2.87 6.67 6.25 5.75 4.07 2.79 2.24 4.57 4.13
0.4
10 12 11
0.3
7 8 9
. -
substance
15
L
1L
6
13
-0 6
( C D I r . rnM
0
4
2
[CD]r.
6
rnM
Figure 3. Plot of llk'against [CD], of 0 - (l), m- (2), and p-nitrophenol (3), 0-(4), m - (5),and p-iodophenol (6), 0 - (7), m - (8),and p-cresol (9). 0 - (lo), m - ( l l ) , and p-fluoroaniline (12). and 0- (13), m - (14), and p-iodoaniline (15). Chromatographic conditions are the same as in Figure 1.
species may exist, either ionized or nonionized. T h e separation factor, a , for the two solutes represented as 1 and 2 is given as
)
K D l - KD2 a = -kz' = - k02'KD2 (7) kl' kOl'KD1 -k KD2 + [CDIT Equation 7 reveals that although the separation factor is also expressed as a hyperbolic function of [CD], in the mobile phase, the shape of the curve is dependent largely on the difference of KD values of the two solutes. The effects of KD and [CD], on a will be discussed later. Effect of Concentration of CD on Capacity Factor. Table I1 lists the capacity factors of some benzene derivatives measured on a column of LiChrosorb RP-8 using a 50% aqueous methanol containing (3-CD of various concentrations as a mobile phase. As expected from eq 6, k'values decreased
('
3.94 2.69 2.11
Table 111. Dissociation Constants of /3-CD Inclusion Complexes Measured in 50% Aqueous Methanol at 23 "C and Estimated Values in 100% Water
0.1
4
2.63 2.69 2.69
20 cm); mobile phase, methanol/water (50/50); flow rate, 0.7 mL/min.
5
2
2.66 2.72 2.77 6.45 6.02 5.43 3.98 2.73 2.17 4.48 3.98 7.46 8.62
0.2
0
4.94 mM
3.92 3.66 3.58
Y
0.1
4.72 mM
2.75 3.44 2.98 6.41 7.69 7.14 4.05 3.44 2.74 3.98 5.99 5.35
8.55 9.80
"Column, LiChrosorb RP-8 (4 mm i.d.
0.4
capacity factors at the following concentration of 0-CD 2.83 mM 3.93 mM 4.42 mM 4.46 mM
o-fluorophenol rn-fluorophenol p-fluorophenol o-iodophenol rn-iodophenol p-iodophenol o-nitrophenol rn-nitrophenol p-nitrophenol o-chlorophenol rn-chlorophenol p-chlorophenol o-cresol rn-cresol p-cresol o-fluoroaniline rn-fluoroaniline p-fluoroaniline o-iodoaniline m-iodoaniline p-iodoaniline o-nitroaniline rn-nitroaniline p-nitroaniline o-chloroaniline rn-chloroaniline p-chloroaniline
K,, mol L-' in 50% MeOH" in 100% water 2.98 X 2.70 X 3.34 x 1.31 X 8.28 x 6.93 x 1.55 X 1.12 x 7.73 x 1.45 x 1.59 X 1.20 x 1.20 x 1.25 x 1.19 x 4.82 x 4.40 x 1.12 x 1.52 x 9.42 x 6-38 x 1.06 x 3.87 x 2.37 x 1.66 x 1.64 x 9.78 x
10-2 10-3 10-3 1.86 x 10-3 2.40 x 10-3 8.71 x 10-4
10-2 10-3 10-2 10-2
7.59 x 10-3 4.57 x 10-3 2.45 x 10-3
10-2 10-2 10-3
10-3 10-I
7.58 x 10-4 6.92 x 10-4 4.89 x 10-4
10-3
"Column, LiChrosorb RP-8 (4 mm id. mL/min.
X
20 cm); flow rate, 0.7
with an increase in the concentration of @-CD([CD],) in the mobile phase, and a linear relationship was observed between l / k ' a n d [CD],. Some typical plots are shown in Figure 3, demonstrating that the assumptions used to derive eq 6 are
2672
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
(B)
(A) 3.0 -
L L
1 .o 100
80
60
LO
1.0 100
80
40
60
Water content, *lo Flgure 4. Plot of pK, of nitrophenols (A) and cresols (B) in aqueous methanol against the water content in the mobile phase: (1) ortho isomer, (2) meta isomer, and (3) para isomer. Chromatographic condnions are the same as in Figure 1. valid, although it should always be borne in mind that the lack of generally accepted substance and/or method for precise measurement of to in RPLC may cause some errors in the measurements of k’ values, especially when they are small. The results obtained above are in good agreement with those reported by Cline Love et al. (21). The apparent dissociation constants, KD, of inclusion complexes calculated from the slope and the intercept of the straight line by the least-squares method are listed in Table I11 (center column). The apparent KD values thus obtained were, however, somewhat larger than those in the literature which had been measured in water; e.g., the literature values of o-, m-,and p-nitrophenols measured in aqueous solution for the ortho isomer, 6.80 of pH 10 a t 20 “C are 2.80 X X for the meta isomer, and 9.80 X lo4 for the para isomer ( I , 24). Based on the facts that the formation of CD inclusion complex proceeds more appreciably in 100% water than in aqueous methanol and that the KD value of the phenolate ion is larger than that of the neutral molecule, the larger KD value obtained in 50% aqueous methanol is considered reasonable. Consequently, KD values listed in Table I11 must be regarded
as being applicable only to neutral 50% aqueous methanol. It is difficult to measure the KD values in water directly in a similar manner as described above by using an aqueous CD solution of various concentrations as a mobile phase, because the retention times of the solute in the mobile phase containing no organic solvent are too long to calculate the k’values exactly. However, as shown in Figure 4, it is possible to estimate the KD values in water from a linear plot of pKD(log l/KD) obtained in aqueous methanol solution against the water content in such solutions by extrapolating to 100% water. The estimated values as KD’s in 100% water are listed also in Table I11 (right column). Effect of the Concentration of CD and KD Value on the Separation Factor. Concerning the dependence of the separation factor, a, on the total concentration of CD, [CDIT, in the mobile phase, three cases can be expected from eq 7 , depending on the difference of magnitude between kOl’and k02/,and between KDl and KD2. When ko2/ > kOl’ and KD, KD2 < 0, the separation factor would increase with an increase of [CD]T;i.e., plots of a vs. [CD], would show a part of rectangular hyperbola of a convex shape, starting from a = kO2’/ko1’,and approaching a = (ko2’/kol’)(KD2/KDl), where KD,/KDl is greater than unity. Alternatively, if k02/ > kol and KD, - K D >~ 0, such plots would indicate a part of a rectangular hyperbola of concave shape starting from a = kO2’/ko1’and approaching a = (k0*’/kO1’)(KDP/KD1), in which KDZ/KDl is smaller than unity. In the third case, where k02/ > kO1’and KD1 - KD2 0, the separation factor cannot be changed by the change of [CD],, i.e., plots of a vs. [CD], would result in a parallel line to the [CD], axis. These expectations are demonstrated in Figure 5 , where typical examples corresponding to such three cases are shown for pairs of m- and p-iodophenols, p- and m-fluoroanilines, and 0-and p-cresols. Thus, it is worth noting that the separation factors between isomers depend not only on the concentration of CD but also on KD values of the inclusion complexes so that the addition of CD in the mobile phase does not necessarily improve the peak resolution if the composition of the mobile phase remains unchanged. However, it is possible to improve the separation factor by changing the mobile phase composition as well as the concentration of CD, because the KD values of the inclusion complexes are based largely on the mobile phase
8
’rs :8(6
1.30 1.20 1.20
1.1 5
t
x 1.10
1.10
1.05 1.00 1.00
L 2 . 4
OO
Concentration
of
p-CD,
mM
Figure 5. Effect of the concentration of P-CD in mobile phase on the separation factor of iodophenol (A), fluoroaniline (B), and cresol (C) isomers. Chromatographic conditions are t h e same as in Figure 1.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
Table IV. Effect of Water Content in Mobile Phase on Capacity Factor and Separation Factor between m - and p -Nitrophenols" mobile phase
substance
30% MeOH
o-cresol p-cresol m-nitrophenol p-nitrophenol o-cresol p-cresol m-nitrophenol p-nitrophenol
40% MeOH
0-
2673
and p-Cresols and
capacity factors and separation factors at the following concentrations of 6-CD 1.0 mM 2.0 mM 3.0 mM 4.0 mM 14.18 13.27 12.65 10.81
1.07
12.99 11.63
1.12
11.90 10.42
1.14
11.05 9.39
1.18
1.17
11.16
1.25
10.00 7.63
1,31
"09 6.67
1.36
9'30
1.07
9.01
1.09
8.37
7.46
1.12
7'85
6.87
1.14
8'14
1.23
7'63
1.25
7'17 5.62
1.28
6'75
1.30
8.66
6.63
8.96
8.23 6.10
5.21
aColumn, LiChrosorb RP-8 (4 mm i.d. X 20 cm); flow rate, 0.7 mL/min. P-
P-
0
4
8
12
16
0
I
E
12
16 ( m i d
Flgwe 6. Separation of cresol (A) and iodoaniline (B) isomers: column, LiChrosorb RP-8, 4 mm i.d. X 20 cm; flow rate, 0.7 mL/min; mobile phase, 40% MeOH containing 6 mM p-CD (A) and 50% MeOH containing 6 mM @-CD.
composition as shown in Figure 4. A typical example is shown in Table IV. Separation of Isomers. Since there is no difference in the dissociation constants of the CD inclusion complexes of three cresol isomers in 50% aqueous methanol, the separation factor could not be altered by adding the CD in the mobile phase. However, it is possible to improve the separation factor by changing the water content in the mobile phase. This is demonstrated in Figure 6A, where the separation of cresol isomers is achieved satisfactorily by using 40% methanol containing 6 mM of @-CD,despite the separation factors of ortho to meta and of meta to para isomers being 1.02 and 1.00, respectively, when (3-CD is absent in the mobile phase. Although the elution order of iodoanilines is ortho < meta < para isomer and the resolution of adjacent peaks is not sufficient in the mobile phase of 50% methanol alone, the addition of p-CD t o the mobile phase results in a complete separation accompanied by the reversion of the elution order, as shown in Figure 6B. Figure 7 shows chromatograms of nitrophenols and nitroanilines. When @-CDis absent in the mobile phase, it is difficult to separate m- and p-nitrophenol completely, the separation factor being 1.10. However, the separation of three isomers becomes possible in the presence of 3 mM of p-CD in the mobile phase. Similarly, base line separation of p- and m-nitroanilines can be achieved by using 50% aqueous methanol containing 3 m M of P-CD, and peak resolution becomes larger with increasing @-CDconcentration. Although the elution order of nitroanilines is meta < ortho < para isomers when @-CDbonded silica and aqueous methanol were used as stationary and mobile phases, respectively (25-29), the present system gives the order para < meta < ortho isomers, which is the same as Hinze and co-workers have found in a TLC system consisting of polyamide sheets and an aqueous a-CD solution ( I ) . The fact that the latter order is
I
0
I
4
8
12(rnin)
o
I
4
8
12 ( m i n )
Flgure 7. Separation of nitrophenol (A) and nitroaniline (B) isomers: mobile phase, 50% MeOH containing 3 mM @ C D . Column and flow rate are the same as in Figure 6.
not exactly the reverse of the former one, or vice versa, implies that some additional factors, other than the easiness of inclusion complex formation, must be taken into account to explain the elution order satisfactorily. More detailed studies on the mechanism of retention on CD-bonded stationary phase will be needed to solve the problem. Registry No. a-CD, 10016-20-3;P-CD, 7585-39-9; P-CD.(ofluorophenol), 104015-39-6;P-CD-(rn-fluorophenol), 98536-99-3; P-CD.(p-fluorophenol), 104015-40-9; P-CD.(o-iodophenol), 104015-41-0; 6-CD.(m-iodophenol), 78096-81-8; P-CD.(p-iodophenol), 104015-42-1;P-CD-(0-nitrophenol),97571-01-2;P-CD(rn-nitrophenol),80065-29-8;P-CD.(p-nitrophenol), 61955-24-6; P-CD.(o-chlorophenol), 70763-75-6; P-CD-(m-chlorophenol), 104015-43-2;P-CD.(p-chlorophenol),89912-74-3;P-CD.(o-cresol), 86563-45-3;P-CD-(m-cresol),86563-46-4;@-CD-(p-cresol), 8656347-5; P-CD.(o-fluoroaniline),104015-44-3;P-CD.(m-fluoroaniline), 104015-45-4;&CD.(p-fluoroaniline), 104015-46-5; P-CD.(o-iodoaniline), 104015-47-6; @-CD.(m-iodoaniline),104015-48-7; 0CD.(p-iodoaniline),85428-93-9;P-CD.(o-nitroaniline),78153-70-5; B-CD.(m-nitroaniline),78153-71-6; @-CD.(p-nitroaniline),7815372-7; P-CD.(o-chloroaniline),104015-49-8;P-CD.(m-chloroaniline), 104015-50-1;P-CD.@-chloroaniline),104015-51-2; D-glucose, 5099-7; o-cresol, 95-48-7; m-cresol, 108-39-4; p-cresol, 106-44-5; m-nitrophenol, 554-84-7; p-nitrophenol, 100-02-7; l-naphthylamine, 134-32-7;2-naphthylamine, 91-59-8;o-fluorophenol, 36712-4; m-fluorophenol, 372-20-3; p-fluorophenol, 371-41-5; oiodophenyl, 533-58-4; m-iodophenyl, 626-02-8; p-iodophenyl, 540-38-5; o-iodophenyl, 88-75-5; o-chlorophenol, 95-57-8; mchlorophenol, 108-43-0;p-chlorophenol, 106-48-9;o-fluoraniline, 348-54-9; m-fluoroaniline, 372-19-0; p-fluoroaniline, 371-40-4;
2674
Anal. Chem. 1986, 58,2674-2680
o-iodoaniline, 615-43-0;m-iodoaniline, 626-01-7; p-iodoaniline, 540-37-4; o-nitroaniline, 88-74-4; n-nitroaniline, 99-09-2; p nitroaniline, 100-01-6;m-chloroaniline, 108-42-9;p-chloroaniline, 106-47-8;o-nitrotoluene, 88-72-2; m-nitrotoluene, 99-08-1.
LITERATURE CITED Hinze. W. L. Sep. Purif. Methods 1981, 10, 195-237. SmoikovB-Keulemansova, E. J. Chromatogr. 1982, 251, 17-34. Uekama, K.; Hirayama, F.; Ikeda, K.; Inaba, K. J. Pharm. Sci. 1977, 66,706-710. Uekama, K.; Hirayama, F.; Nasu, S.; Matsuo, N.; Irie, T. Chem. Pharm. Bull. 1978. 26, 3477-3489. Armstrong, D. W. J. Li9. Chromatogr. 1980, 3 ,895-900. Hinze, W. L.; Armstrong, D. W. Anal. Lett. 1980, 13, 1093-1104. Burkert. W. G.; Owensby, C. N.; Hinze, W. L. J. Li9. Chromatogr. 1981, 4, 1065-1085. Debowski, J.; Sybilska, D.; Jurczak, J. J . Chromatogr. 1982, 237, 303-306. Sybilska. D.; Lipkowski, J.; W6ycikowski, J. J . Chromatogr. 1982, 253,95-100. Debowski, J.; Sybilska, D.; Jurczak, J. Chromatographia 1982, 16, 198-200. Nobuhara, Y.; Hirano, S.; Nakanishi, Y. J. Chromatogr. 1983, 258, 276-279. Debowski, J.; Jurczak, J.; Sybilska, D. J. Chromatogr. 1983, 282, 83-68. Armstrong, D. W.; Stine, G. Y. J. A m . Chem. SOC. 1983, 105, 2962-2964. Sybiiska, D.; Debowski. J.; Jurczak, J.; Zukowski, J. J. Chromatogr. 1984, 286, 163-170. Korpela, T. K.; Himanen, J. P. J. Chromatogr. 1984, 290,351-361
Debowski, J.; Jurczak. J.; Sibilska, D.; Zukowski, J. J. Chromatogr. 1985, 329,206-210. Tanaka, M.; Miki, T.; Shono, T. J. Chromatogr. 1985, 330,253-261. Zukowski, J.; Sybilska, D.; Jurczak, J. Anal. Chem. 1985, 57, 22 15-22 19. Debowski, J.; Grassini-Strazza, G.; Sybiiska, 0.J , Chromatogr. 1985, 349, 131-136. Gazdag, M.; Szepesi, G.; Huszar, L. J . Chromatogr. 1986, 351, 128-135. Cline Love, L. J.; Arunyanart, M. ACS Symp. Ser. 1986, N o . 297, 226-243. Matsui, Y.; Mochida, K. Bull. Chem. SOC.Jpn. 1979, 52,2808-2814. Fujimura, K.: Ueda, T.; Ando, T. Proceedings of 25th Meeting of the Liquid Chromatography Research Society; Kyoto, Japan, Feb 23-24, 1982; pp 47-50. Osa, T.; Matsue, T.; Fujihira, M. Heterocycles 1977, 6 , 1833-1839. Fujimura, K.; Ueda, T.; Ando, T. Anal. Chem. 1983, 55, 446-450. Armstrong, D. W.; DeMond, W. J. Chromatogr. Sci. 1984, 22, 411-415. Kawaguchi, Y.; Tanaka, M.: Nakae, M.; Funazo, K.; Shono, T. Anal. Chem. 1983, 55, 1852-1657. ranaka. M.; Kawaguchi, Y.; Nakae, M.; Mizobuchi, Y.; Shono, T. J. Chromatogr. 1984, 299,341-350. Tanaka, M.; Kawaguchi, Y.; Shono, T.: Uebori, M.; Kuge, Y. J. Chromatogr. 1984, 301,345-353.
RECEIVED for review March 18, 1986. Accepted July 2, 1986. Part of this work was presented a t the 25th Meeting of The Liquid Chromatography Research Society, Kyoto, Japan, Feb 23-24, 1982.
Study of Temperature and Mobile-Phase Effects in Reversed-Phase High-Performance Liquid Chromatography by the Use of the Solvatochromic Comparison Method Peter W. Carr* Department of Chemistry, Kolthoff and Smith Halls, University of Minnesota, 207 Pleasant Street, Minneapolis, Minnesota 55455 Ruth M. Doherty and Mortimer J. Kamlet Naval Surface Weapons Laboratory, White Oak Laboratory, Silver Spring, Maryland 20903-5000 Robert W . Taft Department o f Chemistry, University of California, Irvine, California 9271 7 Wayne Melander and Csaba Horvath Department of Chemical Engineering, Yale Unioersity, New Haven, Connecticut 06520 Retentlon of a serles of aromatic sotutes In reversed-phase HPLC has been studied as a functlon of temperature and moblle-phase composition in acetonltrlle-water mixtures. The chief factors that control retentlon are the solute slre (molar volume) and hydrogen bond basicity. Less Important factors are the polarlzaMIlty4polarltyand hydrogen bond acldlty of the solute. Through the use of the sohratochromlc comparison method and h e a r solvation energy relationships, the key system parameters are seen to decrease In magnitude as the mobile phase becomes rlcher In organlc modlfter. That Is,the endoergk cavity formation energy and the exoerglc hydrogen bond formation processes both become less signHkant as the temperature increases. RelationsMps between the logarlthmlc capacity factor and moblle-phase composltlon are predicted to be more linear for small nonpolar solutes and hydrogen bond bases than for large nonpolar solutes.
In the preceding paper in this series, the solvatochromic comparison method and linear solvation energy relationships 0003-2700/88/0358-2674$0 1.50/0
(LSER) were used to examine the chemical and physical characteristics of a solute that govern retention in reversedphase HPLC ( I ) . In that work, the effects of the type of bonded phase, including hydrocarbon vs. fluorocarbon, and the amount and nature of the organic modifier, were tested as variables. We found that the most significant factors by far were the solute size, as measured by molar volume, and hydrogen bond basicity. Solute polarizability-dipolarity was a statistically significant but less important variable. Due to the nature of the data then available to us, the solute hydrogen bond basicity could not be examined over as large a range as desired. However, in related studies concerning the factors that govern water solubility and octanol-water partition coefficients of organic nonelectrolytes, it was similarly shown that hydrogen bond basicity of the solute is significantly more important than its hydrogen bond acidity (2, 3). I t is clear that temperature is an important variable in reversed-phase HPLC (4-7) and can be used to optimize a separation (8,9). To date, we have not examined the effect of temperature on solubility or phase transfer process by the solvatochromic comparison method. The data set examined 0 1986 American Chemical Society
