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Micellization of bile salts in a formamide solution: A gas liquid chromatography study
Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Micellization of bile salts in a formamide solution: A gas liquid chromatography study Małgorzata Skórka, Monika Asztemborska ∗ Institute of Physical Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 3 June 2008 Received in revised form 21 August 2008 Accepted 22 August 2008 Available online 31 August 2008 Keywords: Gas chromatography Bile salts Sodium cholate Sodium deoxycholate Formamide Micelle Stationary phase Thermodynamics Enthalpy of binding
a b s t r a c t Sodium cholate and sodium deoxycholate dissolved in formamide were applied as stationary phases in gas chromatography. The critical micelle concentration of sodium cholate and deoxycholate in formamide was determined by surface tension measurements. The relation of retention times vs. concentration of bile salts was investigated for isomers of monoterpenes and xylenes. The enthalpy of binding of selected compounds with sodium cholate and sodium deoxycholate monomers and micelles was determined. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Among many surfactants able to form a micellar structure, bile salts play an important role, since they are biosurfactants in mammals and important cholesterol end products. Due to their rigid steroidal structure with hydrophobic and hydrophilic faces, their aggregation behaviour and micellar structure is different from that of traditional linear surfactants. One consequence of planar polarity is that bile salts in aqueous systems form smaller micelles than classical surfactants, with an aggregation number of 2–9 molecules [1]. The structure of bile salt micelles and the mechanism of aggregation are still under discussion. Two main models have been proposed in the literature. According to the Small model [2], primary aggregates of bile salts are formed by hydrophobic interactions, while secondary aggregates form via intermolecular hydrogen bonding between the hydroxyl groups. In contrast to the aggregation model, Giglio et al. [3] proposed a helical structure of bile salt micelles in polar solvents. Surprisingly, the helical model postulates the formation of reverse micelles: the nonpolar faces of the bile salt molecules are oriented outward toward the bulk aqueous solution.
∗ Corresponding author. Tel.: +48 2234 33296. E-mail address: [email protected] (M. Asztemborska). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.08.018
Micelles of various surfactants are applied in a particular mode of capillary electrophoresis—micellar electrokinetic chromatography (MEKC). They have also found application in high performance liquid chromatography as mobile phase additives. This technique is known as micellar liquid chromatography (MLC) [4–6]. Since bile salts are chiral, their micelles have been applied in separation of enantiomers in the MLC and MEKC techniques [6–8]. In this paper we report our study on the use of sodium cholate (NaC) and sodium deoxycholate (NaDC) as a component of the stationary phase in gas liquid chromatography (GLC). To our best knowledge, there is little information on the application of micelles of bile salts in gas chromatography (GC). 2. Theoretical considerations In gas liquid chromatography with micelles of various surfactants in ionic liquids as the stationary phase, a three-phase model was proposed by Armstrong and coworkers [9]. The threephase model has previously been used to describe micellar liquid chromatography and gas liquid chromatography using dissolved cyclodextrins [10,11]. This model does not take into account solute interaction with monomers of the surfactant. However, on the basis of obtained results it was interesting to show the range of concentration of bile salts below critical micelle concentration (cmc) as well as above cmc. Thus four cases will be considered.
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M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
In the first case, the stationary phase consists of pure solvent. The behaviour of this system is described by the equilibrium:
ln k = −
Kgl
S(g) ←→S(l) where S(g) , S(l) , is the solute in the gas and liquid phases, respectively, and Kgl is the partition coefficient. In the second case the stationary phase consists of a solution of bile salts below cmc, where only monomers of bile salts are present. The equilibrium can be expressed as Kgl
K
l mon S(g) ←→S(l) + B(mon) ←→SB (mon)
where B(mon) is monomeric bile salt, SB(mon) is the complex of the solute with bile salt monomer, and Klmon is the equilibrium coefficient. The third case corresponds to cmc, where the surface of solvent is fully covered by monomers of bile salts. The equilibrium at this point is described as follows: Kgl
Klcmc
S(g) ←→S(l) + B(cmc) ←→SB(cmc) where B(cmc) is bile salt at cmc, SB(cmc) is the complex of the solute with bile salt at cmc, and Klcmc is the equilibrium coefficient. Above the cmc of bile salts – the fourth case – the equilibrium is written as Kgl
van’t Hoff equation:
K
K
S(g) ←→S(l) + B(cmc) + B(mic) lcmc ←→lmic SB(cmc) + SB(mic) where B(mic) is a micelle of bile salts, SB(mic) is the complex of the solute with micelle of bile salts, and Klmic is the equilibrium coefficient. Chromatographic retention is often used to calculate the partial molar enthalpy of transfer of a solute from the mobile phase to the stationary phase. The transfer enthalpy can be calculated from the
S H 1 + + ln R T R
(1)
where T is the absolute temperature, k is the retention factor, R is the gas constant, and is the volume ratio of the stationary phase to the mobile phase. When the retention mechanism remains the same over the investigated temperature range the van’t Hoff plot yields a straight line. According to Eq. (1), the partial molar enthalpy HI of transfer of the solute from the mobile phase to the stationary phase (formamide) can be determined (the first case). In the presence of monomers of surfactants in the system, the transfer enthalpy HII can be expressed as −HII = −(HI + Hmon )
(2)
where Hmon is the enthalpy of binding of the solute with bile salt monomers. At the point when the surface becomes fully covered with bile salt monomers (the third case), HIII of binding of the solute with bile salt molecules can be defined by the following equation: −HIII = −(HI + Hcmc ).
(3)
Above the critical micelle concentration, where aggregates of bile salts are formed in the bulk solution, the transfer enthalpy HIV is written as −HIV = −(HI + Hcmc + Hmic ) = −(HIII + Hmic )
(4)
where Hcmc and Hmic are the enthalpies of binding of the solute with surfactant molecules at cmc and in micellar form in the bulk solution, respectively. It is important to note that the presented model of equilibrium does not take into account the influence of solute on micellization
Fig. 1. Structure of bile salt surfactants.
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
of bile salts. It is usually accepted that micellization of bile salts proceeds stepwise over a broad range of concentration [2]. The addition of favourable solutes to the bile salt solution can alter the process of aggregation [12]. However, since the concentration of solute is much less then the concentration of micelles one can assume a negligible effect of solute on aggregation process. In such case the solute does not change significantly the micellar properties of bile salts [13]. 3. Materials and methods 3.1. Chemicals NaC, NaDC and formamide were supplied by Fluka (Buchs, Switzerland). The structures of NaC and NaDC are presented in Fig. 1. The Chromosorb W NAW (60–100 mesh) for GC was a product of Johns-Manville (Litho, USA). The model compounds (+) and (−)-pinene, (+) and (−)--citronellene, myrcene, ␣-phellandrene, ␣-terpinene, ␥-terpinene, ocimene were supplied by Fluka (Buchs, Switzerland). (+)- and (−)-␣-pinene, (+)- and (−)-camphene were from Aldrich (Milwaukee, USA). p-Xylene, m-xylene and o-xylene were product of POCh (Gliwice, Poland). The structures of the model compounds are presented in Table 1. All other substances were of analytical reagent grade and were used without further purification. Table 1 The names and structural formulae of the model compounds
3
Table 2 Column characteristics Column code
Bile salts
Concentration of bile salts in formamide (mm)a
Matrix C1 C2 C3 C4 C5 C6 C7 DC1 DC2 DC3 DC4 DC5 DC6 DC7
– NaC NaC NaC NaC NaC NaC NaC NaDC NaDC NaDC NaDC NaDC NaDC NaDC
0 7 10 14 20 30 60 100 3 7 10 14 20 60 100
a
mm, millimolality (millimoles of solute per 1 kg of solvent).
3.2. Columns Glass columns with dimensions 2 m × 4 mm I.D. were packed with Chromosorb W NAW (60–100 mesh), coated with NaC or NaDC dissolved in formamide or with pure formamide as the matrix column. Detailed information on surfactant concentration is shown in Table 2. 3.3. Apparatus and procedure 3.3.1. Surface tension measurements The surface tensions of formamide/surfactant solutions were measured by the Wilhelmy plate method with Nina Technology equipment (resolution for surface pressure 0.01 mN/m). A rectangular piece of analytical filtering paper (20 mm × 10 mm × 0.1 mm) was used as a surface pressure sensor. The sample was introduced into a double-walled glass cell connected to a water cooling/heating circulating bath thermostat. Temperature was measured with accuracy of ±0.2 ◦ C using two Pt 100 resistance thermometers connected to a Keithley multimeter. All measurements were carried out at 30 ◦ C. The whole system was placed on a laboratorymade antivibration table and closed in a ventilated plexiglas box. 3.3.2. Chromatographic measurements Chromatographic studies were performed using a HewlettPackard Model 5890 series II gas chromatograph (Waldbronn, Germany) equipped with dual flame ionisation detectors. The peak areas and retention time were measured by means of an HP 3396 series II integrator. Each measurement was carried out two or three times. Methane was co-injected as a marker to determine the hold-up time (tM ). The flow rate (40 ± 0.50 ml/min) was carefully maintained. The temperature of the injector and detector was 200 ◦ C and 250 ◦ C, respectively. The amount of injected sample was 0.02 l on column. 4. Results and discussion 4.1. Surface tension measurements At some concentration in a solution, surfactants start to form micellar structures. This concentration is known as the critical micelle concentration. The cmc and the aggregation number (the quantity of surfactant molecules in the aggregate) of NaC and NaDC in water were determined by various methods. According to Terabe
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Fig. 2. Surface tension of a bile salt/formamide solution as a function of surfactant concentration at 30 ◦ C.
et al. [14] the cmc values are 13–15 mM for NaC and 4–6 mM for NaDC at 25 ◦ C. The most natural environment for micelle formation is water. Since water cannot be applied as a stationary phase in GC, it was necessary to choose another solvent. Among many solvents, formamide was chosen as the liquid stationary phase and the solvent of bile salts since its physicochemical properties are close to those of water: the cohesion energy of formamide at 25 ◦ C is 1.579 kJ/cm and the dielectric constant at 20 ◦ C is 109, compared to the corresponding values of 2.291 kJ/cm and 80.0 for water. Akhter and Alawi [15] found that surfactants such as sodium salts of selected
Fig. 3. Separation of the artificial mixture of monoterpenes obtained on columns: matrix (a), DC3 (b) and C6 (c). Peak identification: ␣-phellandrene (1); ␣-terpinene (2); limonene (3); ␥-terpinene (4); terpinolene (5). Conditions: temp. 30 ◦ C, flow rate 40 ml/min.
fatty acids form micellar structures in formamide. Since there is a lack of information about the behaviour of bile salts in a formamide solution, it was necessary to control whatever NaC and NaDC form micellar structures in this particular solvent. From several available methods, surface tension studies were chosen and applied to determine the cmc of NaC and NaDC in the formamide solution.
Fig. 4. Relation of adjusted retention times of the investigated compounds vs. concentration of sodium cholate.
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
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Fig. 5. Relation of adjusted retention times of polar solvents vs. concentration of sodium cholate (a) and sodium deoxycholate (b).
Fig. 2 shows the relation of 0 − vs. the logarithm of molar concentration of NaC and NaDC, where 0 is the surface tension for the formamide/air interface and is the surface tension of the measured solution in the presence of the bile salts. The slope change in the plot allows to estimate the cmc, above which the surface tension for the bile salt/formamide system approaches a constant value. As can be seen in Fig. 2, with increasing surfactant concentration the relative surface tension decreases for NaC as well as for NaDC solutions. As the cmc point is reached, the curve changes its slope and approaches a constant value. Thus the cmc determined
from the plot is 17 mM for NaC and 9 mM for NaDC. The cmc values obtained for the formamide solution in the present study are slightly higher than for a water solution. 4.2. Chromatographic measurements As surface tension measurements showed that sodium cholate and deoxycholate form aggregates also in formamide, gas chromatographic columns were prepared with a formamide solution of NaC and NaDC as stationary phases. The influence of bile salt con-
Fig. 6. Relation of adjusted retention times of the investigated compounds vs. concentration of sodium deoxycholate.
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centration on the retention parameters of the model compounds was investigated. Columns with NaC and NaDC at a concentration below, close to, and above cmc were prepared. A column with pure formamide – the matrix column – was also prepared. Chiral monoterpenes from the acyclic, monocyclic and bicyclic groups, and isomeric ortho-, para- and meta-xylenes (Table 1) served as model tested compounds. Examples of chromatograms of the artificial mixture of monoterpenes obtained on the columns with the formamide solution of NaC, NaDC and pure formamide (matrix column) are presented in Fig. 3. The relations of adjusted retention times of the investigated compounds vs. the concentration of NaC are presented in Fig. 4. For concentrations below cmc, the adjusted retention times of monoterpenes increase quickly with NaC concentration and reach the maximum for concentration equal to 14 mm and then rapidly decrease for concentration close to cmc (20 mm). In the range of concentration above 20 mm the retention times increase almost linearly with NaC concentration, but the slope is lower in comparison with that obtained for the monomeric range of concentrations. Comparing the behaviour of acyclic, monocyclic and bicyclic monoterpenes, the acyclic and monocyclic compounds interact more strongly with NaC than the bicyclic ones. The behaviour of xylene isomers is different. The retention times remain constant in the NaC concentration range of 0–7 mm and unexpectedly decrease for concentration of 14 mm. After that the retention times increase to almost the previous value and remain practically constant with the increase of concentration. Since the adjusted retention times of xylenes for 14 mm are shorter than on the matrix column, this could suggest that some processes unfavourable for aromatic compound sorption occur at the surface of the stationary phase. It seems that the affinity of xylenes to formamide is more favourable than interaction with NaC. A similar tendency of dependence of tR vs. NaC concentration was obtained for polar solvents: acetone and ethylene acetate (Fig. 5a). It seems that a specific arrangement of NaC molecules at the surface of formamide makes the behaviour of nonpolar aromatic hydrocarbons similar to polar solvents. Similar chromatographic measurements were conducted for NaDC. The relations of retention times of the investigated compounds vs. NaDC concentration are presented in Fig. 6. In this case the retention times increase quickly in the 0–7 mm concentration range for most of the monoterpenes. A rapid decrease of retention time analogous to that found for NaC is observed for concentration close to cmc (10 mm). In comparison with NaC, the increase of retention times is greater for NaDC in the micellar range of concentration. In the case of xylene isomers, similar behaviour to that found for NaC is observed. A decrease of retention times is observed for concentration of 7 mm. Also in the case of polar solvents the relation of tR vs. NaDC concentration exhibits a similar tendency as for xylenes (Fig. 5b). The obtained results may be explained by the following hypothesis. Bile salt molecules have a quite rigid and flat structure with hydrophobic and hydrophilic faces. It is possible that in low concentrations (below cmc) they lie flat on the surface of formamide with the hydrophilic face oriented toward the solution (Fig. 7a). When the concentration of the bile salt increases, the layer of formamide becomes more covered with the bile salt molecules (Fig. 7b). Such surface behaviour of bile salts at the air/water interface was described by Tiss et al. [16]. At a concentration close to cmc the surface is too crowded and the bile salt molecules probably reorganize at the surface of formamide from a flat to an upright orientation, with their hydrophobic faces oriented together (Fig. 7c). Afterwards, the increase of concentration of the bile salts induces
Fig. 7. Schematic representation of the behaviour of bile salts: below cmc (a, b); close to cmc (c); above cmc (d).
the formation of aggregates inside the formamide solution (Fig. 7d). Similar behaviour of NaC and NaDC in a water solution was suggested by Almgren and coworkers [17]. The concentration at which the surface of formamide is hypothetically tightly packed with flat-lying molecules of bile salts, corresponds to the point where the adjusted retention times reach the maximum value for monoterpenes and the minimum for xylenes. The subsequent reorganization of bile salts from lying flat to an upright orientation makes the surface of formamide more accessible for xylenes but this position is less convenient for interaction between bile salts and monoterpenes. This could explain the unexpected decrease of retention times for concentration close to the cmc for NaC (20 mm) and NaDC (10 mm). Unfortunately, the investigated systems do not exhibit selective properties towards enantiomers of monoterpenes as well as for constitutional isomers of xylenes. 4.3. Temperature investigations The stability of micelles is temperature-dependent. An increase of temperature may cause a demicellization process. Gas chromatographic measurements are usually performed in temperatures much higher than ambient temperature. It was therefore necessary to check the thermal stability of micellar systems in formamide. To prove the thermal stability of the micellar stationary phase, temperature investigations were performed for selected compounds. The retention factors of compounds selected from the
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
Fig. 8. van’t Hoff plots of ln k of selected compounds vs. 1/T obtained on columns: matrix, C1, C3 and C6.
Fig. 9. van’t Hoff plots of ln k of selected compounds vs. 1/T obtained on columns: matrix, DC1, DC2 and DC6.
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Table 3 Enthalpies of solute transfer (kJ/mol) from the mobile to the stationary phase and enthalpies of binding (kJ/mol) of selected compounds with NaC and NaDC
Without surfactant −HI Sodium cholate (NaC) −HII −HIII −HIV −Hmon −Hcmc −Hmic
Column
-Citronellene
␣-Terpinene
␣-Pinene
o-Xylene
Matrix
37.82 (0.998)
39.07 (0.998)
31.92 (0.992)
35.58 (0.998)
C3 C4 C6
47.55 (0.998) 44.81 (0.998) 47.97 (0.999) 9.73 6.99 3.16
45.88 (0.998) 41.39 (0.998) 44.72 (0.998) 6.81 2.32 3.33
42.81 (0.998) 38.57 (0.998) 41.57 (0.998) 10.89 6.65 3.00
35.75 (0.998) 36.57 (0.996) 37.92 (0.998) 0.17 0.99 1.25
51.21 (0.998) 42.48 (0.997) 49.22 (0.994) 13.39 4.66 6.74
47.96 (0.998) 40.25 (0.998) 45.06 (0.998) 8.89 1.18 4.81
45.56 (0.998) 34.58 (0.997) 40.23 (0.994) 13.64 2.66 5.65
38.48 (0.998) 34.41 (0.999) 34.58 (0.998) 2.90 −1.17 0.17
Sodium deoxycholate (NaDC) DC2 −HII −HIII DC3 −HIV DC6 −Hmon −Hcmc −Hmic R2 = regression coefficient.
acyclic, monocyclic, bicyclic, and aromatic groups were measured in the 303–328 K temperature range on columns with concentration 7, 14, 20 and 60 mm of NaC, 3, 7, 10 and 60 mm of NaDC, and the matrix column. The obtained results are presented as van’t Hoff plots in Figs. 8 and 9. A very good linear variation of ln k vs. 1/T is observed with regression coefficient R2 > 0.992. These results indicate that the investigated stationary phases are stable in the studied temperature range, in the micellar range as well as in the monomeric range of concentrations. In accordance with Eqs. (1)–(4), the partial molar enthalpy of solute transfer from the mobile to the stationary phase was determined from the slopes of relations ln k vs. 1/T for -citronellene, ␣-terpinene, ␣-pinene, and o-xylene on the matrix column and selected columns with bile salt concentration below, at, and above the cmc point. The determined values of HI , HII , HIII and HIV are presented in Table 3. The enthalpies of binding of selected compounds with NaC and NaDC as monomers Hmon at cmc Hcmc and in micellar form Hmic were calculated according to Eqs. (2)–(4). The calculated values of H are presented in Table 3. In the case of -citronellene, ␣-terpinene and ␣-pinene, Hmon and Hmic are larger for NaDC than for NaC, while Hcmc is larger for NaC than for NaDC. The binding enthalpy of the investigated terpenoids is larger for bile salts monomers than micelle ((−Hmon ) > (−Hmic )). The binding enthalpy of bile salts with o-xylene is close to zero independently on concentration of surfactant. 5. Summary The cmc values of NaC and NaDC in a formamide solution were determined from surface tension measurements. The experimentally estimated cmc values are 17 mM for NaC and 9 mM for NaDC. The obtained chromatographic results provide interesting information on the behaviour of bile salts on the surface of a
nonaqueous solvent. Temperature investigations indicate that the studied micellar phase is stable in the range 30–50 ◦ C. The binding enthalpies of selected compounds to bile salt monomers and in aggregate form were also determined. Acknowledgments ˙ ´ The authors wish to thank Andrzej Zywoci nski Ph.D. and Patrycja Milczarczyk-Piwowarczyk M.Sc. for their help in surface tension measurements and interpretation. This work was partially supported by grant no. N204 3702 33 of the Polish Ministry of Science and Higher Education. References [1] A. Coello, F. Meijide, E.R. Nunez, J.V. Tato, J. Pharm. Sci. 85 (1996) 9–15. [2] D.M. Small, P.P. Nair, D. Kritchevsky (Eds.), The Bile Salts, vol. 1, Plenum Press, New York, 1971, p. 249. [3] E. Giglio, S. Loreti, N.V. Pavel, J. Phys. Chem. 92 (1988) 2858–2862. [4] R. Williams, F. ZhengSheng, W. Hinze, J. Chromatogr. Sci. 28 (1990) 292–302. [5] W. Hinze, R. Williams, F. Zheng Sheng, Y. Suzuki, F. Quina, Colloids Surf. 48 (1990) 79–94. [6] M.G. Khaledi, J. Chromatogr. A 780 (1997) 3–40. [7] P.G. Muijselaar, K. Otsuka, S. Terabe, J. Chromatogr. A 780 (1997) 41–61. [8] M.L. Marina, M.A. Garcia, J. Chromatogr. A 780 (1997) 103–116. [9] A.W. Lantz, V. Pino, J.L. Anderson, D.W. Armstrong, J. Chromatogr. A 1115 (2006) 217–224. [10] D.W. Armstrong, F. Nome, Anal. Chem. 52 (1981) 1662–1666. [11] V. Pino, A.W. Lantz, J.L. Anderson, A. Berthod, D.W. Armstrong, Anal. Chem. 78 (2006) 113–119. [12] S. Reis, C. Guimaraes Moutinho, C. Matos, B. de Castro, P. Gameiro, J.L.F.C. Lima, Anal. Biochem. 334 (2004) 117–126. [13] R. Shaw, W.H. Elliott, B.G. Barisas, Microchim. Acta 105 (1991) 137–145. [14] S. Terabe, N. Chen, K. Otsuka, in: A. Chrambach, J. Dunn, B. Radola (Eds.), Advances in Electrophoresis, vol. 7, VCH, Weinheim, 1994, p. 91. [15] M.S. Akhter, S.M. Alawi, Colloids Surf. A 173 (2000) 95–100. [16] A. Tiss, S. Ransac, H. Lengsfeld, P. Hadvary, A. Cagna, R. Verger, Chem. Phys. Lipids 111 (2001) 73–85. [17] M. Swanson-Vethamuthu, M. Almgren, P. Hansson, J. Zhao, Langmuir 12 (1996) 2186–2189.
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Micellization of bile salts in a formamide solution: A gas liquid chromatography study Małgorzata Skórka, Monika Asztemborska ∗ Institute of Physical Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 3 June 2008 Received in revised form 21 August 2008 Accepted 22 August 2008 Available online 31 August 2008 Keywords: Gas chromatography Bile salts Sodium cholate Sodium deoxycholate Formamide Micelle Stationary phase Thermodynamics Enthalpy of binding
a b s t r a c t Sodium cholate and sodium deoxycholate dissolved in formamide were applied as stationary phases in gas chromatography. The critical micelle concentration of sodium cholate and deoxycholate in formamide was determined by surface tension measurements. The relation of retention times vs. concentration of bile salts was investigated for isomers of monoterpenes and xylenes. The enthalpy of binding of selected compounds with sodium cholate and sodium deoxycholate monomers and micelles was determined. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Among many surfactants able to form a micellar structure, bile salts play an important role, since they are biosurfactants in mammals and important cholesterol end products. Due to their rigid steroidal structure with hydrophobic and hydrophilic faces, their aggregation behaviour and micellar structure is different from that of traditional linear surfactants. One consequence of planar polarity is that bile salts in aqueous systems form smaller micelles than classical surfactants, with an aggregation number of 2–9 molecules [1]. The structure of bile salt micelles and the mechanism of aggregation are still under discussion. Two main models have been proposed in the literature. According to the Small model [2], primary aggregates of bile salts are formed by hydrophobic interactions, while secondary aggregates form via intermolecular hydrogen bonding between the hydroxyl groups. In contrast to the aggregation model, Giglio et al. [3] proposed a helical structure of bile salt micelles in polar solvents. Surprisingly, the helical model postulates the formation of reverse micelles: the nonpolar faces of the bile salt molecules are oriented outward toward the bulk aqueous solution.
∗ Corresponding author. Tel.: +48 2234 33296. E-mail address: [email protected] (M. Asztemborska). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.08.018
Micelles of various surfactants are applied in a particular mode of capillary electrophoresis—micellar electrokinetic chromatography (MEKC). They have also found application in high performance liquid chromatography as mobile phase additives. This technique is known as micellar liquid chromatography (MLC) [4–6]. Since bile salts are chiral, their micelles have been applied in separation of enantiomers in the MLC and MEKC techniques [6–8]. In this paper we report our study on the use of sodium cholate (NaC) and sodium deoxycholate (NaDC) as a component of the stationary phase in gas liquid chromatography (GLC). To our best knowledge, there is little information on the application of micelles of bile salts in gas chromatography (GC). 2. Theoretical considerations In gas liquid chromatography with micelles of various surfactants in ionic liquids as the stationary phase, a three-phase model was proposed by Armstrong and coworkers [9]. The threephase model has previously been used to describe micellar liquid chromatography and gas liquid chromatography using dissolved cyclodextrins [10,11]. This model does not take into account solute interaction with monomers of the surfactant. However, on the basis of obtained results it was interesting to show the range of concentration of bile salts below critical micelle concentration (cmc) as well as above cmc. Thus four cases will be considered.
2
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
In the first case, the stationary phase consists of pure solvent. The behaviour of this system is described by the equilibrium:
ln k = −
Kgl
S(g) ←→S(l) where S(g) , S(l) , is the solute in the gas and liquid phases, respectively, and Kgl is the partition coefficient. In the second case the stationary phase consists of a solution of bile salts below cmc, where only monomers of bile salts are present. The equilibrium can be expressed as Kgl
K
l mon S(g) ←→S(l) + B(mon) ←→SB (mon)
where B(mon) is monomeric bile salt, SB(mon) is the complex of the solute with bile salt monomer, and Klmon is the equilibrium coefficient. The third case corresponds to cmc, where the surface of solvent is fully covered by monomers of bile salts. The equilibrium at this point is described as follows: Kgl
Klcmc
S(g) ←→S(l) + B(cmc) ←→SB(cmc) where B(cmc) is bile salt at cmc, SB(cmc) is the complex of the solute with bile salt at cmc, and Klcmc is the equilibrium coefficient. Above the cmc of bile salts – the fourth case – the equilibrium is written as Kgl
van’t Hoff equation:
K
K
S(g) ←→S(l) + B(cmc) + B(mic) lcmc ←→lmic SB(cmc) + SB(mic) where B(mic) is a micelle of bile salts, SB(mic) is the complex of the solute with micelle of bile salts, and Klmic is the equilibrium coefficient. Chromatographic retention is often used to calculate the partial molar enthalpy of transfer of a solute from the mobile phase to the stationary phase. The transfer enthalpy can be calculated from the
S H 1 + + ln R T R
(1)
where T is the absolute temperature, k is the retention factor, R is the gas constant, and is the volume ratio of the stationary phase to the mobile phase. When the retention mechanism remains the same over the investigated temperature range the van’t Hoff plot yields a straight line. According to Eq. (1), the partial molar enthalpy HI of transfer of the solute from the mobile phase to the stationary phase (formamide) can be determined (the first case). In the presence of monomers of surfactants in the system, the transfer enthalpy HII can be expressed as −HII = −(HI + Hmon )
(2)
where Hmon is the enthalpy of binding of the solute with bile salt monomers. At the point when the surface becomes fully covered with bile salt monomers (the third case), HIII of binding of the solute with bile salt molecules can be defined by the following equation: −HIII = −(HI + Hcmc ).
(3)
Above the critical micelle concentration, where aggregates of bile salts are formed in the bulk solution, the transfer enthalpy HIV is written as −HIV = −(HI + Hcmc + Hmic ) = −(HIII + Hmic )
(4)
where Hcmc and Hmic are the enthalpies of binding of the solute with surfactant molecules at cmc and in micellar form in the bulk solution, respectively. It is important to note that the presented model of equilibrium does not take into account the influence of solute on micellization
Fig. 1. Structure of bile salt surfactants.
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
of bile salts. It is usually accepted that micellization of bile salts proceeds stepwise over a broad range of concentration [2]. The addition of favourable solutes to the bile salt solution can alter the process of aggregation [12]. However, since the concentration of solute is much less then the concentration of micelles one can assume a negligible effect of solute on aggregation process. In such case the solute does not change significantly the micellar properties of bile salts [13]. 3. Materials and methods 3.1. Chemicals NaC, NaDC and formamide were supplied by Fluka (Buchs, Switzerland). The structures of NaC and NaDC are presented in Fig. 1. The Chromosorb W NAW (60–100 mesh) for GC was a product of Johns-Manville (Litho, USA). The model compounds (+) and (−)-pinene, (+) and (−)--citronellene, myrcene, ␣-phellandrene, ␣-terpinene, ␥-terpinene, ocimene were supplied by Fluka (Buchs, Switzerland). (+)- and (−)-␣-pinene, (+)- and (−)-camphene were from Aldrich (Milwaukee, USA). p-Xylene, m-xylene and o-xylene were product of POCh (Gliwice, Poland). The structures of the model compounds are presented in Table 1. All other substances were of analytical reagent grade and were used without further purification. Table 1 The names and structural formulae of the model compounds
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Table 2 Column characteristics Column code
Bile salts
Concentration of bile salts in formamide (mm)a
Matrix C1 C2 C3 C4 C5 C6 C7 DC1 DC2 DC3 DC4 DC5 DC6 DC7
– NaC NaC NaC NaC NaC NaC NaC NaDC NaDC NaDC NaDC NaDC NaDC NaDC
0 7 10 14 20 30 60 100 3 7 10 14 20 60 100
a
mm, millimolality (millimoles of solute per 1 kg of solvent).
3.2. Columns Glass columns with dimensions 2 m × 4 mm I.D. were packed with Chromosorb W NAW (60–100 mesh), coated with NaC or NaDC dissolved in formamide or with pure formamide as the matrix column. Detailed information on surfactant concentration is shown in Table 2. 3.3. Apparatus and procedure 3.3.1. Surface tension measurements The surface tensions of formamide/surfactant solutions were measured by the Wilhelmy plate method with Nina Technology equipment (resolution for surface pressure 0.01 mN/m). A rectangular piece of analytical filtering paper (20 mm × 10 mm × 0.1 mm) was used as a surface pressure sensor. The sample was introduced into a double-walled glass cell connected to a water cooling/heating circulating bath thermostat. Temperature was measured with accuracy of ±0.2 ◦ C using two Pt 100 resistance thermometers connected to a Keithley multimeter. All measurements were carried out at 30 ◦ C. The whole system was placed on a laboratorymade antivibration table and closed in a ventilated plexiglas box. 3.3.2. Chromatographic measurements Chromatographic studies were performed using a HewlettPackard Model 5890 series II gas chromatograph (Waldbronn, Germany) equipped with dual flame ionisation detectors. The peak areas and retention time were measured by means of an HP 3396 series II integrator. Each measurement was carried out two or three times. Methane was co-injected as a marker to determine the hold-up time (tM ). The flow rate (40 ± 0.50 ml/min) was carefully maintained. The temperature of the injector and detector was 200 ◦ C and 250 ◦ C, respectively. The amount of injected sample was 0.02 l on column. 4. Results and discussion 4.1. Surface tension measurements At some concentration in a solution, surfactants start to form micellar structures. This concentration is known as the critical micelle concentration. The cmc and the aggregation number (the quantity of surfactant molecules in the aggregate) of NaC and NaDC in water were determined by various methods. According to Terabe
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Fig. 2. Surface tension of a bile salt/formamide solution as a function of surfactant concentration at 30 ◦ C.
et al. [14] the cmc values are 13–15 mM for NaC and 4–6 mM for NaDC at 25 ◦ C. The most natural environment for micelle formation is water. Since water cannot be applied as a stationary phase in GC, it was necessary to choose another solvent. Among many solvents, formamide was chosen as the liquid stationary phase and the solvent of bile salts since its physicochemical properties are close to those of water: the cohesion energy of formamide at 25 ◦ C is 1.579 kJ/cm and the dielectric constant at 20 ◦ C is 109, compared to the corresponding values of 2.291 kJ/cm and 80.0 for water. Akhter and Alawi [15] found that surfactants such as sodium salts of selected
Fig. 3. Separation of the artificial mixture of monoterpenes obtained on columns: matrix (a), DC3 (b) and C6 (c). Peak identification: ␣-phellandrene (1); ␣-terpinene (2); limonene (3); ␥-terpinene (4); terpinolene (5). Conditions: temp. 30 ◦ C, flow rate 40 ml/min.
fatty acids form micellar structures in formamide. Since there is a lack of information about the behaviour of bile salts in a formamide solution, it was necessary to control whatever NaC and NaDC form micellar structures in this particular solvent. From several available methods, surface tension studies were chosen and applied to determine the cmc of NaC and NaDC in the formamide solution.
Fig. 4. Relation of adjusted retention times of the investigated compounds vs. concentration of sodium cholate.
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
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Fig. 5. Relation of adjusted retention times of polar solvents vs. concentration of sodium cholate (a) and sodium deoxycholate (b).
Fig. 2 shows the relation of 0 − vs. the logarithm of molar concentration of NaC and NaDC, where 0 is the surface tension for the formamide/air interface and is the surface tension of the measured solution in the presence of the bile salts. The slope change in the plot allows to estimate the cmc, above which the surface tension for the bile salt/formamide system approaches a constant value. As can be seen in Fig. 2, with increasing surfactant concentration the relative surface tension decreases for NaC as well as for NaDC solutions. As the cmc point is reached, the curve changes its slope and approaches a constant value. Thus the cmc determined
from the plot is 17 mM for NaC and 9 mM for NaDC. The cmc values obtained for the formamide solution in the present study are slightly higher than for a water solution. 4.2. Chromatographic measurements As surface tension measurements showed that sodium cholate and deoxycholate form aggregates also in formamide, gas chromatographic columns were prepared with a formamide solution of NaC and NaDC as stationary phases. The influence of bile salt con-
Fig. 6. Relation of adjusted retention times of the investigated compounds vs. concentration of sodium deoxycholate.
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centration on the retention parameters of the model compounds was investigated. Columns with NaC and NaDC at a concentration below, close to, and above cmc were prepared. A column with pure formamide – the matrix column – was also prepared. Chiral monoterpenes from the acyclic, monocyclic and bicyclic groups, and isomeric ortho-, para- and meta-xylenes (Table 1) served as model tested compounds. Examples of chromatograms of the artificial mixture of monoterpenes obtained on the columns with the formamide solution of NaC, NaDC and pure formamide (matrix column) are presented in Fig. 3. The relations of adjusted retention times of the investigated compounds vs. the concentration of NaC are presented in Fig. 4. For concentrations below cmc, the adjusted retention times of monoterpenes increase quickly with NaC concentration and reach the maximum for concentration equal to 14 mm and then rapidly decrease for concentration close to cmc (20 mm). In the range of concentration above 20 mm the retention times increase almost linearly with NaC concentration, but the slope is lower in comparison with that obtained for the monomeric range of concentrations. Comparing the behaviour of acyclic, monocyclic and bicyclic monoterpenes, the acyclic and monocyclic compounds interact more strongly with NaC than the bicyclic ones. The behaviour of xylene isomers is different. The retention times remain constant in the NaC concentration range of 0–7 mm and unexpectedly decrease for concentration of 14 mm. After that the retention times increase to almost the previous value and remain practically constant with the increase of concentration. Since the adjusted retention times of xylenes for 14 mm are shorter than on the matrix column, this could suggest that some processes unfavourable for aromatic compound sorption occur at the surface of the stationary phase. It seems that the affinity of xylenes to formamide is more favourable than interaction with NaC. A similar tendency of dependence of tR vs. NaC concentration was obtained for polar solvents: acetone and ethylene acetate (Fig. 5a). It seems that a specific arrangement of NaC molecules at the surface of formamide makes the behaviour of nonpolar aromatic hydrocarbons similar to polar solvents. Similar chromatographic measurements were conducted for NaDC. The relations of retention times of the investigated compounds vs. NaDC concentration are presented in Fig. 6. In this case the retention times increase quickly in the 0–7 mm concentration range for most of the monoterpenes. A rapid decrease of retention time analogous to that found for NaC is observed for concentration close to cmc (10 mm). In comparison with NaC, the increase of retention times is greater for NaDC in the micellar range of concentration. In the case of xylene isomers, similar behaviour to that found for NaC is observed. A decrease of retention times is observed for concentration of 7 mm. Also in the case of polar solvents the relation of tR vs. NaDC concentration exhibits a similar tendency as for xylenes (Fig. 5b). The obtained results may be explained by the following hypothesis. Bile salt molecules have a quite rigid and flat structure with hydrophobic and hydrophilic faces. It is possible that in low concentrations (below cmc) they lie flat on the surface of formamide with the hydrophilic face oriented toward the solution (Fig. 7a). When the concentration of the bile salt increases, the layer of formamide becomes more covered with the bile salt molecules (Fig. 7b). Such surface behaviour of bile salts at the air/water interface was described by Tiss et al. [16]. At a concentration close to cmc the surface is too crowded and the bile salt molecules probably reorganize at the surface of formamide from a flat to an upright orientation, with their hydrophobic faces oriented together (Fig. 7c). Afterwards, the increase of concentration of the bile salts induces
Fig. 7. Schematic representation of the behaviour of bile salts: below cmc (a, b); close to cmc (c); above cmc (d).
the formation of aggregates inside the formamide solution (Fig. 7d). Similar behaviour of NaC and NaDC in a water solution was suggested by Almgren and coworkers [17]. The concentration at which the surface of formamide is hypothetically tightly packed with flat-lying molecules of bile salts, corresponds to the point where the adjusted retention times reach the maximum value for monoterpenes and the minimum for xylenes. The subsequent reorganization of bile salts from lying flat to an upright orientation makes the surface of formamide more accessible for xylenes but this position is less convenient for interaction between bile salts and monoterpenes. This could explain the unexpected decrease of retention times for concentration close to the cmc for NaC (20 mm) and NaDC (10 mm). Unfortunately, the investigated systems do not exhibit selective properties towards enantiomers of monoterpenes as well as for constitutional isomers of xylenes. 4.3. Temperature investigations The stability of micelles is temperature-dependent. An increase of temperature may cause a demicellization process. Gas chromatographic measurements are usually performed in temperatures much higher than ambient temperature. It was therefore necessary to check the thermal stability of micellar systems in formamide. To prove the thermal stability of the micellar stationary phase, temperature investigations were performed for selected compounds. The retention factors of compounds selected from the
M. Skórka, M. Asztemborska / Colloids and Surfaces A: Physicochem. Eng. Aspects 332 (2009) 1–8
Fig. 8. van’t Hoff plots of ln k of selected compounds vs. 1/T obtained on columns: matrix, C1, C3 and C6.
Fig. 9. van’t Hoff plots of ln k of selected compounds vs. 1/T obtained on columns: matrix, DC1, DC2 and DC6.
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Table 3 Enthalpies of solute transfer (kJ/mol) from the mobile to the stationary phase and enthalpies of binding (kJ/mol) of selected compounds with NaC and NaDC
Without surfactant −HI Sodium cholate (NaC) −HII −HIII −HIV −Hmon −Hcmc −Hmic
Column
-Citronellene
␣-Terpinene
␣-Pinene
o-Xylene
Matrix
37.82 (0.998)
39.07 (0.998)
31.92 (0.992)
35.58 (0.998)
C3 C4 C6
47.55 (0.998) 44.81 (0.998) 47.97 (0.999) 9.73 6.99 3.16
45.88 (0.998) 41.39 (0.998) 44.72 (0.998) 6.81 2.32 3.33
42.81 (0.998) 38.57 (0.998) 41.57 (0.998) 10.89 6.65 3.00
35.75 (0.998) 36.57 (0.996) 37.92 (0.998) 0.17 0.99 1.25
51.21 (0.998) 42.48 (0.997) 49.22 (0.994) 13.39 4.66 6.74
47.96 (0.998) 40.25 (0.998) 45.06 (0.998) 8.89 1.18 4.81
45.56 (0.998) 34.58 (0.997) 40.23 (0.994) 13.64 2.66 5.65
38.48 (0.998) 34.41 (0.999) 34.58 (0.998) 2.90 −1.17 0.17
Sodium deoxycholate (NaDC) DC2 −HII −HIII DC3 −HIV DC6 −Hmon −Hcmc −Hmic R2 = regression coefficient.
acyclic, monocyclic, bicyclic, and aromatic groups were measured in the 303–328 K temperature range on columns with concentration 7, 14, 20 and 60 mm of NaC, 3, 7, 10 and 60 mm of NaDC, and the matrix column. The obtained results are presented as van’t Hoff plots in Figs. 8 and 9. A very good linear variation of ln k vs. 1/T is observed with regression coefficient R2 > 0.992. These results indicate that the investigated stationary phases are stable in the studied temperature range, in the micellar range as well as in the monomeric range of concentrations. In accordance with Eqs. (1)–(4), the partial molar enthalpy of solute transfer from the mobile to the stationary phase was determined from the slopes of relations ln k vs. 1/T for -citronellene, ␣-terpinene, ␣-pinene, and o-xylene on the matrix column and selected columns with bile salt concentration below, at, and above the cmc point. The determined values of HI , HII , HIII and HIV are presented in Table 3. The enthalpies of binding of selected compounds with NaC and NaDC as monomers Hmon at cmc Hcmc and in micellar form Hmic were calculated according to Eqs. (2)–(4). The calculated values of H are presented in Table 3. In the case of -citronellene, ␣-terpinene and ␣-pinene, Hmon and Hmic are larger for NaDC than for NaC, while Hcmc is larger for NaC than for NaDC. The binding enthalpy of the investigated terpenoids is larger for bile salts monomers than micelle ((−Hmon ) > (−Hmic )). The binding enthalpy of bile salts with o-xylene is close to zero independently on concentration of surfactant. 5. Summary The cmc values of NaC and NaDC in a formamide solution were determined from surface tension measurements. The experimentally estimated cmc values are 17 mM for NaC and 9 mM for NaDC. The obtained chromatographic results provide interesting information on the behaviour of bile salts on the surface of a
nonaqueous solvent. Temperature investigations indicate that the studied micellar phase is stable in the range 30–50 ◦ C. The binding enthalpies of selected compounds to bile salt monomers and in aggregate form were also determined. Acknowledgments ˙ ´ The authors wish to thank Andrzej Zywoci nski Ph.D. and Patrycja Milczarczyk-Piwowarczyk M.Sc. for their help in surface tension measurements and interpretation. This work was partially supported by grant no. N204 3702 33 of the Polish Ministry of Science and Higher Education. References [1] A. Coello, F. Meijide, E.R. Nunez, J.V. Tato, J. Pharm. Sci. 85 (1996) 9–15. [2] D.M. Small, P.P. Nair, D. Kritchevsky (Eds.), The Bile Salts, vol. 1, Plenum Press, New York, 1971, p. 249. [3] E. Giglio, S. Loreti, N.V. Pavel, J. Phys. Chem. 92 (1988) 2858–2862. [4] R. Williams, F. ZhengSheng, W. Hinze, J. Chromatogr. Sci. 28 (1990) 292–302. [5] W. Hinze, R. Williams, F. Zheng Sheng, Y. Suzuki, F. Quina, Colloids Surf. 48 (1990) 79–94. [6] M.G. Khaledi, J. Chromatogr. A 780 (1997) 3–40. [7] P.G. Muijselaar, K. Otsuka, S. Terabe, J. Chromatogr. A 780 (1997) 41–61. [8] M.L. Marina, M.A. Garcia, J. Chromatogr. A 780 (1997) 103–116. [9] A.W. Lantz, V. Pino, J.L. Anderson, D.W. Armstrong, J. Chromatogr. A 1115 (2006) 217–224. [10] D.W. Armstrong, F. Nome, Anal. Chem. 52 (1981) 1662–1666. [11] V. Pino, A.W. Lantz, J.L. Anderson, A. Berthod, D.W. Armstrong, Anal. Chem. 78 (2006) 113–119. [12] S. Reis, C. Guimaraes Moutinho, C. Matos, B. de Castro, P. Gameiro, J.L.F.C. Lima, Anal. Biochem. 334 (2004) 117–126. [13] R. Shaw, W.H. Elliott, B.G. Barisas, Microchim. Acta 105 (1991) 137–145. [14] S. Terabe, N. Chen, K. Otsuka, in: A. Chrambach, J. Dunn, B. Radola (Eds.), Advances in Electrophoresis, vol. 7, VCH, Weinheim, 1994, p. 91. [15] M.S. Akhter, S.M. Alawi, Colloids Surf. A 173 (2000) 95–100. [16] A. Tiss, S. Ransac, H. Lengsfeld, P. Hadvary, A. Cagna, R. Verger, Chem. Phys. Lipids 111 (2001) 73–85. [17] M. Swanson-Vethamuthu, M. Almgren, P. Hansson, J. Zhao, Langmuir 12 (1996) 2186–2189.