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Szymon Bocian Ewelina Dziubakiewicz Bogusław Buszewski Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Torun, Poland Received January 21, 2015 Revised May 8, 2015 Accepted May 9, 2015
Research Article
Influence of the charge distribution on the stationary phases zeta potential A set of seven home-made silica based bonded phases with different functional groups was investigated. Their zeta potential data in methanol and acetonitrile as well as in methanol/water and acetonitrile/water solution were obtained by using a Zetasizer. The influence of polar functional groups on a zeta potential was investigated. The results show that the amines incorporated in the structure of chemically bonded phases of reversed-phase materials are protonated during chromatographic analysis, resulting in changes of the zeta potential from negative to positive values. Acetonitrile causes more negative values and methanol provides positive (or less negative) values of the zeta potential. Keywords: Charge distribution / Polar functional groups / Stationary phases / Zeta potential DOI 10.1002/jssc.201500072
1 Introduction The surface of silica-based stationary bonded phases usually contains a more or less significant portion of residual silanol groups, which cannot be removed or blocked due to steric effects of bonded ligands, even if the stationary bonded phase is completed with an end-capping [1]. These residual silanol groups on the silica surface in the solution are partially ionized, if the contacting liquid solvates protons split off from silanols. The solvation of hydronium ions in water is also an important issue, even though it is known that the hydronium ion solvates six or even more (up to 20) water molecules [2]. For this reason, under chromatographic conditions, residual silanols are always partially ionized, if the mobile phase can solvate protons [3]. Thus, even alkyl stationary bonded phases poses some charges on the surface [4]. The electric field created by charges on the stationary phase surface has the highest intensity at this surface. This intensity is expressed by surface potential ⌽O . In the Stern layer, the potential of the electric field ⌽S linearly decreases with distance from the solid surface to the zeta potential [5]. The zeta potential gives the potential of the electric field created by silica gel charges in the point from which the liquid phase can move either by action of pressure gradient or by the action of outer electric field. For these reasons, the zeta potential is the key characteristic of electric double layer from a practical point of view [6–9]. When the stationary phase is packed into capillaries, and with an application of electric field across them, the zeta potential causes the EOF used as the driving force in CEC [10]. Correspondence: Dr. Szymon Bocian, Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7 st., 87-100 Torun, Poland E-mail: [email protected] Fax: +48 56 611 48 37
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Negative charges on the silica surface in the bonded phase used in LC are sufficient to create the electric double-layer in the solution adjacent to this surface [11,12]. This effect makes stationary bonded phases useful in CEC. The opposite situation may be observed when the stationary phase possess some specific functional groups, which consist of other elements than carbon and hydrogen. Many specific phases are known, for example: N-acylamide, amine, diol, or stationary phases with embedded phosphate groups. In such stationary phases the oxygen, nitrogen, or phosphorus atoms are presented. These atoms are significantly electronegative and thus the ligands are more or less polarized. As a result of polar functionalities, different molecular interactions can take place, not only hydrophobic interactions that is the most common in the case of alkyl adsorbents. The presence of ionized silanols and electronegative atoms in the ligands may create a surface charge on the chromatographic adsorbents. The most evident charges on the stationary phase surface are present in the stationary phases used in ion-exchange chromatography, where the ionized functional groups are incorporated into the ligands [13]. In materials used for aqueous normal-phase chromatography, residual silanols are blocked through the formation of a silica hydride surface. Such surface modification strongly influences the zeta potential of the stationary phase [14–16]. The silica hydride surface in acidic conditions provide negative values of zeta potential [17] similar to that of bare silica gel. In the case of octadecyl adsorbents, the zeta potential of the stationary bonded phases, changes with the coverage density of bonded ligands as well as with the presence of trimethylsilyl groups connected to the surface as an endcapping. The highest zeta potential is observed in water-rich mobile phases and it usually decreases with the increase of the organic modifier content in the mobile phase. The significant influence of solvent on the zeta potential of the chemically bonded phases may results from two cases: the differences in
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Figure 1. Structures of stationary phases used in the study: octadecyl (A1), octadecyl end-capped (A2), Amino (B), Amino-AP-C12 (C), Amino-Chol (D), Di-amino (E), Di-amino-AP-C12 (F), Di-amino-Chol (G), Amino-P-C10 (H), Amino-P-C18 (I), and Phenyl-propyl (J).
the dielectric properties of methanol, acetonitrile and water, and from differences, how different solvent molecules solvate the bonded phase particles. Although information about the zeta potential of octadecyl adsorbents has been presented in literature, the zeta potential of stationary bonded phases still requires further investigations. In this work we determined the zeta potentials for characterization of the surface properties of silica-based stationary phase for LC that poses specific polar groups in their structures. The goal of our study was to determine the influence of the presence of the electronegative atoms (oxygen, nitrogen, phosphorus) in the structure of bonded ligands and the solution composition on the zeta potential of the stationary bonded phases used in LC.
2 Materials and methods
Table 1. Physicochemical properties of stationary phases used in the study
Stationary phase
Structure in Fig. 1
Carbon load [%]
Nitrogen load [%]
Octadecyl Octadecyl EC
A1 A2
18.7 18.8
– –
Amino Amino-AP-C12 Amino-Chol Di-amino Di-amino-AP-C12 Di-amino-Chol Amino-P-C10 Amino-P-C18 Phenyl-propyl
B C D E F G H I J
2.62 11.24 17.82 3.84 15.58 22.39 8.43 9.32 11.75
1.56 1.50 0.83 1.85 1.86 1.43 0.91 1.25 –
Coverage density [mol/m2 ] 3.27 3.27 (3.34 including EC) 2.57 2.14 1.77 2.30 3.05 2.28 1.64 1.04 3.39
2.1 Instruments The zeta potential measurement was performed using Zetasizer nano ZS (Malvern Instrument, UK) equipped with dip cell. The degree of the silica surface coverage was calculated based on the carbon percentage determined with a Model 240 CHN analyzer (Perkin Elmer, Norwalk, USA).
2.2 Materials A series of stationary phases that contain specific functional groups were tested. Structures of materials used are shown C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in Fig. 1. All stationary phases were synthesized in the lab and described elsewhere [18–22]. As a support for the synthesis, the Kromasil 100 silica gel (Akzo Nobel, Bohus, Sweden) with a particle diameter 5 m and pore size 100 Å was used. For the comparison, alkyl C18 stationary phases with different coverage were used. As it can be seen, tested stationary phases possess different functional groups in their structures. The presence of those groups and electronegative atoms influences significantly chromatographic properties of these materials. The physicochemical properties of synthesized stationary phases are listed in Table 1. www.jss-journal.com
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Table 2. The zeta potential of stationary bonded phases. Standard deviation were calculated for n = 9
2.4 Methods For each measurement 0.5 mg of the stationary bonded phase was suspended in 5 mL organic solvent or organic solvent/water mixture (50% v/v) using an ultrasonic bath for 10 min to help obtaining a stable suspension due to removing air from pores. The measurement of zeta potential was done immediately after removing the suspension from the bath. Before measurement the stability of the suspension was routinely tested by Zetasizer. In the case of satisfactory suspension, the zeta potential of the stationary bonded phase in solutions were automatically calculated by Zetasizer from electrophoretic mobility using the Smoluchowski’s formula [23]: =
grgo
(1)
where is the viscosity of the solution, g r is the relative permittivity of the medium, and g o is the absolute permittivity of vacuum [23, 24]. The viscosity and permittivity data were taken from the literature [25–27]. The dielectric constant of electrolytes solution were calculated according to methodology present by Wang and Anderko [28]. The zeta potential measurements were repeated to obtain nine data points for each sample. Only the results reported as reproducibly obtained were taken. Using a zetasizer, different values of electrophoretic mobility were obtained from different correlations between the scattering pattern of a laser beam after passing through the medium and that of reference sample. The intensity of the scattering pattern significantly depends on the refractive index. This implies the need to provide the correct values of the refractive index of the reference medium in which the stationary phase was suspended in because the measured electrophoretic mobility, and thus the zeta potential of a sample, depends on the refractive index of the medium. The refractive indices of all the studied solvents were corrected by using the corrected refractive index value for each solvent before the measurement. The values for 100% v/v ACN, 50% v/v ACN, 50% v/v MeOH, and 100% v/v MeOH at 25⬚C were 1.3422, 1.3454, 1.3412, and 1.3269, respectively [29].
3 Results and discussion The results of stationary phases zeta potential measurement in four different mobile phases are listed in Table 2. In contrast to previously published results [4], in the case of polar stationary phases, both positive and negative values of zeta potential was observed. In the case of octadecyl adsorbents with different coverage densities usually negative values were obtained regardless of the type of organic modifier or the water content in aqueous-organic solvent mixtures (except C18 3.27EC in 50% of MeOH). Negative values of zeta potential were probably caused by the presence of the partially ionized residual silanols. Various activities of silanol groups can be C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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C18 3.27 C18 3.27EC Amino Amino-AP-C12 Amino-Chol Di-amino Di-amino-AP-C12 Di-amino-Chol Amino-P-C10 Amino-P-C18 Phenyl-propyl SiO2
100% ACN
50% ACN
50% MeOH 100% MeOH
−33.9 ± 2.09 −39.3 ± 1.95 −23.4 ± 4.28 −27.6 ± 4.04 −28.2 ± 5.92 −22.5 ± 4.50 20.0 ± 5.06 14.6 ± 2.31 −29.7 ± 3.04 −24.9 ± 3.56 −28.8 ± 2.19 −22.8 ± 1.53
−35.2 ± 1.18 −12.1 ± 0.89 22.9 ± 3.56 −15.8 ± 4.84 −9.3 ± 3.46 20.5 ± 2.53 18.9 ± 2.48 22.2 ± 4.57 21.9 ± 3.94 −11.1 ± 4.51 −29.5 ± 3.58 −24.3 ± 1.28
−13.2 ± 0.87 0.725 ± 0.03 21.4 ± 4.50 −14.1 ± 5.61 4.2 ± 8.72 20.4 ± 3.79 14.7 ± 5.56 12.4 ± 5.04 16.0 ± 3.25 −11.5 ± 3.58 −13.3 ± 4.92 −21.2 ± 1.25
−23.8 ± 1.24 −17.3 ± 1.32 25.6 ± 2.68 −3.8 ± 7.85 18.3 ± 2.11 30.9 ± 2.99 34.6 ± 3.24 42.1 ± 3.91 30.0 ± 3.52 −22.7 ± 4.74 −23.2 ± 5.91 −19.5 ± 1.17
manifested in their acidities [30]. One type is very acidic and it has a pKa value between 3.5 and 4.6 (for vicinal silanols) and the other type is less acidic with a pKa between 6.2 and 6.8 (single silanols) [31, 32]. Since all tested stationary phases were synthesized on a silica support, negatively charged surface will influence the zeta potential of all stationary phases. However, different functional groups used for silica gel modification enhanced or weekend the influence of silica support on the zeta potential of chromatographic stationary phases. In Fig. 2, stationary phases were ordered from those that provide positive values of the zeta potential on the left to material that provides only negative values. Two stationary phases (Di-amino-Chol and Di-amino-AP-C12) provide only positive values of zeta potential, independent of the mobile phase composition. These stationary phases contain an amido group and secondary amines in the structure and residual primary amines. In the second step, these amines are modified to form an amide bond, but more than 50% of amine groups are unmodified. Unmodified amines with a pKa in the range 9.0–9.6 (in pure water) are protonated in the mobile phase used. The scheme of protonated stationary phases is shown in Fig. 3. It shows that these materials are positively charged and it causes the positive values of zeta potential. Positive charges in the structure of bonded ligands have a more significant influence on the stationary phase zeta potential than negatively charged ionized silanols on the support surface. A similar situation is observed in the case of the unmodified Di-amino stationary phase, however, in this case the negative values of zeta potential are observed in pure acetonitrile. An important observation from this study is that Di-amino, Di-amino-Chol and Di-amino-AP-C12 may act in a reversedphase system as a weak anion exchanger. Another group of stationary phases exhibit similar values. In this group one can find Amino-P-C10, Amino, and Amino-Chol. This group of stationary phases provides negative values of zeta potential in pure ACN and positive values in other mobile phases used in the study. The positive values of zeta potential in the case of Amino-P-C10, Amino may www.jss-journal.com
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Figure 2. Changes of stationary phases zeta potential in different reversed-phase mobile phases.
Figure 3. Structures of stationary phases that exhibit positive values of zeta potential (Di-amino-AP-C12, Di-Amino-Chol) in different pH range (A: 1–4, B: 4–9, C: above 9.6). Models based on pKa values in pure water.
be explained by protonation of nitrogen atom, that has pKa around 6.8–7 (in pure water). Bare silica gel and alkyl and phenyl stationary phases exhibit only negative values of zeta potential in all tested mobile phases. These materials do not possess any nitrogen atoms C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
that may be protonated. Their zeta potential is influenced only by negative charges on the silica support. The zeta potential of bare silica gel is almost independent on the type of organic modifier as well as on the composition of the solvent mixture. Modified silica gel exhibits changes of www.jss-journal.com
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the zeta potential with the mobile phase composition. A more negative zeta potential is observed in an ACN environment. It may be explained by the higher ACN dipole moment than the methanol dipole moment. In this case, the creation of a double layer in a non-electrolyte solution is easier because of the higher polarity of the solution. A higher dipole moment and higher polarizability of ACN in the comparison with MeOH may be the reason for the higher absolute values of zeta potential. The solvation process of polar stationary phases is also different in MeOH and ACN environments, which may also influences the zeta potential. A series of polar stationary phases provide positive values of the zeta potential in the present of the solvent with hydroxyl groups (methanol or water) whereas in the case of ACN the zeta potential is negative. It confirms the observation [4], that solvation process influence significantly zeta potential of the stationary phases in RP conditions. Usually, ACN causes values that are more negative whereas the presence of methanol moves these values to a positive range.
[15] Kulsing, C., Yang, Y., Matyska, M. T., Pesek, J. J., Boysen, R. I., Hearn, M. T. W., Anal. Chim. Acta 2014, 859, 79–86.
4 Conclusions
[16] Kulsing, C., Yang, Y., Boysen, R. I., Matyska, M. T., Pesek, J. J., Hearn, M. T. W., Anal. Method. 2015, 7, 1578– 1585.
The zeta potential of the polar stationary bonded phases, measured by Zetasizer, depends on the protonation of the nitrogen atoms. Materials with amine group may be protonated under RP conditions (ACN, MeOH and its mixtures with water) and provide positive values of zeta potential. Materials without proton acceptors provide negative values of zeta potential. However, the type of the organic modifier has significant influence on the zeta potential of the chemically bonded phases. In ACN the zeta potential is more negative and in methanol it is more positive. This influence may result from the differences in the dielectric properties of methanol, acetonitrile and water, and from differences in solvation of bonded phase particles in different solvents. This work was supported by Ministry of Science and Higher Education, grant no. NCN 2013/09/D/ST4/03807 for period 2014-2017. Authors thank Akzo Nobel (Bohus, Sweden) for kind donation of silica gel Kromasil 100 used in the study. The authors have declared no conflict of interest.
[5] Hunter, R. J., Zeta Potential in Colloid Science: Principles and Applications, Academic Press, London, 1981. [6] Stern, O., Z. Elektrochem. 1924, 30, 508–514. [7] Shaw, D. J., Electrophoresis, Academic Press, London 1969. [8] Foret, F., Bocek, P., Adv. Electrophoresis 1990, 3, 272. [9] Salomon, K., Burgi, D. S., Helmer, J. C., J. Chromatogr. 1991, 559, 69–80. [10] Pretorius, V., Hopkins, B. J., Schiecke, J. D., J. Chromatogr. 1974, 99, 23–30. ´ S., Top. Biochem. Bioenerg. 1978, 2, 89. [11] Hjerten, [12] Grahame, D. C., Chem. Rev. 1947, 41, 441–501. ´ [13] Buszewski, B., Jackowska, M., Bocian, S., Dziubakiewicz, E., J. Sep. Sci. 2013, 36, 156–163. [14] Kulsing, C., Yang, Y., Munera, C., Tse, C., Matyska, M. T., Pesek, J. J., Boysen, R. I., Hearn, M. T. W., Anal. Chim. Acta 2014, 817, 48–60.
[17] Yang, Y., Boysen, R. I., Kulsing, C., Matyska, M. T., Pesek, J. J., Hearn, M. T. W., J. Sep. Sci. 2013, 36, 3019– 3025. [18] Bocian, S., Vajda, P., Felinger, A., Buszewski, B., Anal. Chem. 2009, 81, 6334–6346. [19] Bocian, S., Nowaczyk, A., Buszewski, B., Anal. Bioanal. Chem. 2012, 404, 731–740. [20] Buszewski, B., Bocian, S., Matyska, M. T., Pesek, J. J., J. Chromatogr. A 2011, 1218, 441–448. [21] Bocian, S., Buszewski, B., J. Sep. Sci. 2014, 37, 3435– 3442. [22] Buszewski, B., Schmid, J., Albert, K., Bayer, E., J. Chromatogr. 1991, 552, 415–427. [23] vonSmoluchowski, M., Handbuch der Elektrizitat und des Magnetismus, Barth, J. A. (Ed.), Liepzig 1921, p. 366. [24] Dearie, H. S., Spikmans, V., Smith, N. W., Moffatt, F., Wren, S. A. C., Evans, K. P., J. Chromatogr. A 2001, 929, 123–131. [25] Thompson, J. W., Kaiser, T. J., Jorgenson, J. W., J. Chromatogr. A 2006, 1134, 201–209. [26] Akerlof, G., J. Am. Chem. Soc. 1932, 54, 4125–4139. [27] Atkins, P. W., Physical Chemistry-6th ed., W.H. Freeman and Company, New York 1997.
5 References
[28] Wang, P., Anderko, A., Fluid Phase Equilibr. 2001, 186, 103–122.
´ M., Buszewski, B., [1] Jandera, P., Bocian, S., Mol´ıkova, J. Chromatogr. A 2009, 1216, 237–248.
[29] Openhaim, G., Grushka, E., J. Chromatogr. A 2002, 942, 63–71.
[2] Yang, Y., Matyska, M. T., Boysen, R. I., Pesek, J. J., Hearn, M. T. W., J. Sep. Sci. 2013, 36, 1209–1216.
[30] Snyder, L. R., Principles of Adsorption Chromatography, Marcel Dekker, New York 1968.
[3] Unger, K. K., Porous Silica, Elsevier, Amsterdam 1979.
[31] Mendez, A., Bosch, E., Roses, M., Neue, U. D., J. Chromatogr. A 2003, 986, 33–44.
[4] Buszewski, B., Bocian, S., Dziubakiewicz, E., J. Sep. Sci. 2010, 33, 1529–1537.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[32] Nawrocki, J., J. Chromatogr. A 1997, 779, 29–71.
www.jss-journal.com
J. Sep. Sci. 2015, 38, 2625–2629
Szymon Bocian Ewelina Dziubakiewicz Bogusław Buszewski Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Torun, Poland Received January 21, 2015 Revised May 8, 2015 Accepted May 9, 2015
Research Article
Influence of the charge distribution on the stationary phases zeta potential A set of seven home-made silica based bonded phases with different functional groups was investigated. Their zeta potential data in methanol and acetonitrile as well as in methanol/water and acetonitrile/water solution were obtained by using a Zetasizer. The influence of polar functional groups on a zeta potential was investigated. The results show that the amines incorporated in the structure of chemically bonded phases of reversed-phase materials are protonated during chromatographic analysis, resulting in changes of the zeta potential from negative to positive values. Acetonitrile causes more negative values and methanol provides positive (or less negative) values of the zeta potential. Keywords: Charge distribution / Polar functional groups / Stationary phases / Zeta potential DOI 10.1002/jssc.201500072
1 Introduction The surface of silica-based stationary bonded phases usually contains a more or less significant portion of residual silanol groups, which cannot be removed or blocked due to steric effects of bonded ligands, even if the stationary bonded phase is completed with an end-capping [1]. These residual silanol groups on the silica surface in the solution are partially ionized, if the contacting liquid solvates protons split off from silanols. The solvation of hydronium ions in water is also an important issue, even though it is known that the hydronium ion solvates six or even more (up to 20) water molecules [2]. For this reason, under chromatographic conditions, residual silanols are always partially ionized, if the mobile phase can solvate protons [3]. Thus, even alkyl stationary bonded phases poses some charges on the surface [4]. The electric field created by charges on the stationary phase surface has the highest intensity at this surface. This intensity is expressed by surface potential ⌽O . In the Stern layer, the potential of the electric field ⌽S linearly decreases with distance from the solid surface to the zeta potential [5]. The zeta potential gives the potential of the electric field created by silica gel charges in the point from which the liquid phase can move either by action of pressure gradient or by the action of outer electric field. For these reasons, the zeta potential is the key characteristic of electric double layer from a practical point of view [6–9]. When the stationary phase is packed into capillaries, and with an application of electric field across them, the zeta potential causes the EOF used as the driving force in CEC [10]. Correspondence: Dr. Szymon Bocian, Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7 st., 87-100 Torun, Poland E-mail: [email protected] Fax: +48 56 611 48 37
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Negative charges on the silica surface in the bonded phase used in LC are sufficient to create the electric double-layer in the solution adjacent to this surface [11,12]. This effect makes stationary bonded phases useful in CEC. The opposite situation may be observed when the stationary phase possess some specific functional groups, which consist of other elements than carbon and hydrogen. Many specific phases are known, for example: N-acylamide, amine, diol, or stationary phases with embedded phosphate groups. In such stationary phases the oxygen, nitrogen, or phosphorus atoms are presented. These atoms are significantly electronegative and thus the ligands are more or less polarized. As a result of polar functionalities, different molecular interactions can take place, not only hydrophobic interactions that is the most common in the case of alkyl adsorbents. The presence of ionized silanols and electronegative atoms in the ligands may create a surface charge on the chromatographic adsorbents. The most evident charges on the stationary phase surface are present in the stationary phases used in ion-exchange chromatography, where the ionized functional groups are incorporated into the ligands [13]. In materials used for aqueous normal-phase chromatography, residual silanols are blocked through the formation of a silica hydride surface. Such surface modification strongly influences the zeta potential of the stationary phase [14–16]. The silica hydride surface in acidic conditions provide negative values of zeta potential [17] similar to that of bare silica gel. In the case of octadecyl adsorbents, the zeta potential of the stationary bonded phases, changes with the coverage density of bonded ligands as well as with the presence of trimethylsilyl groups connected to the surface as an endcapping. The highest zeta potential is observed in water-rich mobile phases and it usually decreases with the increase of the organic modifier content in the mobile phase. The significant influence of solvent on the zeta potential of the chemically bonded phases may results from two cases: the differences in
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Figure 1. Structures of stationary phases used in the study: octadecyl (A1), octadecyl end-capped (A2), Amino (B), Amino-AP-C12 (C), Amino-Chol (D), Di-amino (E), Di-amino-AP-C12 (F), Di-amino-Chol (G), Amino-P-C10 (H), Amino-P-C18 (I), and Phenyl-propyl (J).
the dielectric properties of methanol, acetonitrile and water, and from differences, how different solvent molecules solvate the bonded phase particles. Although information about the zeta potential of octadecyl adsorbents has been presented in literature, the zeta potential of stationary bonded phases still requires further investigations. In this work we determined the zeta potentials for characterization of the surface properties of silica-based stationary phase for LC that poses specific polar groups in their structures. The goal of our study was to determine the influence of the presence of the electronegative atoms (oxygen, nitrogen, phosphorus) in the structure of bonded ligands and the solution composition on the zeta potential of the stationary bonded phases used in LC.
2 Materials and methods
Table 1. Physicochemical properties of stationary phases used in the study
Stationary phase
Structure in Fig. 1
Carbon load [%]
Nitrogen load [%]
Octadecyl Octadecyl EC
A1 A2
18.7 18.8
– –
Amino Amino-AP-C12 Amino-Chol Di-amino Di-amino-AP-C12 Di-amino-Chol Amino-P-C10 Amino-P-C18 Phenyl-propyl
B C D E F G H I J
2.62 11.24 17.82 3.84 15.58 22.39 8.43 9.32 11.75
1.56 1.50 0.83 1.85 1.86 1.43 0.91 1.25 –
Coverage density [mol/m2 ] 3.27 3.27 (3.34 including EC) 2.57 2.14 1.77 2.30 3.05 2.28 1.64 1.04 3.39
2.1 Instruments The zeta potential measurement was performed using Zetasizer nano ZS (Malvern Instrument, UK) equipped with dip cell. The degree of the silica surface coverage was calculated based on the carbon percentage determined with a Model 240 CHN analyzer (Perkin Elmer, Norwalk, USA).
2.2 Materials A series of stationary phases that contain specific functional groups were tested. Structures of materials used are shown C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in Fig. 1. All stationary phases were synthesized in the lab and described elsewhere [18–22]. As a support for the synthesis, the Kromasil 100 silica gel (Akzo Nobel, Bohus, Sweden) with a particle diameter 5 m and pore size 100 Å was used. For the comparison, alkyl C18 stationary phases with different coverage were used. As it can be seen, tested stationary phases possess different functional groups in their structures. The presence of those groups and electronegative atoms influences significantly chromatographic properties of these materials. The physicochemical properties of synthesized stationary phases are listed in Table 1. www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2015, 38, 2625–2629
Table 2. The zeta potential of stationary bonded phases. Standard deviation were calculated for n = 9
2.4 Methods For each measurement 0.5 mg of the stationary bonded phase was suspended in 5 mL organic solvent or organic solvent/water mixture (50% v/v) using an ultrasonic bath for 10 min to help obtaining a stable suspension due to removing air from pores. The measurement of zeta potential was done immediately after removing the suspension from the bath. Before measurement the stability of the suspension was routinely tested by Zetasizer. In the case of satisfactory suspension, the zeta potential of the stationary bonded phase in solutions were automatically calculated by Zetasizer from electrophoretic mobility using the Smoluchowski’s formula [23]: =
grgo
(1)
where is the viscosity of the solution, g r is the relative permittivity of the medium, and g o is the absolute permittivity of vacuum [23, 24]. The viscosity and permittivity data were taken from the literature [25–27]. The dielectric constant of electrolytes solution were calculated according to methodology present by Wang and Anderko [28]. The zeta potential measurements were repeated to obtain nine data points for each sample. Only the results reported as reproducibly obtained were taken. Using a zetasizer, different values of electrophoretic mobility were obtained from different correlations between the scattering pattern of a laser beam after passing through the medium and that of reference sample. The intensity of the scattering pattern significantly depends on the refractive index. This implies the need to provide the correct values of the refractive index of the reference medium in which the stationary phase was suspended in because the measured electrophoretic mobility, and thus the zeta potential of a sample, depends on the refractive index of the medium. The refractive indices of all the studied solvents were corrected by using the corrected refractive index value for each solvent before the measurement. The values for 100% v/v ACN, 50% v/v ACN, 50% v/v MeOH, and 100% v/v MeOH at 25⬚C were 1.3422, 1.3454, 1.3412, and 1.3269, respectively [29].
3 Results and discussion The results of stationary phases zeta potential measurement in four different mobile phases are listed in Table 2. In contrast to previously published results [4], in the case of polar stationary phases, both positive and negative values of zeta potential was observed. In the case of octadecyl adsorbents with different coverage densities usually negative values were obtained regardless of the type of organic modifier or the water content in aqueous-organic solvent mixtures (except C18 3.27EC in 50% of MeOH). Negative values of zeta potential were probably caused by the presence of the partially ionized residual silanols. Various activities of silanol groups can be C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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C18 3.27 C18 3.27EC Amino Amino-AP-C12 Amino-Chol Di-amino Di-amino-AP-C12 Di-amino-Chol Amino-P-C10 Amino-P-C18 Phenyl-propyl SiO2
100% ACN
50% ACN
50% MeOH 100% MeOH
−33.9 ± 2.09 −39.3 ± 1.95 −23.4 ± 4.28 −27.6 ± 4.04 −28.2 ± 5.92 −22.5 ± 4.50 20.0 ± 5.06 14.6 ± 2.31 −29.7 ± 3.04 −24.9 ± 3.56 −28.8 ± 2.19 −22.8 ± 1.53
−35.2 ± 1.18 −12.1 ± 0.89 22.9 ± 3.56 −15.8 ± 4.84 −9.3 ± 3.46 20.5 ± 2.53 18.9 ± 2.48 22.2 ± 4.57 21.9 ± 3.94 −11.1 ± 4.51 −29.5 ± 3.58 −24.3 ± 1.28
−13.2 ± 0.87 0.725 ± 0.03 21.4 ± 4.50 −14.1 ± 5.61 4.2 ± 8.72 20.4 ± 3.79 14.7 ± 5.56 12.4 ± 5.04 16.0 ± 3.25 −11.5 ± 3.58 −13.3 ± 4.92 −21.2 ± 1.25
−23.8 ± 1.24 −17.3 ± 1.32 25.6 ± 2.68 −3.8 ± 7.85 18.3 ± 2.11 30.9 ± 2.99 34.6 ± 3.24 42.1 ± 3.91 30.0 ± 3.52 −22.7 ± 4.74 −23.2 ± 5.91 −19.5 ± 1.17
manifested in their acidities [30]. One type is very acidic and it has a pKa value between 3.5 and 4.6 (for vicinal silanols) and the other type is less acidic with a pKa between 6.2 and 6.8 (single silanols) [31, 32]. Since all tested stationary phases were synthesized on a silica support, negatively charged surface will influence the zeta potential of all stationary phases. However, different functional groups used for silica gel modification enhanced or weekend the influence of silica support on the zeta potential of chromatographic stationary phases. In Fig. 2, stationary phases were ordered from those that provide positive values of the zeta potential on the left to material that provides only negative values. Two stationary phases (Di-amino-Chol and Di-amino-AP-C12) provide only positive values of zeta potential, independent of the mobile phase composition. These stationary phases contain an amido group and secondary amines in the structure and residual primary amines. In the second step, these amines are modified to form an amide bond, but more than 50% of amine groups are unmodified. Unmodified amines with a pKa in the range 9.0–9.6 (in pure water) are protonated in the mobile phase used. The scheme of protonated stationary phases is shown in Fig. 3. It shows that these materials are positively charged and it causes the positive values of zeta potential. Positive charges in the structure of bonded ligands have a more significant influence on the stationary phase zeta potential than negatively charged ionized silanols on the support surface. A similar situation is observed in the case of the unmodified Di-amino stationary phase, however, in this case the negative values of zeta potential are observed in pure acetonitrile. An important observation from this study is that Di-amino, Di-amino-Chol and Di-amino-AP-C12 may act in a reversedphase system as a weak anion exchanger. Another group of stationary phases exhibit similar values. In this group one can find Amino-P-C10, Amino, and Amino-Chol. This group of stationary phases provides negative values of zeta potential in pure ACN and positive values in other mobile phases used in the study. The positive values of zeta potential in the case of Amino-P-C10, Amino may www.jss-journal.com
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Figure 2. Changes of stationary phases zeta potential in different reversed-phase mobile phases.
Figure 3. Structures of stationary phases that exhibit positive values of zeta potential (Di-amino-AP-C12, Di-Amino-Chol) in different pH range (A: 1–4, B: 4–9, C: above 9.6). Models based on pKa values in pure water.
be explained by protonation of nitrogen atom, that has pKa around 6.8–7 (in pure water). Bare silica gel and alkyl and phenyl stationary phases exhibit only negative values of zeta potential in all tested mobile phases. These materials do not possess any nitrogen atoms C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
that may be protonated. Their zeta potential is influenced only by negative charges on the silica support. The zeta potential of bare silica gel is almost independent on the type of organic modifier as well as on the composition of the solvent mixture. Modified silica gel exhibits changes of www.jss-journal.com
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the zeta potential with the mobile phase composition. A more negative zeta potential is observed in an ACN environment. It may be explained by the higher ACN dipole moment than the methanol dipole moment. In this case, the creation of a double layer in a non-electrolyte solution is easier because of the higher polarity of the solution. A higher dipole moment and higher polarizability of ACN in the comparison with MeOH may be the reason for the higher absolute values of zeta potential. The solvation process of polar stationary phases is also different in MeOH and ACN environments, which may also influences the zeta potential. A series of polar stationary phases provide positive values of the zeta potential in the present of the solvent with hydroxyl groups (methanol or water) whereas in the case of ACN the zeta potential is negative. It confirms the observation [4], that solvation process influence significantly zeta potential of the stationary phases in RP conditions. Usually, ACN causes values that are more negative whereas the presence of methanol moves these values to a positive range.
[15] Kulsing, C., Yang, Y., Matyska, M. T., Pesek, J. J., Boysen, R. I., Hearn, M. T. W., Anal. Chim. Acta 2014, 859, 79–86.
4 Conclusions
[16] Kulsing, C., Yang, Y., Boysen, R. I., Matyska, M. T., Pesek, J. J., Hearn, M. T. W., Anal. Method. 2015, 7, 1578– 1585.
The zeta potential of the polar stationary bonded phases, measured by Zetasizer, depends on the protonation of the nitrogen atoms. Materials with amine group may be protonated under RP conditions (ACN, MeOH and its mixtures with water) and provide positive values of zeta potential. Materials without proton acceptors provide negative values of zeta potential. However, the type of the organic modifier has significant influence on the zeta potential of the chemically bonded phases. In ACN the zeta potential is more negative and in methanol it is more positive. This influence may result from the differences in the dielectric properties of methanol, acetonitrile and water, and from differences in solvation of bonded phase particles in different solvents. This work was supported by Ministry of Science and Higher Education, grant no. NCN 2013/09/D/ST4/03807 for period 2014-2017. Authors thank Akzo Nobel (Bohus, Sweden) for kind donation of silica gel Kromasil 100 used in the study. The authors have declared no conflict of interest.
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