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J. Sep. Sci. 2014, 37, 1937–1943
Danielle N. Bassanese1 Arianne Soliven2 Paul G. Stevenson1 Gary R. Dennis2 Neil W. Barnett1 Ross A. Shalliker2 Xavier A. Conlan1 1 Faculty
of Science, Engineering and Built Environment, Centre for Chemistry and Biotechnology, Deakin University, Geelong, Victoria, Australia 2 Australian Centre for Research on Separation Science (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), Sydney, NSW, Australia Received February 23, 2014 Revised April 29, 2014 Accepted April 30, 2014
Research Article
Investigating retention characteristics of a mixed-mode stationary phase and the enhancement of monolith selectivity for high-performance liquid chromatography The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance -type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the -type selectivities afforded by the two different ligands on the mixed-mode column. Keywords: Aromatic Selectivity / Cyano columns / Methylene Selectivity / Mixedmode columns / Phenyl columns DOI 10.1002/jssc.201400201
1 Introduction The complexity of samples derived from a natural origin requires their analysis by means of LC to exploit the various attributes that describe the sample, at least the dominant factors [1]. Many natural samples contain a degree of conjugation, an ability to undergo either resonance or nonresonance -type bonding that can be exploited in chromatographic separations [2–4], many stationary phases have been designed for exactly this purpose [5]. For example, phenyl-type phases exploit resonance -type bonding, while the cyano (CN)-type phases exploit nonresonance -type bonding. Separations on these two phases often display vastly different selectivities, for example, in the 2D analysis of coffee the correlation in retention against a C18 column was greater for the phenyl-type phases than for CN-type phases when the analysis was compared to the sample as a whole [6]. However, when the analysis of separation selectivity was broken down into smaller subunits within the sample, the phenyl-type phases showed more divergent correlation against a C18 phase than the CN
Correspondence: Dr. Xavier A. Conlan, Faculty of Science, Engineering and Built Environment, Centre for Chemistry and Biotechnology, Deakin University, Geelong Waurn Ponds Campus, Locked Bag 20000, Geelong, Victoria 3220, Australia E-mail: [email protected] Fax: +61-3-5227-1040
Abbreviations: ACN, acetonitrile; CN, cyano; methanol; PAH, polycyclic aromatic hydrocarbon C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
phase [6]. In 1D studies, which involved the analysis of a homologous series of polycyclic aromatic hydrocarbons (PAHs), selectivity correlation between the phenyl and CN phases depended on the number of aromatic rings [7, 8]. Therefore, the complexity of samples derived from natural origin is in themselves so complex that any single phase cannot provide a separation environment that may be described as “ideal.” The resolution of complex mixtures requires the power of 2D-HPLC, which allows for multiple separations that are tuned to specific sample attributes [1]. Two different dimensions attempt to individually exploit the dominant attributes of the sample, providing an expanded separation space and a far greater peak capacity. Although there are exceptions [9,10], multidimensional separations are typically limited to two dimensions as: (i) sample dilution causes problems with limits of detection in the third dimension, and (ii), for on-line analysis the speed of the third dimension must be very much faster than the first dimension in order to minimize any wraparound effects. It is conceivable that the third dimension may need to be more than 25 times faster than the first [11], depending on the nature and complexity of the sample. In an effort to overcome these limitations a mixed mode, a -type selective stationary phase that incorporates both phenyl and CN functionality has been designed. Monolithic HPLC columns offer high-throughput capabilities that may be useful for high-speed multidimensional applications
MeOH, Colour Online: See the article online to view Figs. 1 and 3 in colour. www.jss-journal.com
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[12–15]. In addition, monolithic columns are a useful platform onto which mixed-mode surfaces can be built [7,16–19]. The aim of this work was to characterize the selectivity of monolithic columns with mixed functionality that was bonded in situ. This was achieved through a study detailing the methylene and aromatic selectivities in typical RP solvents and comparing the data to individual columns prepared with these ligands.
2 Materials and methods 2.1 Theory The linear solvent strength theory is commonly used to assess the chromatographic behavior of columns [20]. This theory provides the chromatographer with a better understanding of the types of interactions that are taking place on the surface of the column [20–22]. The relationship between logk and solvent composition (ϕ) is usually linear in accordance with Eq. (1): log k = log kw − S
(1)
where k is the retention factor of a solute, kw is the extrapolated retention factor in pure water, S is a representation of the sites that can interact directly with the solute molecule and ϕ is the proportion of organic solvent in the isocratic mobile phase. S is used as a predictive measure of the ease of optimization of separations via the construction of S versus n plots, where n is the number of repeating units in a homolog [21, 23]. Linearity in these relationships should be observed across all members of the homologous series because the hydrophobic contact surface area increases in a consistent manner with the addition of each subsequent repeating unit [24].
2.2 Chemicals Deionized water (Continental Water Systems, Victoria, Australia) filtered through a 0.45 m membrane filter (SigmaAldrich, Castle Hill, NSW, Australia) and analytical grade reagents were used unless otherwise stated. HPLC-grade methanol (MeOH) and acetonitrile (ACN) were obtained from Ajax Finechem (Taren point, NSW, Australia); HPLCgrade tetrahydrofuran was obtained from Chem-Supply (Gillman, SA, Australia). HPLC-grade isopropanol and heptane were purchased from Merck (Kilsyth, Victoria, Australia). Heptane was dried by reflux over sodium. Chloro(3cyanopropyl)dimethyl silane, chlorodimethyl propyl phenyl silane and chlorotrimethyl silane were obtained from Gelest (USA). Thiourea was obtained from BDH Chemical (Poole, UK). Linear PAHs (benzene, naphthalene, anthracene, 2,3benzanthracene and pentacene) and n-alkyl benzenes (benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene, heptylbenzene and octylben C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
zene) were obtained from the Aldrich Chemical Company (Castle Hill, NSW, Australia). 2.3 Instrumentation Chromatographic tests were performed on an Agilent 1260 system (Agilent Technologies, Mulgrave, Vic, Australia), incorporating a quaternary pump with solvent degasser, an auto-sampler, a thermostatted column compartment, and a DAD module that monitored the absorbance at 254 nm. Chromatographic data were obtained and processed with Agilent ChemStation software. The temperature of the column was thermostatted at 30⬚C. All injections were 2 L and elution performed in various isocratic modes. 2.4 In situ modification method Monolithic stationary phases were prepared according to the methods described in references [7, 16]. Neat silica monoliths (Onyx, 100 × 4.6 mm, 130 Å pore size) were purchased from Phenomenex (Lane Cove, NSW, Australia) for the in situ modifications. Prior to surface modification, dried heptane (50 mL) was pumped through the monolith. A 1% v/v solution of chlorodimethyl propyl phenyl silane was used as the phenyl ligand bonding silane solution and a 1% v/v solution of chloro-(3-cyanopropyl)dimethyl silane in dried heptane was used as the CN ligand bonding silane. End-capping was achieved using a 1% v/v solution of chlorotrimethyl silane. Column modification was undertaken using 100 mL of each silane solution that was pumped through the monolith at 30 min intervals (at flow rates of up to 4 mL/min) using each time five column volumes of the silane solution. Between each pump cycle the silane solution was allowed to sit static within the column for 30 min. This step was completed once, in both the forward (50 mL) and reverse (50 mL) directions. First for the phenyl silane solution, then repeated with the CN silane solution and finally the end-capping silane solution. These solutions were pumped through the monolith using a Waters 501 HPLC pump thermostatted at 80⬚C using a HPLC column heater (Thermasphere TS-130) from Phenomenex (Lane Cove, NSW, Australia). After completion of the silylation, the surface of the monolith was deactivated and removed of any remnants of the silane solution by eluting pure solutions of heptane (50 mL), isopropanol (30 mL) and MeOH (30 mL) using flow rates of 4 mL/min at room temperature. 2.5 Chromatographic separations n-Alkyl benzene test solutes were dissolved in either MeOH/water or ACN/water (80:20), and made up to concentrations between 7 and 14 mmol/L. Because of the poor solubility, PAH test solutes were first dissolved in tetrahydrofuran and then diluted further in either MeOH/water or ACN/water (80:20), and made up to concentrations between 0.1 and 1.5 mmol/L. Chromatographic behavior was www.jss-journal.com
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assessed in at least five mobile phases (five MeOH/water and five ACN/water compositions), with the column equilibrated with 20 column volumes of mobile phase prior to analysis. Mobile phases were mixed at the appropriate compositions by the quaternary pump. The column was thermally equilibrated for 1 h prior to analysis and then maintained at a constant temperature (30 ± 0.2⬚C) and a flow rate of 3mL/min was employed for all analyses. Experiments were randomized, and duplicates were performed for each injection. Void volumes were measured using the minor disturbance method [25, 26].
3 Results and discussion 3.1 Methylene selectivity
Figure 1. Log k versus ϕ at 30⬚C for the n-alkyl benzenes in (A) MeOH solvent system (R2 > 0.999) and (B) ACN solvent system (R2 > 0.998).
The methylene selectivity was measured with a homologous series of n-alkyl benzenes with alkyl chains between zero and eight carbon atoms. Plots of log k versus ϕ were constructed for the retention factors of each n-alkyl benzene with both ACN and MeOH mobile phases as shown in Fig. 1A (MeOH) and Fig. 1B (ACN). These plots show excellent linearity with correlation coefficients above 0.998 for ACN and above 0.999 for MeOH. The S values derived from the logk versus ϕ plots for the n-alkyl benzenes are shown in Table 1. Plots of S versus the substituent alkyl chain length (n) are shown in Fig. 2A (MeOH) and Fig. 2B (ACN). This type of plot represents methylene selectivity across all mobile-phase compositions. Both S versus n plots show a similar trend of a linear increase of S as the alkyl chains increase, which fits with the linear solvent strength theory.
3.2 Aromatic selectivity The aromatic selectivity was measured with a homologous series of linear PAHs with the number of benzene rings
Table 1. S values for alkyl benzenes and linear PAHs
n-Alkyl benzenes Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene Pentylbenzene Hexylbenzene Heptylbenzene Octylbenzene Linear PAHs Benzene Naphthalene Anthracene 2,3-Benzanthracene Pentacene
Mixed-mode MeOH
Mixed-mode ACN
CN MeOH
CN ACN
Phenyl MeOH
Phenyl ACN
1.93 2.31 2.75 3.17 3.71 4.26 4.82 5.42 6.13
1.12 1.60 2.11 2.67 3.33 4.03 4.82 5.48 6.30
2.44 2.77 3.22 3.67 4.13 4.63 5.16 5.69 6.29
2.26 2.56 2.92 3.22 3.57 3.93 4.36 4.70 5.04
2.50 2.88 3.27 3.72 4.17 4.64 5.12 5.63 6.13
2.28 2.55 2.84 3.15 3.45 3.75 4.04 4.34 4.62
2.03 2.97 4.20 4.96 6.36
1.16 2.59 4.27 5.34 7.19
2.44 3.34 4.28 4.62 5.70
2.17 3.10 4.06 3.93 4.85
2.50 3.35 4.23 4.42 5.70
2.33 3.30 4.20 4.12 4.99
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Figure 3. Log k versus ϕ at 30⬚C for the PAHs in (A) MeOH solvent system (R2 > 0.998) and (B) ACN solvent system (R2 > 0.992). Figure 2. Plots of S versus tail length (RCH2 ) for the n-alkyl benzenes in (A) MeOH solvent system and (B) ACN solvent system.
3.3 S value correlation between one and five. Plots of logk versus ϕ were constructed for the retention of each PAH in both ACN and MeOH mobile-phase compositions, illustrated in Fig. 3A (MeOH) and 3B (ACN). These plots display a high degree of linearity with correlation coefficients above 0.992 for ACN and above 0.998 for MeOH. Notwithstanding the excellent degree of linearity seen in the log k versus ϕ plots, it was observed that the plots of anthracene and 2,3-benzanthracene begin to converge as the amount of ACN increased. An intersection of these lines indicates a possible loss of resolution or coelution of the analytes. The S values derived from the logk versus ϕ plots for the PAHs are shown in Table 1. Plots of S versus the number of repeating aromatic rings (nrings ) are illustrated in Fig. 4A (MeOH) and Fig. 4B (ACN). The S versus nrings plots for both the MeOH and ACN solvent systems show a great degree of linearity despite the convergence of the anthracene and 2,3-benzanthrace lines on the log k versus ϕ plot in the ACN system. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The S values obtained for the mixed-mode monolith were compared to the S values for monolith columns that were functionalized using the CN and phenyl ligands separately (Table 1). Correlation plots were constructed for the alkyl benzene and PAH S values measured on the mixed mode, CN, and phenyl monoliths. A selection of these plots is shown in Fig. 5A–C. The alkyl benzene homologs show a similar trend in retention for the phenyl monolith and the CN monolith using MeOH as the organic modifier, as evinced by the strong correlation of the S values seen in Fig. 5A. A similar degree of correlation was seen when comparing these two columns using ACN as the organic modifier (plot not shown). The high degree of correlation suggests that the different -type bonding afforded by the phenyl and CN ligands will not be fully exploited by the alkyl benzene homologs. This is demonstrated on the mixed-mode column, where the selectivity was very similar to the individual phenyl and CN columns. This result was expected as the retention of the alkyl benzenes www.jss-journal.com
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Figure 4. Plots of S versus nrings for the PAHs in (A) MeOH solvent system and (B) ACN solvent system.
change due to the changing tail length, which does not undergo -type bonding. Correlation plots for the S values of the PAH homologs highlight the advantage of the different -type selectivities of the mixed-mode column compared to columns that contain the individual CN or phenyl ligands. Figure 5B shows a correlation plot of the S values measured on the individual CN and phenyl columns using a MeOH solvent system. It can be seen that in both the individual CN and phenyl phases, the three ring and four ring PAH members (anthracene and 2,3-benzanthracene, respectively) have very similar S values that led to a discontinuous relationship between S and n. This discontinuity has an important impact on the optimization process. When the relationship between S and n does not have a monotonic increase the retention behavior of the molecules is unpredictable. This may lead to a loss of resolution, coelution, or even a change in the elution order, with larger molecules eluting before smaller molecules. A reversal of the elution order is demonstrated in the correlation plot of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the PAH S values measured on the individual CN and phenyl columns using the ACN solvent system (Fig. 5C) with 2,3benzanthracene (four rings) eluting from the column before anthracene (three rings). This discontinuity makes optimization much more difficult for complex separation problems where the analytes are unrelated. This trend in discontinuity has been observed before in the work of Soliven et al. [7], Kayillo et al. [23, 27], and Stevenson et al. [24] on CN, phenyl-type and C18 phases, respectively. The trend has also previously been observed in both MeOH and ACN mobile phases [28]. The cause of the discontinuity is still under investigation, however it is most likely related to the physical aspects of the solute retention on the stationary phase. Two important physical characteristics that have shown to affect aromatic selectivity of PAHs are ligand density [24] and ligand spacer chain length [29]. Stevenson et al. [24] proposed that at higher ligand densities, it becomes more difficult for the stationary phase to undergo reordering to allow larger molecules to penetrate the stationary phase, leaving only the outer surface of the bonded phase to be available for solute interactions, therefore resulting in a change in retention behavior and also the S coefficient. Rafferty et al. [30] also suggested that in stationary phases with high ligand densities, the solvent is effectively excluded from the bonded phase, limiting the interactions between the solute and the bonded ligands. Measurement of the ligand density could confirm this postulation [24] for the observed discontinuity, however, evaluation the ligand density is destructive to the monolithic column bed and further experimental work is required on these columns. The retention of the larger PAH molecules may also be affected by the spacer chain that binds the ligand to the stationary-phase media. If the spacer chain is short, this restricts the flexibility of the ligand that can prevent the PAH molecules with a larger number of rings from moving in the space between the ligands. Unlike n-alkyl benzenes, which have a tail that is free to rotate, the linear PAHs are rigid and if the space between the stationary-phase ligands cannot accommodate the PAH molecule, this forces a change in the retention mechanism of the solute, resulting in the observed discontinuity. While discontinuity was seen with the PAH homologs on the individual CN and phenyl columns, no such discontinuity was observed on the mixed-mode column (Fig. 4A and B). This suggests that both the CN and phenyl selectivities on the mixed-mode column are being fully exploited by the larger, more conjugated PAH molecules.
3.4 Organic modifiers Two different solvent systems were employed in this study to assess the effect that organic modifiers have on the interactions between solutes and stationary phases. ACN proved to be a much stronger eluent than MeOH, which is clearly illustrated in Figs. 1 and 2. This is a direct result of ACN containing electrons in the nitrile group, which enables it to www.jss-journal.com
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Figure 5. (A) Correlation plot of S (phenol MeOH) versus S (CN MeOH) for n-alkyl benzene homologs in MeOH solvent system. (B) Correlation plot of S (phenyl MeOH) versus S (CN MeOH) for PAH homologs in MeOH solvent system. (C) Correlation plot of S (phenyl ACN) versus S (CN ACN) for PAH homologs in ACN solvent system.
competitively bind to the stationary phase or the solutes that also contain electrons. This in turn inhibits interactions between the solutes and the bonding sites on the column bed. Inhibition of these interactions between the solute and stationary phase can cause unpredictable retention behavior of the solutes such as the reversal of elution order demonstrated in Fig 5C. As mentioned previously, unpredictable retention of molecules can make optimization of complex separations difficult. Interference with – bonding has been previously reported for phenyl [28, 31] and CN [32, 33] stationary phases.
4 Concluding remarks
on the mixed-mode column. Additionally, the use of ACN as the organic modifier was shown to act as a stronger eluent, evinced particularly in the reversal of elution order of the three- and four-ring PAHs on the CN and phenyl columns, due to its ability to reduce the – interactions occurring between the solute and the stationary phase. D.N.B. acknowledges the support of a Deakin University postgraduate research award and P.G.S. acknowledges the support of an Alfred Deakin postdoctoral research fellowship. The authors also wish to acknowledge funding from Deakin University’s Strategic Research Centre (Chemistry and Biotechnology). The authors have declared no conflict of interest.
A mixed-mode—CN and phenyl—silica-based monolith was successfully functionalized and selectivity tests with two different solvent systems were performed. The mixed-mode monolith demonstrated excellent methylene and aromatic selectivity, with a high degree of linearity for the plots of S versus n (n = member of the series) in both MeOH and ACN solvent systems, which fits the linear solvent strength theory. The individual CN and phenyl monolith columns displayed excellent methylene selectivity; however, discontinuity was seen in the S versus nrings plots. This had been observed before on CN, phenyl and C18 phases and is likely due to the physical characteristics of the stationary phase, in particular the ligand spacer chain length and the ligand density. Comparison of the selectivity of the individual CN and phenyl columns to the mixed-mode column illustrated that larger, more conjugated PAH molecules were able to exploit both of the -type selectivities afforded by the two different ligands C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5 References [1] Giddings, J. C., J. Chromatogr. A 1995, 703, 3–15. [2] Kalili, K. M., de Villiers, A., J. Sep. Sci. 2011, 34, 854–876. [3] Slavin, M., Yu, L., Food Chem. 2012, 135, 2789–2795. [4] D’Ambrosio, M., Food Chem. 2013, 138, 2079–2088. [5] Snyder, L. R., Anal. Chem. 2007, 79, 3254–3262. [6] Mnatsakanyan, M., Stevenson, P. G., Shock, D., Conlan, X. A., Goodie, T. A., Spencer, K. N., Barnett, N. W., Francis, P. S., Shalliker, R. A., Talanta 2010, 82, 1349–1357. [7] Soliven, A., Dennis, G. R., Guiochon, G., Hilder, E. F., Haddad, P. R., Shalliker, R. A., J. Chromatogr. A 2010, 1217, 6085–6091. [8] Stevenson, P. G., Mnatsakanyan, M., Dennis, G. R., Shalliker, R. A., J. Sep. Sci. 2011, 34, 21–26.
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[9] Moore, A. W., Jorgenson, J. W., Anal. Chem. 1995, 67, 3456–3463.
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[21] Quarry, M. A., Grob, R. L., Snyder, L. R., Anal. Chem. 1986, 58, 907–917.
[10] Simpkins, S. W., Bedard, J. W., Groskreutz, S. R., Swenson, M. M., Liskutin, T. E., Stoll, D. R., J. Chromatogr. A 2010, 1217, 7648–7660.
[22] Zhao, J., Carr, P. W., Anal. Chem. 1999, 71, 5217–5224.
[11] Guiochon, G., Marchetti, N., Mriziq, K., Shalliker, R. A., J. Chromatogr. A 2008, 1189, 109–168.
[24] Stevenson, P. G., Soliven, A., Dennis, G. R., Gritti, F., Guiochon, G., Shalliker, R. A., J. Chromatogr. A 2010, 1217, 5377–5383.
[12] Cabrera, K., J. Sep. Sci. 2004, 27, 843–852.
[23] Kayillo, S., Dennis, G. R., Shalliker, R. A., J. Chromatogr. A 2006, 1126, 283–297.
[13] Tanaka, N., Kimura, H., Tokuda, D., Hosoya, K., Ikegami, T., Ishizuka, N., Minakuchi, H., Nakanishi, K., Shintani, Y., Furuno, M., Cabrera, K., Anal. Chem. 2004, 76, 1273– 1281.
[25] Gritti, F., Kazakevich, Y., Guiochon, G., J. Chromatogr. A 2007, 1161, 157–169.
[14] Kimura, H., Tanigawa, T., Morisaka, H., Ikegami, T., Hosoya, K., Ishizuka, N., Minakuchi, H., Nakanishi, K., Ueda, M., Cabrera, K., Tanaka, N., J. Sep. Sci. 2004, 27, 897–904.
[27] Kayillo, S., Dennis, G. R., Shalliker, R. A., J. Chromatogr. A 2007, 1148, 168–176.
[15] Guiochon, G., J. Chromatogr. A 2007, 1168, 101–168.
[26] Rimmer, C. A., Simmons, C. R., Dorsey, J. G., J. Chromatogr. A 2002, 965, 219–232.
[28] Stevenson, P. G., Kayillo, S., Dennis, G. R., Shalliker, R. A., J. Liq. Chromatogr. Relat. Technol. 2007, 31, 324–347.
[16] Soliven, A., Dennis, G. R., Hilder, E. F., Shalliker, R. A., Stevenson, P. G., Chromatographia 2014, 77, 663–671.
[29] Stevenson, P. G., Mayfield, K. J., Soliven, A., Dennis, G. R., Gritti, F., Guiochon, G., Shalliker, R. A., J. Chromatogr. A 2010, 1217, 5358–5364.
[17] Lubda, D., Lindner, W., J. Chromatogr. A 2004, 1036, 135– 143.
[30] Rafferty, J. L., Siepmann, J. I., Schure, M. R., J. Chromatogr. A 2008, 1204, 11–19.
[18] Sugrue, E., Nesterenko, P., Paull, B., J. Sep. Sci. 2004, 27, 921–930.
[31] Yang, M., Fazio, S., Munch, D., Drumm, P., J. Chromatogr. A 2005, 1097, 124–129.
[19] Nogueira, R., Lubda, D., Leitner, A., Bicker, W., Maier, N. ¨ M., Lammerhofer, M., Lindner, W., J. Sep. Sci. 2006, 29, 966–978.
[32] Croes, K., Steffens, A., Marchand, D. H., Snyder, L. R., J. Chromatogr. A 2005, 1098, 123–130.
[20] Tate, P. A., Dorsey, J. G., J. Chromatogr. A 2004, 1042, 37–48.
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[33] Marchand, D. H., Croes, K., Dolan, J. W., Snyder, L. R., J. Chromatogr. A 2005, 1062, 57–64.
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J. Sep. Sci. 2014, 37, 1937–1943
Danielle N. Bassanese1 Arianne Soliven2 Paul G. Stevenson1 Gary R. Dennis2 Neil W. Barnett1 Ross A. Shalliker2 Xavier A. Conlan1 1 Faculty
of Science, Engineering and Built Environment, Centre for Chemistry and Biotechnology, Deakin University, Geelong, Victoria, Australia 2 Australian Centre for Research on Separation Science (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), Sydney, NSW, Australia Received February 23, 2014 Revised April 29, 2014 Accepted April 30, 2014
Research Article
Investigating retention characteristics of a mixed-mode stationary phase and the enhancement of monolith selectivity for high-performance liquid chromatography The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance -type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the -type selectivities afforded by the two different ligands on the mixed-mode column. Keywords: Aromatic Selectivity / Cyano columns / Methylene Selectivity / Mixedmode columns / Phenyl columns DOI 10.1002/jssc.201400201
1 Introduction The complexity of samples derived from a natural origin requires their analysis by means of LC to exploit the various attributes that describe the sample, at least the dominant factors [1]. Many natural samples contain a degree of conjugation, an ability to undergo either resonance or nonresonance -type bonding that can be exploited in chromatographic separations [2–4], many stationary phases have been designed for exactly this purpose [5]. For example, phenyl-type phases exploit resonance -type bonding, while the cyano (CN)-type phases exploit nonresonance -type bonding. Separations on these two phases often display vastly different selectivities, for example, in the 2D analysis of coffee the correlation in retention against a C18 column was greater for the phenyl-type phases than for CN-type phases when the analysis was compared to the sample as a whole [6]. However, when the analysis of separation selectivity was broken down into smaller subunits within the sample, the phenyl-type phases showed more divergent correlation against a C18 phase than the CN
Correspondence: Dr. Xavier A. Conlan, Faculty of Science, Engineering and Built Environment, Centre for Chemistry and Biotechnology, Deakin University, Geelong Waurn Ponds Campus, Locked Bag 20000, Geelong, Victoria 3220, Australia E-mail: [email protected] Fax: +61-3-5227-1040
Abbreviations: ACN, acetonitrile; CN, cyano; methanol; PAH, polycyclic aromatic hydrocarbon C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
phase [6]. In 1D studies, which involved the analysis of a homologous series of polycyclic aromatic hydrocarbons (PAHs), selectivity correlation between the phenyl and CN phases depended on the number of aromatic rings [7, 8]. Therefore, the complexity of samples derived from natural origin is in themselves so complex that any single phase cannot provide a separation environment that may be described as “ideal.” The resolution of complex mixtures requires the power of 2D-HPLC, which allows for multiple separations that are tuned to specific sample attributes [1]. Two different dimensions attempt to individually exploit the dominant attributes of the sample, providing an expanded separation space and a far greater peak capacity. Although there are exceptions [9,10], multidimensional separations are typically limited to two dimensions as: (i) sample dilution causes problems with limits of detection in the third dimension, and (ii), for on-line analysis the speed of the third dimension must be very much faster than the first dimension in order to minimize any wraparound effects. It is conceivable that the third dimension may need to be more than 25 times faster than the first [11], depending on the nature and complexity of the sample. In an effort to overcome these limitations a mixed mode, a -type selective stationary phase that incorporates both phenyl and CN functionality has been designed. Monolithic HPLC columns offer high-throughput capabilities that may be useful for high-speed multidimensional applications
MeOH, Colour Online: See the article online to view Figs. 1 and 3 in colour. www.jss-journal.com
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[12–15]. In addition, monolithic columns are a useful platform onto which mixed-mode surfaces can be built [7,16–19]. The aim of this work was to characterize the selectivity of monolithic columns with mixed functionality that was bonded in situ. This was achieved through a study detailing the methylene and aromatic selectivities in typical RP solvents and comparing the data to individual columns prepared with these ligands.
2 Materials and methods 2.1 Theory The linear solvent strength theory is commonly used to assess the chromatographic behavior of columns [20]. This theory provides the chromatographer with a better understanding of the types of interactions that are taking place on the surface of the column [20–22]. The relationship between logk and solvent composition (ϕ) is usually linear in accordance with Eq. (1): log k = log kw − S
(1)
where k is the retention factor of a solute, kw is the extrapolated retention factor in pure water, S is a representation of the sites that can interact directly with the solute molecule and ϕ is the proportion of organic solvent in the isocratic mobile phase. S is used as a predictive measure of the ease of optimization of separations via the construction of S versus n plots, where n is the number of repeating units in a homolog [21, 23]. Linearity in these relationships should be observed across all members of the homologous series because the hydrophobic contact surface area increases in a consistent manner with the addition of each subsequent repeating unit [24].
2.2 Chemicals Deionized water (Continental Water Systems, Victoria, Australia) filtered through a 0.45 m membrane filter (SigmaAldrich, Castle Hill, NSW, Australia) and analytical grade reagents were used unless otherwise stated. HPLC-grade methanol (MeOH) and acetonitrile (ACN) were obtained from Ajax Finechem (Taren point, NSW, Australia); HPLCgrade tetrahydrofuran was obtained from Chem-Supply (Gillman, SA, Australia). HPLC-grade isopropanol and heptane were purchased from Merck (Kilsyth, Victoria, Australia). Heptane was dried by reflux over sodium. Chloro(3cyanopropyl)dimethyl silane, chlorodimethyl propyl phenyl silane and chlorotrimethyl silane were obtained from Gelest (USA). Thiourea was obtained from BDH Chemical (Poole, UK). Linear PAHs (benzene, naphthalene, anthracene, 2,3benzanthracene and pentacene) and n-alkyl benzenes (benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene, heptylbenzene and octylben C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
zene) were obtained from the Aldrich Chemical Company (Castle Hill, NSW, Australia). 2.3 Instrumentation Chromatographic tests were performed on an Agilent 1260 system (Agilent Technologies, Mulgrave, Vic, Australia), incorporating a quaternary pump with solvent degasser, an auto-sampler, a thermostatted column compartment, and a DAD module that monitored the absorbance at 254 nm. Chromatographic data were obtained and processed with Agilent ChemStation software. The temperature of the column was thermostatted at 30⬚C. All injections were 2 L and elution performed in various isocratic modes. 2.4 In situ modification method Monolithic stationary phases were prepared according to the methods described in references [7, 16]. Neat silica monoliths (Onyx, 100 × 4.6 mm, 130 Å pore size) were purchased from Phenomenex (Lane Cove, NSW, Australia) for the in situ modifications. Prior to surface modification, dried heptane (50 mL) was pumped through the monolith. A 1% v/v solution of chlorodimethyl propyl phenyl silane was used as the phenyl ligand bonding silane solution and a 1% v/v solution of chloro-(3-cyanopropyl)dimethyl silane in dried heptane was used as the CN ligand bonding silane. End-capping was achieved using a 1% v/v solution of chlorotrimethyl silane. Column modification was undertaken using 100 mL of each silane solution that was pumped through the monolith at 30 min intervals (at flow rates of up to 4 mL/min) using each time five column volumes of the silane solution. Between each pump cycle the silane solution was allowed to sit static within the column for 30 min. This step was completed once, in both the forward (50 mL) and reverse (50 mL) directions. First for the phenyl silane solution, then repeated with the CN silane solution and finally the end-capping silane solution. These solutions were pumped through the monolith using a Waters 501 HPLC pump thermostatted at 80⬚C using a HPLC column heater (Thermasphere TS-130) from Phenomenex (Lane Cove, NSW, Australia). After completion of the silylation, the surface of the monolith was deactivated and removed of any remnants of the silane solution by eluting pure solutions of heptane (50 mL), isopropanol (30 mL) and MeOH (30 mL) using flow rates of 4 mL/min at room temperature. 2.5 Chromatographic separations n-Alkyl benzene test solutes were dissolved in either MeOH/water or ACN/water (80:20), and made up to concentrations between 7 and 14 mmol/L. Because of the poor solubility, PAH test solutes were first dissolved in tetrahydrofuran and then diluted further in either MeOH/water or ACN/water (80:20), and made up to concentrations between 0.1 and 1.5 mmol/L. Chromatographic behavior was www.jss-journal.com
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assessed in at least five mobile phases (five MeOH/water and five ACN/water compositions), with the column equilibrated with 20 column volumes of mobile phase prior to analysis. Mobile phases were mixed at the appropriate compositions by the quaternary pump. The column was thermally equilibrated for 1 h prior to analysis and then maintained at a constant temperature (30 ± 0.2⬚C) and a flow rate of 3mL/min was employed for all analyses. Experiments were randomized, and duplicates were performed for each injection. Void volumes were measured using the minor disturbance method [25, 26].
3 Results and discussion 3.1 Methylene selectivity
Figure 1. Log k versus ϕ at 30⬚C for the n-alkyl benzenes in (A) MeOH solvent system (R2 > 0.999) and (B) ACN solvent system (R2 > 0.998).
The methylene selectivity was measured with a homologous series of n-alkyl benzenes with alkyl chains between zero and eight carbon atoms. Plots of log k versus ϕ were constructed for the retention factors of each n-alkyl benzene with both ACN and MeOH mobile phases as shown in Fig. 1A (MeOH) and Fig. 1B (ACN). These plots show excellent linearity with correlation coefficients above 0.998 for ACN and above 0.999 for MeOH. The S values derived from the logk versus ϕ plots for the n-alkyl benzenes are shown in Table 1. Plots of S versus the substituent alkyl chain length (n) are shown in Fig. 2A (MeOH) and Fig. 2B (ACN). This type of plot represents methylene selectivity across all mobile-phase compositions. Both S versus n plots show a similar trend of a linear increase of S as the alkyl chains increase, which fits with the linear solvent strength theory.
3.2 Aromatic selectivity The aromatic selectivity was measured with a homologous series of linear PAHs with the number of benzene rings
Table 1. S values for alkyl benzenes and linear PAHs
n-Alkyl benzenes Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene Pentylbenzene Hexylbenzene Heptylbenzene Octylbenzene Linear PAHs Benzene Naphthalene Anthracene 2,3-Benzanthracene Pentacene
Mixed-mode MeOH
Mixed-mode ACN
CN MeOH
CN ACN
Phenyl MeOH
Phenyl ACN
1.93 2.31 2.75 3.17 3.71 4.26 4.82 5.42 6.13
1.12 1.60 2.11 2.67 3.33 4.03 4.82 5.48 6.30
2.44 2.77 3.22 3.67 4.13 4.63 5.16 5.69 6.29
2.26 2.56 2.92 3.22 3.57 3.93 4.36 4.70 5.04
2.50 2.88 3.27 3.72 4.17 4.64 5.12 5.63 6.13
2.28 2.55 2.84 3.15 3.45 3.75 4.04 4.34 4.62
2.03 2.97 4.20 4.96 6.36
1.16 2.59 4.27 5.34 7.19
2.44 3.34 4.28 4.62 5.70
2.17 3.10 4.06 3.93 4.85
2.50 3.35 4.23 4.42 5.70
2.33 3.30 4.20 4.12 4.99
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Figure 3. Log k versus ϕ at 30⬚C for the PAHs in (A) MeOH solvent system (R2 > 0.998) and (B) ACN solvent system (R2 > 0.992). Figure 2. Plots of S versus tail length (RCH2 ) for the n-alkyl benzenes in (A) MeOH solvent system and (B) ACN solvent system.
3.3 S value correlation between one and five. Plots of logk versus ϕ were constructed for the retention of each PAH in both ACN and MeOH mobile-phase compositions, illustrated in Fig. 3A (MeOH) and 3B (ACN). These plots display a high degree of linearity with correlation coefficients above 0.992 for ACN and above 0.998 for MeOH. Notwithstanding the excellent degree of linearity seen in the log k versus ϕ plots, it was observed that the plots of anthracene and 2,3-benzanthracene begin to converge as the amount of ACN increased. An intersection of these lines indicates a possible loss of resolution or coelution of the analytes. The S values derived from the logk versus ϕ plots for the PAHs are shown in Table 1. Plots of S versus the number of repeating aromatic rings (nrings ) are illustrated in Fig. 4A (MeOH) and Fig. 4B (ACN). The S versus nrings plots for both the MeOH and ACN solvent systems show a great degree of linearity despite the convergence of the anthracene and 2,3-benzanthrace lines on the log k versus ϕ plot in the ACN system. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The S values obtained for the mixed-mode monolith were compared to the S values for monolith columns that were functionalized using the CN and phenyl ligands separately (Table 1). Correlation plots were constructed for the alkyl benzene and PAH S values measured on the mixed mode, CN, and phenyl monoliths. A selection of these plots is shown in Fig. 5A–C. The alkyl benzene homologs show a similar trend in retention for the phenyl monolith and the CN monolith using MeOH as the organic modifier, as evinced by the strong correlation of the S values seen in Fig. 5A. A similar degree of correlation was seen when comparing these two columns using ACN as the organic modifier (plot not shown). The high degree of correlation suggests that the different -type bonding afforded by the phenyl and CN ligands will not be fully exploited by the alkyl benzene homologs. This is demonstrated on the mixed-mode column, where the selectivity was very similar to the individual phenyl and CN columns. This result was expected as the retention of the alkyl benzenes www.jss-journal.com
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Figure 4. Plots of S versus nrings for the PAHs in (A) MeOH solvent system and (B) ACN solvent system.
change due to the changing tail length, which does not undergo -type bonding. Correlation plots for the S values of the PAH homologs highlight the advantage of the different -type selectivities of the mixed-mode column compared to columns that contain the individual CN or phenyl ligands. Figure 5B shows a correlation plot of the S values measured on the individual CN and phenyl columns using a MeOH solvent system. It can be seen that in both the individual CN and phenyl phases, the three ring and four ring PAH members (anthracene and 2,3-benzanthracene, respectively) have very similar S values that led to a discontinuous relationship between S and n. This discontinuity has an important impact on the optimization process. When the relationship between S and n does not have a monotonic increase the retention behavior of the molecules is unpredictable. This may lead to a loss of resolution, coelution, or even a change in the elution order, with larger molecules eluting before smaller molecules. A reversal of the elution order is demonstrated in the correlation plot of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the PAH S values measured on the individual CN and phenyl columns using the ACN solvent system (Fig. 5C) with 2,3benzanthracene (four rings) eluting from the column before anthracene (three rings). This discontinuity makes optimization much more difficult for complex separation problems where the analytes are unrelated. This trend in discontinuity has been observed before in the work of Soliven et al. [7], Kayillo et al. [23, 27], and Stevenson et al. [24] on CN, phenyl-type and C18 phases, respectively. The trend has also previously been observed in both MeOH and ACN mobile phases [28]. The cause of the discontinuity is still under investigation, however it is most likely related to the physical aspects of the solute retention on the stationary phase. Two important physical characteristics that have shown to affect aromatic selectivity of PAHs are ligand density [24] and ligand spacer chain length [29]. Stevenson et al. [24] proposed that at higher ligand densities, it becomes more difficult for the stationary phase to undergo reordering to allow larger molecules to penetrate the stationary phase, leaving only the outer surface of the bonded phase to be available for solute interactions, therefore resulting in a change in retention behavior and also the S coefficient. Rafferty et al. [30] also suggested that in stationary phases with high ligand densities, the solvent is effectively excluded from the bonded phase, limiting the interactions between the solute and the bonded ligands. Measurement of the ligand density could confirm this postulation [24] for the observed discontinuity, however, evaluation the ligand density is destructive to the monolithic column bed and further experimental work is required on these columns. The retention of the larger PAH molecules may also be affected by the spacer chain that binds the ligand to the stationary-phase media. If the spacer chain is short, this restricts the flexibility of the ligand that can prevent the PAH molecules with a larger number of rings from moving in the space between the ligands. Unlike n-alkyl benzenes, which have a tail that is free to rotate, the linear PAHs are rigid and if the space between the stationary-phase ligands cannot accommodate the PAH molecule, this forces a change in the retention mechanism of the solute, resulting in the observed discontinuity. While discontinuity was seen with the PAH homologs on the individual CN and phenyl columns, no such discontinuity was observed on the mixed-mode column (Fig. 4A and B). This suggests that both the CN and phenyl selectivities on the mixed-mode column are being fully exploited by the larger, more conjugated PAH molecules.
3.4 Organic modifiers Two different solvent systems were employed in this study to assess the effect that organic modifiers have on the interactions between solutes and stationary phases. ACN proved to be a much stronger eluent than MeOH, which is clearly illustrated in Figs. 1 and 2. This is a direct result of ACN containing electrons in the nitrile group, which enables it to www.jss-journal.com
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Figure 5. (A) Correlation plot of S (phenol MeOH) versus S (CN MeOH) for n-alkyl benzene homologs in MeOH solvent system. (B) Correlation plot of S (phenyl MeOH) versus S (CN MeOH) for PAH homologs in MeOH solvent system. (C) Correlation plot of S (phenyl ACN) versus S (CN ACN) for PAH homologs in ACN solvent system.
competitively bind to the stationary phase or the solutes that also contain electrons. This in turn inhibits interactions between the solutes and the bonding sites on the column bed. Inhibition of these interactions between the solute and stationary phase can cause unpredictable retention behavior of the solutes such as the reversal of elution order demonstrated in Fig 5C. As mentioned previously, unpredictable retention of molecules can make optimization of complex separations difficult. Interference with – bonding has been previously reported for phenyl [28, 31] and CN [32, 33] stationary phases.
4 Concluding remarks
on the mixed-mode column. Additionally, the use of ACN as the organic modifier was shown to act as a stronger eluent, evinced particularly in the reversal of elution order of the three- and four-ring PAHs on the CN and phenyl columns, due to its ability to reduce the – interactions occurring between the solute and the stationary phase. D.N.B. acknowledges the support of a Deakin University postgraduate research award and P.G.S. acknowledges the support of an Alfred Deakin postdoctoral research fellowship. The authors also wish to acknowledge funding from Deakin University’s Strategic Research Centre (Chemistry and Biotechnology). The authors have declared no conflict of interest.
A mixed-mode—CN and phenyl—silica-based monolith was successfully functionalized and selectivity tests with two different solvent systems were performed. The mixed-mode monolith demonstrated excellent methylene and aromatic selectivity, with a high degree of linearity for the plots of S versus n (n = member of the series) in both MeOH and ACN solvent systems, which fits the linear solvent strength theory. The individual CN and phenyl monolith columns displayed excellent methylene selectivity; however, discontinuity was seen in the S versus nrings plots. This had been observed before on CN, phenyl and C18 phases and is likely due to the physical characteristics of the stationary phase, in particular the ligand spacer chain length and the ligand density. Comparison of the selectivity of the individual CN and phenyl columns to the mixed-mode column illustrated that larger, more conjugated PAH molecules were able to exploit both of the -type selectivities afforded by the two different ligands C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5 References [1] Giddings, J. C., J. Chromatogr. A 1995, 703, 3–15. [2] Kalili, K. M., de Villiers, A., J. Sep. Sci. 2011, 34, 854–876. [3] Slavin, M., Yu, L., Food Chem. 2012, 135, 2789–2795. [4] D’Ambrosio, M., Food Chem. 2013, 138, 2079–2088. [5] Snyder, L. R., Anal. Chem. 2007, 79, 3254–3262. [6] Mnatsakanyan, M., Stevenson, P. G., Shock, D., Conlan, X. A., Goodie, T. A., Spencer, K. N., Barnett, N. W., Francis, P. S., Shalliker, R. A., Talanta 2010, 82, 1349–1357. [7] Soliven, A., Dennis, G. R., Guiochon, G., Hilder, E. F., Haddad, P. R., Shalliker, R. A., J. Chromatogr. A 2010, 1217, 6085–6091. [8] Stevenson, P. G., Mnatsakanyan, M., Dennis, G. R., Shalliker, R. A., J. Sep. Sci. 2011, 34, 21–26.
www.jss-journal.com
J. Sep. Sci. 2014, 37, 1937–1943
[9] Moore, A. W., Jorgenson, J. W., Anal. Chem. 1995, 67, 3456–3463.
Liquid Chromatography
1943
[21] Quarry, M. A., Grob, R. L., Snyder, L. R., Anal. Chem. 1986, 58, 907–917.
[10] Simpkins, S. W., Bedard, J. W., Groskreutz, S. R., Swenson, M. M., Liskutin, T. E., Stoll, D. R., J. Chromatogr. A 2010, 1217, 7648–7660.
[22] Zhao, J., Carr, P. W., Anal. Chem. 1999, 71, 5217–5224.
[11] Guiochon, G., Marchetti, N., Mriziq, K., Shalliker, R. A., J. Chromatogr. A 2008, 1189, 109–168.
[24] Stevenson, P. G., Soliven, A., Dennis, G. R., Gritti, F., Guiochon, G., Shalliker, R. A., J. Chromatogr. A 2010, 1217, 5377–5383.
[12] Cabrera, K., J. Sep. Sci. 2004, 27, 843–852.
[23] Kayillo, S., Dennis, G. R., Shalliker, R. A., J. Chromatogr. A 2006, 1126, 283–297.
[13] Tanaka, N., Kimura, H., Tokuda, D., Hosoya, K., Ikegami, T., Ishizuka, N., Minakuchi, H., Nakanishi, K., Shintani, Y., Furuno, M., Cabrera, K., Anal. Chem. 2004, 76, 1273– 1281.
[25] Gritti, F., Kazakevich, Y., Guiochon, G., J. Chromatogr. A 2007, 1161, 157–169.
[14] Kimura, H., Tanigawa, T., Morisaka, H., Ikegami, T., Hosoya, K., Ishizuka, N., Minakuchi, H., Nakanishi, K., Ueda, M., Cabrera, K., Tanaka, N., J. Sep. Sci. 2004, 27, 897–904.
[27] Kayillo, S., Dennis, G. R., Shalliker, R. A., J. Chromatogr. A 2007, 1148, 168–176.
[15] Guiochon, G., J. Chromatogr. A 2007, 1168, 101–168.
[26] Rimmer, C. A., Simmons, C. R., Dorsey, J. G., J. Chromatogr. A 2002, 965, 219–232.
[28] Stevenson, P. G., Kayillo, S., Dennis, G. R., Shalliker, R. A., J. Liq. Chromatogr. Relat. Technol. 2007, 31, 324–347.
[16] Soliven, A., Dennis, G. R., Hilder, E. F., Shalliker, R. A., Stevenson, P. G., Chromatographia 2014, 77, 663–671.
[29] Stevenson, P. G., Mayfield, K. J., Soliven, A., Dennis, G. R., Gritti, F., Guiochon, G., Shalliker, R. A., J. Chromatogr. A 2010, 1217, 5358–5364.
[17] Lubda, D., Lindner, W., J. Chromatogr. A 2004, 1036, 135– 143.
[30] Rafferty, J. L., Siepmann, J. I., Schure, M. R., J. Chromatogr. A 2008, 1204, 11–19.
[18] Sugrue, E., Nesterenko, P., Paull, B., J. Sep. Sci. 2004, 27, 921–930.
[31] Yang, M., Fazio, S., Munch, D., Drumm, P., J. Chromatogr. A 2005, 1097, 124–129.
[19] Nogueira, R., Lubda, D., Leitner, A., Bicker, W., Maier, N. ¨ M., Lammerhofer, M., Lindner, W., J. Sep. Sci. 2006, 29, 966–978.
[32] Croes, K., Steffens, A., Marchand, D. H., Snyder, L. R., J. Chromatogr. A 2005, 1098, 123–130.
[20] Tate, P. A., Dorsey, J. G., J. Chromatogr. A 2004, 1042, 37–48.
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[33] Marchand, D. H., Croes, K., Dolan, J. W., Snyder, L. R., J. Chromatogr. A 2005, 1062, 57–64.
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