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Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase Maressa D. Dolzan a,b , Daniel A. Spudeit a,b , Zachary S. Breitbach a , William E. Barber c , Gustavo A. Micke b , Daniel W. Armstrong a,d,∗ a
Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil c Agilent Technologies Inc., 2850 Centerville Road, Wilmington, DE 19808, USA d AZYP LLC, 700 Planetarium Place, Arlington, TX, 76019, USA b
a r t i c l e
i n f o
Article history: Received 6 June 2014 Received in revised form 28 August 2014 Accepted 2 September 2014 Available online xxx Keywords: Superficially porous particles HILIC Cyclofructan 6 FRULIC Core–shell
a b s t r a c t A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7 m) was developed. Its performance was evaluated and compared to fully porous silica particles with 5 m (commercially available as FRULIC-N) and 3 m diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin ) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Superficially porous particles (SPPs), also called core–shell, porous shell or fused core particles [1,2], are state-of-the-art support materials used in the production of HPLC columns. Historically, the concept of shell particles (pellicular particles) was firstly proposed by Horvath et al. during the 1960s and they were developed as ion exchange materials for the analysis of large biological molecules [2–4]. SPP technology was advanced by Kirkland, who prepared 50 m particles in the 1970s and 5 m particles in the 1990s [5–7]. Concurrent improvements in the manufacturing of high-quality fully porous particles (FPPs) inhibited the application
∗ Corresponding author at: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76019, United States. Tel.: +1 817 272 0632; fax: +1 817 272 0619. E-mail address: [email protected] (D.W. Armstrong).
of SPPs [8]. FPPs with diameters of 3 m (1990s) and sub 2 m (2004) came in vogue along with liquid chromatographs that could operate at higher pressures (i.e., ≥1000 bar) [3]. However, recent improvements to SPP technology have moved them to the forefront of HPLC packing materials. These, more successful core–shell particles have thicker porous shells compared to the early pellicular particles. For example, columns are now available with SPP sizes of 1.7, 2.6 or 2.7 m and porous shell thicknesses of 0.23, 0.35 and 0.5 m, respectively [9]. This generation of SPPs markedly improved its chromatographic performance, due to its morphology, which consists of a solid inner core surrounded by a porous layer, where analytes and mobile phase can diffuse [2]. The presence of the solid core results in a shorter path for diffusion and decreases band broadening caused by poor mass transfer for systems with slow mass transfer kinetics, such as large molecule separations and some chiral separations. These factors permit analyses at high flow rates without a significant loss in efficiency. Further, SPP columns can be very well packed (particularly from the wall to the center
http://dx.doi.org/10.1016/j.chroma.2014.09.010 0021-9673/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: M.D. Dolzan, et al., Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.09.010
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Table 1 Physical parameters and bonded selector loading of the stationary phases.
FPP 5 m FPP 3 m SPP 2.7 m b
Particle diameter (m)
Porosity %
Pore size (Å)
Surface area (m2 /g)
CF6 content (mol/m2 )a
CF6 content (mass%)a
4.3 3.0 2.7
100% 100% 75%
93 100 120
465 300 120
0.72 0.91 0.86
32.2 27.9 12.8
Legend: FFP and SPP mean fully and superficially porous particles, respectively. a Values obtained from the percentage of carbon. b FRULIC-N
of the column) and therefore exhibit decreased band broadening due to eddy diffusion [3]. Columns packed with superficially porous particles have been used for high throughput separations by improving efficiency while keeping methods robust [8–10]. In recent years, the number of publications involving HPLC columns based on SPP has increased [1,3,8,11–16]. Many SPP HILIC columns can be purchased from different companies, but the majority of the marketed HILIC packing material is simply unmodified silica [9]. Silica gel does not always offer acceptable HILIC separations [17,18]. Thus, it is both timely and important to produce and evaluate newer, more promising, HILIC separating agents bound to SPPs. Native cyclofructan-6 (CF6) has been reported to be a powerful selector in separation of polar compounds in the HILIC mode [17,18]. The column based on CF6 chemically bonded to FPPs is commercially available (FRULIC-N) and it has demonstrated advantages over other popular commercial columns in separating several compounds such as nucleic acid bases, nucleosides, nucleotides, xanthines, -blockers, carbohydrates, etc. [17,18]. Further, the native CF6 phase is hydrolytically stable, whereas evidence of dissolution of silica and polar/polar embedded phases in the HILIC mode has been reported [19,20]. In this work, a HILIC stationary phase based on native CF6 was evaluated when bonded to SPPs as the support material (2.7 m), and it was compared with 3 m and 5 m FPP based columns. The columns were also tested in the normal phase (NP) mode, to evaluate the influence of aqueous and non-aqueous containing mobile phases in performance. Results in terms of efficiency, analysis time and resolution were evaluated, demonstrating clear advantages of the new CF6 HILIC column based on SPPs.
diameter) were obtained from Agilent Technologies (Santa Clara, CA, USA). 2.2. Synthesis of native cyclofructan 6 (CF6) based stationary phases Native CF6 was chemically bonded to silica gel according to literature [17]. The same procedure was used to develop all the stationary phases applied in this work, just changing the silica particles used as supporting material (previously described on Section 2.1). The products were characterized by elemental analysis (CHN), and the loading of CF6 in the stationary phases could be calculated. Some physical parameters of the silica particles and the developed stationary phases are listed in Table 1. 2.3. Instruments A total of three columns were prepared in this work (150 mm × 4.6 mm i.d.). All the chromatographic separations were conducted on an Agilent HPLC series 1200 system (Agilent Technologies, Santa Clara, CA), equipped with a quaternary pump, an autosampler and a multiwavelength UV–vis detector. For data acquisition and analysis, the Chemstation software version Rev. B.03.02 [341] was used. The injection volume was 0.5 L for all analyses. The temperature was maintained at 30 ◦ C. The mobile phases (MP) used in the HILIC mode were composed of 75–95% of ACN and 5–25% of 25 mM NH4 OAc, except for the cyclic nucleotides, for which the MP was composed of ACN/100 mM NH4 OAc (70/30, v/v). For the separations carried out in the normal phase, Hep/IPA and Hep/EtOH in different ratios were used and 0.1% of TFA was added to the EtOH phase for the analysis of ferulic acid. Efficiencies were measured using the peak width at half height.
2. Experimental 3. Results and discussions 2.1. Materials 3.1. Preparation of the stationary phases Anhydrous N,N-dimethylformamide (DMF), anhydrous toluene, anhydrous pyridine, 3-(triethoxysilyl)propylisocyanate, ammonium acetate (NH4 OAc), trifluoracetic acid (TFA) and all analytes tested in this work (5-phenylvaleric acid, ferulic acid, pyridoxine, l-ascorbic acid, uracil, adenosine, cytosine, thymidine 3 :5 cyclic monophosphate (cTMP), adenosine 2 :3 cyclic monophosphate (cAMP), guanosine 2 :3 cyclic monophosphate (cGMP), cytidine 2 :3 cyclic monophosphate (cCMP), 1,3-dinitrobenzene (1,3-DNB), ␣-tocopherol, (R)-(+)-2 -amino-1,1 -binaphthalen-2-ol (NOBIN) and 1,3,5-tri-t-butylbenzene) were purchased from Sigma–Aldrich (Milwaukee, WI). The CF6 was provided by AZYP, LLC (Arlington, TX). Acetonitrile (ACN), heptane (Hep), isopropyl alcohol (IPA) and ethanol (EtOH), used for the chromatographic separations, were obtained from EMD (Gibbstown, NJ). Water was purified by a MilliQ Water Purification System (Millipore, Billerica, MA). The FPPs with 5 m of diameter (dp ) were purchased from DAISO (Osaka, Japan), the FPPs with 3 m of dp were obtained from Glantreo (Cork, Ireland), and the SPPs with 2.7 m of dp (0.5 m of thickness of the porous layer and a solid core having a 1.7 m
The results obtained for the elemental analysis of the packing materials are important in understanding the performance of the columns and also to evaluate synthetic implications of the varying particle morphologies. The CF6 content on the stationary phase was calculated from the percentage of carbon obtained in the elemental analysis. The mol/m2 loading (CF6/surface area of the silica particle) is important in understanding the effective/relative coverage of each phase (Table 1). As can be seen, the effective coverage (mol/m2 ) ranged from 0.72 for the FPP 5 m media to 0.91 for the FPP 3 m media. The SPP material had an effective coverage of 0.86 which was greater than the commercial 5 m FPP column (0.72) indicating that the selector density was slightly higher and that sufficient coverage was achieved. It is important to consider coverage/surface area for a bonded/modified SPP based HILIC phase in order to know how much silica character has been masked by the bonded HILIC selector. As can be seen (Table 1), the stationary phase particles of the SPP have less than a half of the absolute amount of CF6 present
Please cite this article in press as: M.D. Dolzan, et al., Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.09.010
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Fig. 1. Separation of polar compounds in HILIC mode using CF6 based columns packed with different types of silica particles. The values on the top of the peaks correspond to the efficiency in terms of number of plates (N) on column. General conditions: column dimensions, 150 mm × 4.6 mm; isocratic mode composed of component A = ACN and, B = 25 mM NH4 OAc and/or C = 100 mM NH4 OAc; flow rate = 750 L/min; T = 30 ◦ C; injected volume = 0.5 L. Specific experimental conditions: A) mobile phase (MP): 75/25 (VA /VB ), = 254 nm; B) MP: 85/15 (VA /VB ), = 210 nm; C) MP: 75/25 (VA /VB ), = 280 nm and; D) MP: 70/30 (Va /Vc ), = 254 nm. The numbers 1–4 represent the peaks in order of elution (1 and 4 correspond the earliest and latest eluted analyte, respectively).
on the 5 m FPP and the 3 m FPP phases. This absolute loading certainly could affect chromatographic properties as analyte bands traverse the length of the column.
3.2. Chromatographic evaluation of CF6 based SPP and FPP columns The main goal of this study was to develop and evaluate the chromatographic performance of native CF6 based columns prepared with SPPs in the HILIC mode. Therefore, a comparison with the 3 m and 5 m FPP based columns was necessary. A total of 11 polar compounds were evaluated in the HILIC mode in terms of efficiency, analysis time and resolution. Further, the efficiency of four different compounds was evaluated in the normal phase mode, and the results were compared.
3.2.1. Performance evaluation of the columns in HILIC The chromatographic performance of the columns was evaluated in the HILIC mode using 11 polar compounds, corresponding to four different groups: 5-phenylvaleric acid and ferulic acid; uracil, adenosine and cytosine; pyridoxine and l-ascorbic acid; and the cyclic nucleotides cTMP, cAMP, cGMP and cCMP (Fig. 1). As can be seen, all columns produced exceptional peak shapes for the tested compounds, indicating that the columns were well packed. The efficiencies obtained for the SPP based column were considerably higher for all analytes. The SPP column resulted in an efficiency improvement of 25–65% compared to the 3 m FPP and efficiencies values 2–4× higher than those obtained using the 5 m FPP column. For example, cCMP gave 7700 plates on the 5 m FPP commercial FRULIC-N column, while cCMP gave 28,000 plates on the SPP
column. The efficiencies and retention times remained constant for more than 600 injections. Under constant mobile phase conditions (Fig. 1), the retention factors were always higher for the 5 m FPP followed by the 3 m FPP, with the SPP column resulting in the shortest analysis times. These results were expected for two reasons: (i) the 5 m FPP contains 32% CF6, while the 3 m FPP contains 28% CF6 and the SPP column only 13% CF6 (w/w) and (ii) the morphology of the superficially porous silica results in a slightly lower dead volume in HPLC, which produces slightly faster analyses [1,11,21]. However, it should be noted that although the analysis times are shorter for the SPP column, the peak capacity (between first and last eluted peak in each chromatogram in Fig. 1) is actually highest on the 3 m FPP (estimated using Giddings approach (i.e. peak capacity = (1 + (N1/2 /4)ln(1 + kn /1 + k1 ))). However, it has been demonstrated that, if the mobile phase conditions (i.e. ratio of weak:strong eluent) are manipulated to give constant retention comparisons between the SPPs and FPPs, the peak capacity of the SPP is the greatest due to the increased efficiency of the SPP column [25]. In order to more specifically probe differences in band broadening on each column, the dependence of the HETP on the mobile phase flow rate was evaluated (Fig. 2) and is discussed in terms of the van Deemter equation:
HETP =
B
v
+ Cs v + Csm v +
1 A
+
1 Cm v
−1 (1)
where v is the velocity of the mobile phase and B, Cs , Csm , A, and Cm are constants, determined by the magnitude of band broadening due to longitudinal diffusion, mass transfer in the stationary phase,
Please cite this article in press as: M.D. Dolzan, et al., Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.09.010
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Fig. 2. Dependence of efficiency (in terms of HETP) on the flow rate of the mobile phase (HILIC) for the compounds and columns indicated. D, E and F show just the data for the SPP and 3 m FPP columns to clarify the comparison between columns packed with similar particle sizes. See Fig. 1 for other experimental conditions.
mass transfer in the stagnant mobile phase, eddy dispersion, and mass transfer in the mobile phase. As can be seen in Fig. 2, the minimum HETP (HETPmin ) ranged from 500 to 750 L/min for the SPP column, 250–500 L/min for the 3 m FPP and from 100 to 500 L/min for the 5 m FPP. In general, the SPP column had the HETPmin at higher flow rates compared to the FPP columns. This is consistent with other SPP materials (e.g. RPLC) and has proven to be due to the reduction of the “B” term caused by the obstruction of flow paths by the solid core [3]. At all tested flow rates, the efficiency of the SPP column was the greatest among the tested columns. However, flatter rises in the van Deemter curves at higher flow rates often associated with SPPs were not obvious. In fact, the slope of the van Deemter curve for adenosine (Fig. 2D) and pyridoxine (Fig. 2F) on the 3 m FPP column was essentially the same as the slope on the SPP column at flow rates between 0.5 and 2.5 mL/min. Only the ferulic acid test (Fig. 2E) showed a slight improvement in the band broadening effects at high flow rates on the SPP column. The fact that the SPP and FPPs appear to have similar van Deemter curve slopes at higher flows, points to the overall efficiency gains largely resulting from generally better packed columns with the SPPs resulting in decreased eddy diffusion contributions to band broadening [3,9,12].
Gritti et al. [16] as well as Heaton et al. [24] have studied FPPs in HILIC, finding the solid–liquid mass transfer resistance HETP is larger in HILIC than it is in the reversed phase mode. This is related to the velocity of adsorption–desorption kinetics, which is slow in HILIC. Our results indicate this HILIC characteristic is also true when using SPPs in the HILIC mode. This is further supported by reports from Gritti et al. [15] which suggest mass transfer advantages of SPPs may be affected by a water enriched adsorbed phase. The postulation that the adsorbed water layer, often present in HILIC separations, effectively dampens “Cs ” term gains of SPPs will be further discussed in Section 3.2.2. It should be noted that “Cs ” term gains due to the use of SPPs are accentuated for analytes with high molar mass (>600 g/mol) or other systems that exhibit slower adsorption/desorption kinetics (e.g. some chiral separations) for small molecules [1,22,23,25]. The analytes used in this study had molar masses generally less than 300 g/mol, but the CF6 selector is the base macrocycle for many other chiral stationary phases which are known to have somewhat slower mass transfer kinetics [25]. Also, as is mentioned in the following Section 3.2.2, the water layer on the CF6 phase is more substantial than that of an unmodified silica HILIC column [26]. Thus, there are at least three possible contributing factors to the mass transfer kinetics of CF6 based HILIC columns: (i) the CF6 molecule can strongly interact
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Fig. 3. Dependence of plate numbers per minute on the flow rate of the mobile phase. For all other experimental conditions, see Fig. 1.
with analytes (similar to a chiral phase) through multiple different simultaneous interactions, (ii) the CF6 stationary phase is essentially “thickened” (note: Cs is directly proportional to the square of the stationary phase film thickness) by a stagnant water layer,
and (iii) the adsorbed water layer will have a significantly higher viscosity than the bulk water. All these factors contribute to the decreased diffusivity of analytes in the HILIC mode. The first contribution may occur in all separation modes, where the second and
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first time, that bonded HILIC phases on SPPs, having an equivalent relative coverage (i.e. mol/m2 ), produces equivalent selectivities. This is in agreement with recent findings of Spudeit et al. in the evaluation of SPP bonded chiral stationary phases [25].
Fig. 4. Selectivity values of neighboring peaks for all the analytes separated in the Fig. 1. 1) Uracil-adenosine; 2) adenosine-cytosine; 3) 5-phenylvaleric acid-ferulic acid; 4) pyridoxine-ascorbic acid; 5) cTMP-cAMP; 6) cAMP-cGMP; 7) cGMP-cCMP.
third contributions should be unique to the HILIC mode. Therefore, at high flow rates, the stationary phase mass transfer contribution from CF6 based columns is greater than might be expected for small molecules. As was noted earlier, the SPP column leads to a decrease in the total analysis time. In order to more clearly show this, the number of plates per unit time was plotted versus the mobile phase flow rate for each compound (Fig. 3). As can be seen, the number of plates afforded per time spent on the analysis is much higher for the SPP column than the FPP columns. Lastly, a comparison of selectivity differences between the FPP columns and the SPP column was made. Fig. 4 represents the selectivity values of neighboring peaks for all the analytes separated in Fig. 1. It was found that selectivity values are essentially the same for most separations when comparing the 5 m FPP column, the 3 m FPP column and the SPP column. These results indicate, for the
3.2.2. Evaluation of the columns in normal phase: a comparison To further support the previously stated postulation, that the flatter rising van Deemter curves often associated with the use of SPPs may be dampened by the presence of an adsorbed multi-layer of water, the columns were also tested under non-aqueous conditions using the normal phase mode. The following compounds were evaluated: ␣-tocopherol, (R)-(+)-NOBIN, 1,3-dinitrobenzene, and ferulic acid (which also was analyzed in the HILIC mode). The dependence of plate numbers on the mobile phase flow rate for these analytes is shown in Fig. 5. As can be seen, chromatographic separations carried out in the normal phase resulted in flatter rising van Deemter curves for the SPP columns compared to the FPP columns. This is in contrast to what was observed in the HILIC mode indicating the influence of the mobile phase and separation mechanisms in the mass transfer process. Previously, Gritti et al. compared the performance of columns packed with SPP bare-silica in HILIC (with high ACN concentration) and per aqueous liquid chromatography (PALC, with low ACN concentration) for the analysis of caffeine. The van Deemter plots for both HILIC and PALC were studied in a narrow range from 0.2 to 1 mL/min and the authors didn’t discuss the influence of these different modes on the mass transfer process and “Cs ” term at high flow rates [15]. Further, Dihn et al. showed that silica has a relatively small water uptake in relation to other HILIC stationary phases [26]. Therefore, the CF6 phase, which is likely to have a more substantial water layer, may not be treated identically to silica in terms of band broadening effects due to mass transfer. To exclude the influence of the nature of the analyte on “Cs ” term effects, ferulic acid was also analyzed in the NP as it is able
Fig. 5. Dependence of efficiency (in terms of HETP) on the flow rate of the mobile phase (NP) for the compounds and columns indicated. Conditions: A and B) Hep/IPA 98/2, = 280 nm; C) Hep/EtOH 80/20, = 254 nm; D) Hep/EtOH 0.1% TFA, = 280 nm.
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to undergo similar strong interactions as in the HILIC mode. When comparing the van Deemter plots for ferulic acid in the HILIC mode (Fig. 2B) and the NP mode, a significant improvement in the HETP at high flow rates can be seen for the SPP column tested in NP mode. For example, under the HILIC conditions, the difference in on-column plate numbers at 2.0 mL/min between the 3 m FPP and the SPP is 4100 whereas the difference in the NP mode increased to 6500. Apparently, non-aqueous conditions can allow for improved exploitation of the “Cs ” term advantages for this SPP based HILIC phase. This implies that, amongst other factors, the increased viscosity and stationary phase thickness resulting from the adsorbed water layer have a significant effect on band broadening at high flow rates. Further, “Cs ” is influenced by diffusion distance inside particles, which is inherently shortened in SPPs [27]. An implication of a substantial water layer may be an increase in the stationary phase diffusional pathlength in the HILIC mode; decreasing one potential advantage of the SPP material. The results obtained in HILIC and NP modes in this work indicate that the mass transfer is dependent on a set of factors, such as the nature of the mobile phase, separation mechanism, nature of the analyte, morphology of the particle used as support material on the stationary phase, and possibly other unknown parameters [1,28–30]. Further experiments are necessary to establish a better interpretation of the results found in this study. 4. Conclusions This study indicates, for the first time, that bonded HILIC phases using SPPs show clear advantages in both efficiency and analysis time without a loss in selectivity. The van Deemter studies demonstrated that the SPP column had higher optimum flow rates than the 3 m FPP column and the 5 m FPP column. It was determined that efficiency gains in the HILIC mode using the SPP column resulted mainly due to a better packed column (i.e., a decrease in eddy diffusion). In HILIC, mass transfer advantages for the SPP column were decreased due to the presence of an adsorbed water multilayer. This was supported by comparing the separations of the same compound in the normal phase mode, and finding that there were increased efficiency gains at high flow rates for the SPP column and a flatter rise (slope) of the curve. In short, SPPs are found to be advantageous in both HILIC and in the NP, but under non-aqueous conditions, more of the benefits of SPPs can be taken advantage of. Acknowledgments DWA acknowledges financial supporting from the Welch Foundation (Y-0026). The authors would like to thank Agilent Technologies (Santa Clara, CA, USA) for providing the superficially porous silica particles. Maressa D. Dolzan and Daniel A. Spudeit thank the Brazilian Program “Science Without Borders” for the interchange from Brazil to USA. References [1] J.J. Destefano, T.J. Langlois, J.J. Kirkland, Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-m particles, J. Chromatogr. Sci. 46 (2008) 254–260. [2] X.L. Wang, W.E. Barber, W.J. Long, Applications of superficially porous particles: high speed, high efficiency or both? J. Chromatogr. A 1228 (2012) 72–88. [3] F. Gritti, G. Guiochon, Facts and legends on columns packed with sub-3-m core-shell particles, LCGC N. Am. 7 (2012) 586–595. [4] C.G. Horvath, B.A. Preiss, S.R. Lipsky, Fast liquid chromatography investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers, Anal. Chem. 39 (1967) 1422–1428.
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[5] J.J. Kirkland, Controlled surface porosity supports for high-speed gas and liquid chromatography, Anal. Chem. 41 (1969) 218–220. [6] J.J. Kirkland, Columns for modern analytical liquid chromatography, Anal. Chem. 43 (1971) 36A–48A. [7] J.J. Kirkland, Superficially porous silica microspheres for the fast highperformance liquid chromatography of macromolecules, Anal. Chem. 64 (1992) 1239–1245. [8] B. Chauve, D. Guillarme, P. Cleon, J.L. Veuthey, Evaluation of various HILIC materials for the fast separation of polar compounds, J. Sep. Sci. 33 (2010) 752– 764. [9] S. Fekete, E. Olah, J. Fekete, Fast liquid chromatography: the domination of core–shell and very fine particles review, J. Chromatogr. A 1228 (2012) 57–71. [10] A. Periat, I. Kohler, J.L. Veuthey, D. Guillarme, Advances in hydrophilic interaction liquid chromatography for pharmaceutical analysis, LCGC Eur. 5 (2013) 17–23. [11] J. Ruta, D. Zurlino, C. Grivel, S. Heinisch, J.L.D. Veuthey, Guillarme evaluation of columns packed with shell particles with compounds of pharmaceutical interest, J. Chromatogr. A 1228 (2012) 221–231. [12] J.J. Kirkland, S.A. Schuster, W.L. Johnson, B.E. Boyes, Fused-core particle technology in high-performance liquid chromatography: an overview, J. Pharma. Anal. 3 (2013) 303–312. [13] D.V. McCalley, Evaluation of the properties of a superficially porous silica stationary phase in hydrophilic interaction chromatography, J. Chromatogr. A 1193 (2008) 85–91. [14] E. Olah, S. Fekete, J. Fekete, K. Ganzler, Comparative study of new shell-type, sub-2 m fully porous and monolith stationary phases, focusing on masstransfer resistance, J. Chromatogr. A 1217 (2010) 3642–3653. [15] F. Gritti, A.D. Pereira, P. Sandra, G. Guiochon, Efficiency of the same neat silica column in hydrophilic interaction chromatography and per aqueous liquid chromatography, J. Chromatogr. A 1217 (2010) 683–688. [16] F. Gritti, I. Leonardis, D. Shock, P. Stevenson, A. Shalliker, G. Guiochon, Performance of columns packed with the new shell particles, Kinetex-C18, J. Chromatogr. A 1217 (2010) 1589–1603. [17] H.X. Qiu, L. Loukotkova, P. Sun, E. Tesarova, Z. Bosakova, D.W. Armstrong, Cyclofructan 6 based stationary phases for hydrophilic interaction liquid chromatography, J. Chromatogr. A 1218 (2011) 270–279. [18] N.L.T. Padivitage, M.K. Dissanayake, D.W. Armstrong, Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column, Anal. Bioanal. Chem. 405 (2013) 8837–8848. [19] J. Nawrocki, The silanol group and its role in liquid chromatography, J. Chromatogr. A 779 (1997) 29–71. [20] J.E. O’Gara, B.A. Alden, C.A. Gendreau, P.C. Iraneta, T.H. Walter, Dependence of yano bonded phase hydrolytic stability on ligand structure and solution pH, J. Chromatogr. A 893 (2000) 245–251. [21] K. Lomsadze, G. Jibuti, T. Farkas, B. Chankvetadze, Comparative highperformance liquid chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by coating totally porous and core–shell silica particles, J. Chromatogr. A 1234 (2012) 50–55. [22] F. Gritti, G. Guiochon, Mass transfer mechanism in chiral reversed phase liquid chromatography, J. Chromatogr. A 1332 (2014) 35–45. [23] F. Gritti, G. Guiochon, The van Deemter equation: assumptions, limits, and adjustment to modern high performance liquid chromatography, J. Chromatogr. A 1302 (2013) 1–13. [24] J.C. Heaton, X. Wang, W.E. Barber, S.M. Buckenmaier, D.V. McCalley, Practical observations on the performance of bare silica in hydrophilic interaction compared with C18 reversed-phase liquid chromatography, J. Chromatogr. A 1328 (2014) 7–15. [25] D.A. Spudeit, M.D. Dolzan, Z.S. Breitbach, W.E. Barber, G.A. Micke, D.W. Armstrong, Superficially porous particles vs. fully porous particles for bonded high performance liquid chromatographic chiral stationary phases: isopropyl cyclofructan 6, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma. 2014.08.022, in press. [26] N.P. Dihn, T. Jonsson, K. Irgum, Water uptakeon polar stationary phases under conditions for hydrophilic interaction chromatography and its relation to solute retention, J. Chromatogr. A 1320 (2013) 33–47. [27] K. Broeckhoven, D. Cabooter, G. Desmet, Kinetic performance of fully and superficially porous partilces with sizes ranging beween 2.7 m and 5 m: intrinsic evaluatio and applicationto a pharmaceutical test compound, J. Pharma. Anal. 3 (2013) 313–323. [28] F. Gritti, G. Guiochon, Unusual behavior of the height equivalent to a theoretical plate of a new poroshell stationary phase at high temperatures, J. Chromatogr. A 1169 (2007) 125–138. [29] F. Gritti, G. Guiochon, Mass transfer mechanism in hydrophilic interaction chromatography, J. Chromatogr. A 1302 (2013) 55–64. [30] F. Gritti, G. Guiochon, Impact of the nature and composition of the mobile phase on the mass transfer mechanism in chiral reversed phase liquid chromatography. Application to the minimization of the solvent cost in chiral separations, J. Chromatogr. A 1327 (2014) 57–65.
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Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase Maressa D. Dolzan a,b , Daniel A. Spudeit a,b , Zachary S. Breitbach a , William E. Barber c , Gustavo A. Micke b , Daniel W. Armstrong a,d,∗ a
Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil c Agilent Technologies Inc., 2850 Centerville Road, Wilmington, DE 19808, USA d AZYP LLC, 700 Planetarium Place, Arlington, TX, 76019, USA b
a r t i c l e
i n f o
Article history: Received 6 June 2014 Received in revised form 28 August 2014 Accepted 2 September 2014 Available online xxx Keywords: Superficially porous particles HILIC Cyclofructan 6 FRULIC Core–shell
a b s t r a c t A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7 m) was developed. Its performance was evaluated and compared to fully porous silica particles with 5 m (commercially available as FRULIC-N) and 3 m diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin ) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Superficially porous particles (SPPs), also called core–shell, porous shell or fused core particles [1,2], are state-of-the-art support materials used in the production of HPLC columns. Historically, the concept of shell particles (pellicular particles) was firstly proposed by Horvath et al. during the 1960s and they were developed as ion exchange materials for the analysis of large biological molecules [2–4]. SPP technology was advanced by Kirkland, who prepared 50 m particles in the 1970s and 5 m particles in the 1990s [5–7]. Concurrent improvements in the manufacturing of high-quality fully porous particles (FPPs) inhibited the application
∗ Corresponding author at: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76019, United States. Tel.: +1 817 272 0632; fax: +1 817 272 0619. E-mail address: [email protected] (D.W. Armstrong).
of SPPs [8]. FPPs with diameters of 3 m (1990s) and sub 2 m (2004) came in vogue along with liquid chromatographs that could operate at higher pressures (i.e., ≥1000 bar) [3]. However, recent improvements to SPP technology have moved them to the forefront of HPLC packing materials. These, more successful core–shell particles have thicker porous shells compared to the early pellicular particles. For example, columns are now available with SPP sizes of 1.7, 2.6 or 2.7 m and porous shell thicknesses of 0.23, 0.35 and 0.5 m, respectively [9]. This generation of SPPs markedly improved its chromatographic performance, due to its morphology, which consists of a solid inner core surrounded by a porous layer, where analytes and mobile phase can diffuse [2]. The presence of the solid core results in a shorter path for diffusion and decreases band broadening caused by poor mass transfer for systems with slow mass transfer kinetics, such as large molecule separations and some chiral separations. These factors permit analyses at high flow rates without a significant loss in efficiency. Further, SPP columns can be very well packed (particularly from the wall to the center
http://dx.doi.org/10.1016/j.chroma.2014.09.010 0021-9673/© 2014 Elsevier B.V. All rights reserved.
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Table 1 Physical parameters and bonded selector loading of the stationary phases.
FPP 5 m FPP 3 m SPP 2.7 m b
Particle diameter (m)
Porosity %
Pore size (Å)
Surface area (m2 /g)
CF6 content (mol/m2 )a
CF6 content (mass%)a
4.3 3.0 2.7
100% 100% 75%
93 100 120
465 300 120
0.72 0.91 0.86
32.2 27.9 12.8
Legend: FFP and SPP mean fully and superficially porous particles, respectively. a Values obtained from the percentage of carbon. b FRULIC-N
of the column) and therefore exhibit decreased band broadening due to eddy diffusion [3]. Columns packed with superficially porous particles have been used for high throughput separations by improving efficiency while keeping methods robust [8–10]. In recent years, the number of publications involving HPLC columns based on SPP has increased [1,3,8,11–16]. Many SPP HILIC columns can be purchased from different companies, but the majority of the marketed HILIC packing material is simply unmodified silica [9]. Silica gel does not always offer acceptable HILIC separations [17,18]. Thus, it is both timely and important to produce and evaluate newer, more promising, HILIC separating agents bound to SPPs. Native cyclofructan-6 (CF6) has been reported to be a powerful selector in separation of polar compounds in the HILIC mode [17,18]. The column based on CF6 chemically bonded to FPPs is commercially available (FRULIC-N) and it has demonstrated advantages over other popular commercial columns in separating several compounds such as nucleic acid bases, nucleosides, nucleotides, xanthines, -blockers, carbohydrates, etc. [17,18]. Further, the native CF6 phase is hydrolytically stable, whereas evidence of dissolution of silica and polar/polar embedded phases in the HILIC mode has been reported [19,20]. In this work, a HILIC stationary phase based on native CF6 was evaluated when bonded to SPPs as the support material (2.7 m), and it was compared with 3 m and 5 m FPP based columns. The columns were also tested in the normal phase (NP) mode, to evaluate the influence of aqueous and non-aqueous containing mobile phases in performance. Results in terms of efficiency, analysis time and resolution were evaluated, demonstrating clear advantages of the new CF6 HILIC column based on SPPs.
diameter) were obtained from Agilent Technologies (Santa Clara, CA, USA). 2.2. Synthesis of native cyclofructan 6 (CF6) based stationary phases Native CF6 was chemically bonded to silica gel according to literature [17]. The same procedure was used to develop all the stationary phases applied in this work, just changing the silica particles used as supporting material (previously described on Section 2.1). The products were characterized by elemental analysis (CHN), and the loading of CF6 in the stationary phases could be calculated. Some physical parameters of the silica particles and the developed stationary phases are listed in Table 1. 2.3. Instruments A total of three columns were prepared in this work (150 mm × 4.6 mm i.d.). All the chromatographic separations were conducted on an Agilent HPLC series 1200 system (Agilent Technologies, Santa Clara, CA), equipped with a quaternary pump, an autosampler and a multiwavelength UV–vis detector. For data acquisition and analysis, the Chemstation software version Rev. B.03.02 [341] was used. The injection volume was 0.5 L for all analyses. The temperature was maintained at 30 ◦ C. The mobile phases (MP) used in the HILIC mode were composed of 75–95% of ACN and 5–25% of 25 mM NH4 OAc, except for the cyclic nucleotides, for which the MP was composed of ACN/100 mM NH4 OAc (70/30, v/v). For the separations carried out in the normal phase, Hep/IPA and Hep/EtOH in different ratios were used and 0.1% of TFA was added to the EtOH phase for the analysis of ferulic acid. Efficiencies were measured using the peak width at half height.
2. Experimental 3. Results and discussions 2.1. Materials 3.1. Preparation of the stationary phases Anhydrous N,N-dimethylformamide (DMF), anhydrous toluene, anhydrous pyridine, 3-(triethoxysilyl)propylisocyanate, ammonium acetate (NH4 OAc), trifluoracetic acid (TFA) and all analytes tested in this work (5-phenylvaleric acid, ferulic acid, pyridoxine, l-ascorbic acid, uracil, adenosine, cytosine, thymidine 3 :5 cyclic monophosphate (cTMP), adenosine 2 :3 cyclic monophosphate (cAMP), guanosine 2 :3 cyclic monophosphate (cGMP), cytidine 2 :3 cyclic monophosphate (cCMP), 1,3-dinitrobenzene (1,3-DNB), ␣-tocopherol, (R)-(+)-2 -amino-1,1 -binaphthalen-2-ol (NOBIN) and 1,3,5-tri-t-butylbenzene) were purchased from Sigma–Aldrich (Milwaukee, WI). The CF6 was provided by AZYP, LLC (Arlington, TX). Acetonitrile (ACN), heptane (Hep), isopropyl alcohol (IPA) and ethanol (EtOH), used for the chromatographic separations, were obtained from EMD (Gibbstown, NJ). Water was purified by a MilliQ Water Purification System (Millipore, Billerica, MA). The FPPs with 5 m of diameter (dp ) were purchased from DAISO (Osaka, Japan), the FPPs with 3 m of dp were obtained from Glantreo (Cork, Ireland), and the SPPs with 2.7 m of dp (0.5 m of thickness of the porous layer and a solid core having a 1.7 m
The results obtained for the elemental analysis of the packing materials are important in understanding the performance of the columns and also to evaluate synthetic implications of the varying particle morphologies. The CF6 content on the stationary phase was calculated from the percentage of carbon obtained in the elemental analysis. The mol/m2 loading (CF6/surface area of the silica particle) is important in understanding the effective/relative coverage of each phase (Table 1). As can be seen, the effective coverage (mol/m2 ) ranged from 0.72 for the FPP 5 m media to 0.91 for the FPP 3 m media. The SPP material had an effective coverage of 0.86 which was greater than the commercial 5 m FPP column (0.72) indicating that the selector density was slightly higher and that sufficient coverage was achieved. It is important to consider coverage/surface area for a bonded/modified SPP based HILIC phase in order to know how much silica character has been masked by the bonded HILIC selector. As can be seen (Table 1), the stationary phase particles of the SPP have less than a half of the absolute amount of CF6 present
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Fig. 1. Separation of polar compounds in HILIC mode using CF6 based columns packed with different types of silica particles. The values on the top of the peaks correspond to the efficiency in terms of number of plates (N) on column. General conditions: column dimensions, 150 mm × 4.6 mm; isocratic mode composed of component A = ACN and, B = 25 mM NH4 OAc and/or C = 100 mM NH4 OAc; flow rate = 750 L/min; T = 30 ◦ C; injected volume = 0.5 L. Specific experimental conditions: A) mobile phase (MP): 75/25 (VA /VB ), = 254 nm; B) MP: 85/15 (VA /VB ), = 210 nm; C) MP: 75/25 (VA /VB ), = 280 nm and; D) MP: 70/30 (Va /Vc ), = 254 nm. The numbers 1–4 represent the peaks in order of elution (1 and 4 correspond the earliest and latest eluted analyte, respectively).
on the 5 m FPP and the 3 m FPP phases. This absolute loading certainly could affect chromatographic properties as analyte bands traverse the length of the column.
3.2. Chromatographic evaluation of CF6 based SPP and FPP columns The main goal of this study was to develop and evaluate the chromatographic performance of native CF6 based columns prepared with SPPs in the HILIC mode. Therefore, a comparison with the 3 m and 5 m FPP based columns was necessary. A total of 11 polar compounds were evaluated in the HILIC mode in terms of efficiency, analysis time and resolution. Further, the efficiency of four different compounds was evaluated in the normal phase mode, and the results were compared.
3.2.1. Performance evaluation of the columns in HILIC The chromatographic performance of the columns was evaluated in the HILIC mode using 11 polar compounds, corresponding to four different groups: 5-phenylvaleric acid and ferulic acid; uracil, adenosine and cytosine; pyridoxine and l-ascorbic acid; and the cyclic nucleotides cTMP, cAMP, cGMP and cCMP (Fig. 1). As can be seen, all columns produced exceptional peak shapes for the tested compounds, indicating that the columns were well packed. The efficiencies obtained for the SPP based column were considerably higher for all analytes. The SPP column resulted in an efficiency improvement of 25–65% compared to the 3 m FPP and efficiencies values 2–4× higher than those obtained using the 5 m FPP column. For example, cCMP gave 7700 plates on the 5 m FPP commercial FRULIC-N column, while cCMP gave 28,000 plates on the SPP
column. The efficiencies and retention times remained constant for more than 600 injections. Under constant mobile phase conditions (Fig. 1), the retention factors were always higher for the 5 m FPP followed by the 3 m FPP, with the SPP column resulting in the shortest analysis times. These results were expected for two reasons: (i) the 5 m FPP contains 32% CF6, while the 3 m FPP contains 28% CF6 and the SPP column only 13% CF6 (w/w) and (ii) the morphology of the superficially porous silica results in a slightly lower dead volume in HPLC, which produces slightly faster analyses [1,11,21]. However, it should be noted that although the analysis times are shorter for the SPP column, the peak capacity (between first and last eluted peak in each chromatogram in Fig. 1) is actually highest on the 3 m FPP (estimated using Giddings approach (i.e. peak capacity = (1 + (N1/2 /4)ln(1 + kn /1 + k1 ))). However, it has been demonstrated that, if the mobile phase conditions (i.e. ratio of weak:strong eluent) are manipulated to give constant retention comparisons between the SPPs and FPPs, the peak capacity of the SPP is the greatest due to the increased efficiency of the SPP column [25]. In order to more specifically probe differences in band broadening on each column, the dependence of the HETP on the mobile phase flow rate was evaluated (Fig. 2) and is discussed in terms of the van Deemter equation:
HETP =
B
v
+ Cs v + Csm v +
1 A
+
1 Cm v
−1 (1)
where v is the velocity of the mobile phase and B, Cs , Csm , A, and Cm are constants, determined by the magnitude of band broadening due to longitudinal diffusion, mass transfer in the stationary phase,
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Fig. 2. Dependence of efficiency (in terms of HETP) on the flow rate of the mobile phase (HILIC) for the compounds and columns indicated. D, E and F show just the data for the SPP and 3 m FPP columns to clarify the comparison between columns packed with similar particle sizes. See Fig. 1 for other experimental conditions.
mass transfer in the stagnant mobile phase, eddy dispersion, and mass transfer in the mobile phase. As can be seen in Fig. 2, the minimum HETP (HETPmin ) ranged from 500 to 750 L/min for the SPP column, 250–500 L/min for the 3 m FPP and from 100 to 500 L/min for the 5 m FPP. In general, the SPP column had the HETPmin at higher flow rates compared to the FPP columns. This is consistent with other SPP materials (e.g. RPLC) and has proven to be due to the reduction of the “B” term caused by the obstruction of flow paths by the solid core [3]. At all tested flow rates, the efficiency of the SPP column was the greatest among the tested columns. However, flatter rises in the van Deemter curves at higher flow rates often associated with SPPs were not obvious. In fact, the slope of the van Deemter curve for adenosine (Fig. 2D) and pyridoxine (Fig. 2F) on the 3 m FPP column was essentially the same as the slope on the SPP column at flow rates between 0.5 and 2.5 mL/min. Only the ferulic acid test (Fig. 2E) showed a slight improvement in the band broadening effects at high flow rates on the SPP column. The fact that the SPP and FPPs appear to have similar van Deemter curve slopes at higher flows, points to the overall efficiency gains largely resulting from generally better packed columns with the SPPs resulting in decreased eddy diffusion contributions to band broadening [3,9,12].
Gritti et al. [16] as well as Heaton et al. [24] have studied FPPs in HILIC, finding the solid–liquid mass transfer resistance HETP is larger in HILIC than it is in the reversed phase mode. This is related to the velocity of adsorption–desorption kinetics, which is slow in HILIC. Our results indicate this HILIC characteristic is also true when using SPPs in the HILIC mode. This is further supported by reports from Gritti et al. [15] which suggest mass transfer advantages of SPPs may be affected by a water enriched adsorbed phase. The postulation that the adsorbed water layer, often present in HILIC separations, effectively dampens “Cs ” term gains of SPPs will be further discussed in Section 3.2.2. It should be noted that “Cs ” term gains due to the use of SPPs are accentuated for analytes with high molar mass (>600 g/mol) or other systems that exhibit slower adsorption/desorption kinetics (e.g. some chiral separations) for small molecules [1,22,23,25]. The analytes used in this study had molar masses generally less than 300 g/mol, but the CF6 selector is the base macrocycle for many other chiral stationary phases which are known to have somewhat slower mass transfer kinetics [25]. Also, as is mentioned in the following Section 3.2.2, the water layer on the CF6 phase is more substantial than that of an unmodified silica HILIC column [26]. Thus, there are at least three possible contributing factors to the mass transfer kinetics of CF6 based HILIC columns: (i) the CF6 molecule can strongly interact
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Fig. 3. Dependence of plate numbers per minute on the flow rate of the mobile phase. For all other experimental conditions, see Fig. 1.
with analytes (similar to a chiral phase) through multiple different simultaneous interactions, (ii) the CF6 stationary phase is essentially “thickened” (note: Cs is directly proportional to the square of the stationary phase film thickness) by a stagnant water layer,
and (iii) the adsorbed water layer will have a significantly higher viscosity than the bulk water. All these factors contribute to the decreased diffusivity of analytes in the HILIC mode. The first contribution may occur in all separation modes, where the second and
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first time, that bonded HILIC phases on SPPs, having an equivalent relative coverage (i.e. mol/m2 ), produces equivalent selectivities. This is in agreement with recent findings of Spudeit et al. in the evaluation of SPP bonded chiral stationary phases [25].
Fig. 4. Selectivity values of neighboring peaks for all the analytes separated in the Fig. 1. 1) Uracil-adenosine; 2) adenosine-cytosine; 3) 5-phenylvaleric acid-ferulic acid; 4) pyridoxine-ascorbic acid; 5) cTMP-cAMP; 6) cAMP-cGMP; 7) cGMP-cCMP.
third contributions should be unique to the HILIC mode. Therefore, at high flow rates, the stationary phase mass transfer contribution from CF6 based columns is greater than might be expected for small molecules. As was noted earlier, the SPP column leads to a decrease in the total analysis time. In order to more clearly show this, the number of plates per unit time was plotted versus the mobile phase flow rate for each compound (Fig. 3). As can be seen, the number of plates afforded per time spent on the analysis is much higher for the SPP column than the FPP columns. Lastly, a comparison of selectivity differences between the FPP columns and the SPP column was made. Fig. 4 represents the selectivity values of neighboring peaks for all the analytes separated in Fig. 1. It was found that selectivity values are essentially the same for most separations when comparing the 5 m FPP column, the 3 m FPP column and the SPP column. These results indicate, for the
3.2.2. Evaluation of the columns in normal phase: a comparison To further support the previously stated postulation, that the flatter rising van Deemter curves often associated with the use of SPPs may be dampened by the presence of an adsorbed multi-layer of water, the columns were also tested under non-aqueous conditions using the normal phase mode. The following compounds were evaluated: ␣-tocopherol, (R)-(+)-NOBIN, 1,3-dinitrobenzene, and ferulic acid (which also was analyzed in the HILIC mode). The dependence of plate numbers on the mobile phase flow rate for these analytes is shown in Fig. 5. As can be seen, chromatographic separations carried out in the normal phase resulted in flatter rising van Deemter curves for the SPP columns compared to the FPP columns. This is in contrast to what was observed in the HILIC mode indicating the influence of the mobile phase and separation mechanisms in the mass transfer process. Previously, Gritti et al. compared the performance of columns packed with SPP bare-silica in HILIC (with high ACN concentration) and per aqueous liquid chromatography (PALC, with low ACN concentration) for the analysis of caffeine. The van Deemter plots for both HILIC and PALC were studied in a narrow range from 0.2 to 1 mL/min and the authors didn’t discuss the influence of these different modes on the mass transfer process and “Cs ” term at high flow rates [15]. Further, Dihn et al. showed that silica has a relatively small water uptake in relation to other HILIC stationary phases [26]. Therefore, the CF6 phase, which is likely to have a more substantial water layer, may not be treated identically to silica in terms of band broadening effects due to mass transfer. To exclude the influence of the nature of the analyte on “Cs ” term effects, ferulic acid was also analyzed in the NP as it is able
Fig. 5. Dependence of efficiency (in terms of HETP) on the flow rate of the mobile phase (NP) for the compounds and columns indicated. Conditions: A and B) Hep/IPA 98/2, = 280 nm; C) Hep/EtOH 80/20, = 254 nm; D) Hep/EtOH 0.1% TFA, = 280 nm.
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to undergo similar strong interactions as in the HILIC mode. When comparing the van Deemter plots for ferulic acid in the HILIC mode (Fig. 2B) and the NP mode, a significant improvement in the HETP at high flow rates can be seen for the SPP column tested in NP mode. For example, under the HILIC conditions, the difference in on-column plate numbers at 2.0 mL/min between the 3 m FPP and the SPP is 4100 whereas the difference in the NP mode increased to 6500. Apparently, non-aqueous conditions can allow for improved exploitation of the “Cs ” term advantages for this SPP based HILIC phase. This implies that, amongst other factors, the increased viscosity and stationary phase thickness resulting from the adsorbed water layer have a significant effect on band broadening at high flow rates. Further, “Cs ” is influenced by diffusion distance inside particles, which is inherently shortened in SPPs [27]. An implication of a substantial water layer may be an increase in the stationary phase diffusional pathlength in the HILIC mode; decreasing one potential advantage of the SPP material. The results obtained in HILIC and NP modes in this work indicate that the mass transfer is dependent on a set of factors, such as the nature of the mobile phase, separation mechanism, nature of the analyte, morphology of the particle used as support material on the stationary phase, and possibly other unknown parameters [1,28–30]. Further experiments are necessary to establish a better interpretation of the results found in this study. 4. Conclusions This study indicates, for the first time, that bonded HILIC phases using SPPs show clear advantages in both efficiency and analysis time without a loss in selectivity. The van Deemter studies demonstrated that the SPP column had higher optimum flow rates than the 3 m FPP column and the 5 m FPP column. It was determined that efficiency gains in the HILIC mode using the SPP column resulted mainly due to a better packed column (i.e., a decrease in eddy diffusion). In HILIC, mass transfer advantages for the SPP column were decreased due to the presence of an adsorbed water multilayer. This was supported by comparing the separations of the same compound in the normal phase mode, and finding that there were increased efficiency gains at high flow rates for the SPP column and a flatter rise (slope) of the curve. In short, SPPs are found to be advantageous in both HILIC and in the NP, but under non-aqueous conditions, more of the benefits of SPPs can be taken advantage of. Acknowledgments DWA acknowledges financial supporting from the Welch Foundation (Y-0026). The authors would like to thank Agilent Technologies (Santa Clara, CA, USA) for providing the superficially porous silica particles. Maressa D. Dolzan and Daniel A. Spudeit thank the Brazilian Program “Science Without Borders” for the interchange from Brazil to USA. References [1] J.J. Destefano, T.J. Langlois, J.J. Kirkland, Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-m particles, J. Chromatogr. Sci. 46 (2008) 254–260. [2] X.L. Wang, W.E. Barber, W.J. Long, Applications of superficially porous particles: high speed, high efficiency or both? J. Chromatogr. A 1228 (2012) 72–88. [3] F. Gritti, G. Guiochon, Facts and legends on columns packed with sub-3-m core-shell particles, LCGC N. Am. 7 (2012) 586–595. [4] C.G. Horvath, B.A. Preiss, S.R. Lipsky, Fast liquid chromatography investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers, Anal. Chem. 39 (1967) 1422–1428.
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[5] J.J. Kirkland, Controlled surface porosity supports for high-speed gas and liquid chromatography, Anal. Chem. 41 (1969) 218–220. [6] J.J. Kirkland, Columns for modern analytical liquid chromatography, Anal. Chem. 43 (1971) 36A–48A. [7] J.J. Kirkland, Superficially porous silica microspheres for the fast highperformance liquid chromatography of macromolecules, Anal. Chem. 64 (1992) 1239–1245. [8] B. Chauve, D. Guillarme, P. Cleon, J.L. Veuthey, Evaluation of various HILIC materials for the fast separation of polar compounds, J. Sep. Sci. 33 (2010) 752– 764. [9] S. Fekete, E. Olah, J. Fekete, Fast liquid chromatography: the domination of core–shell and very fine particles review, J. Chromatogr. A 1228 (2012) 57–71. [10] A. Periat, I. Kohler, J.L. Veuthey, D. Guillarme, Advances in hydrophilic interaction liquid chromatography for pharmaceutical analysis, LCGC Eur. 5 (2013) 17–23. [11] J. Ruta, D. Zurlino, C. Grivel, S. Heinisch, J.L.D. Veuthey, Guillarme evaluation of columns packed with shell particles with compounds of pharmaceutical interest, J. Chromatogr. A 1228 (2012) 221–231. [12] J.J. Kirkland, S.A. Schuster, W.L. Johnson, B.E. Boyes, Fused-core particle technology in high-performance liquid chromatography: an overview, J. Pharma. Anal. 3 (2013) 303–312. [13] D.V. McCalley, Evaluation of the properties of a superficially porous silica stationary phase in hydrophilic interaction chromatography, J. Chromatogr. A 1193 (2008) 85–91. [14] E. Olah, S. Fekete, J. Fekete, K. Ganzler, Comparative study of new shell-type, sub-2 m fully porous and monolith stationary phases, focusing on masstransfer resistance, J. Chromatogr. A 1217 (2010) 3642–3653. [15] F. Gritti, A.D. Pereira, P. Sandra, G. Guiochon, Efficiency of the same neat silica column in hydrophilic interaction chromatography and per aqueous liquid chromatography, J. Chromatogr. A 1217 (2010) 683–688. [16] F. Gritti, I. Leonardis, D. Shock, P. Stevenson, A. Shalliker, G. Guiochon, Performance of columns packed with the new shell particles, Kinetex-C18, J. Chromatogr. A 1217 (2010) 1589–1603. [17] H.X. Qiu, L. Loukotkova, P. Sun, E. Tesarova, Z. Bosakova, D.W. Armstrong, Cyclofructan 6 based stationary phases for hydrophilic interaction liquid chromatography, J. Chromatogr. A 1218 (2011) 270–279. [18] N.L.T. Padivitage, M.K. Dissanayake, D.W. Armstrong, Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column, Anal. Bioanal. Chem. 405 (2013) 8837–8848. [19] J. Nawrocki, The silanol group and its role in liquid chromatography, J. Chromatogr. A 779 (1997) 29–71. [20] J.E. O’Gara, B.A. Alden, C.A. Gendreau, P.C. Iraneta, T.H. Walter, Dependence of yano bonded phase hydrolytic stability on ligand structure and solution pH, J. Chromatogr. A 893 (2000) 245–251. [21] K. Lomsadze, G. Jibuti, T. Farkas, B. Chankvetadze, Comparative highperformance liquid chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by coating totally porous and core–shell silica particles, J. Chromatogr. A 1234 (2012) 50–55. [22] F. Gritti, G. Guiochon, Mass transfer mechanism in chiral reversed phase liquid chromatography, J. Chromatogr. A 1332 (2014) 35–45. [23] F. Gritti, G. Guiochon, The van Deemter equation: assumptions, limits, and adjustment to modern high performance liquid chromatography, J. Chromatogr. A 1302 (2013) 1–13. [24] J.C. Heaton, X. Wang, W.E. Barber, S.M. Buckenmaier, D.V. McCalley, Practical observations on the performance of bare silica in hydrophilic interaction compared with C18 reversed-phase liquid chromatography, J. Chromatogr. A 1328 (2014) 7–15. [25] D.A. Spudeit, M.D. Dolzan, Z.S. Breitbach, W.E. Barber, G.A. Micke, D.W. Armstrong, Superficially porous particles vs. fully porous particles for bonded high performance liquid chromatographic chiral stationary phases: isopropyl cyclofructan 6, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma. 2014.08.022, in press. [26] N.P. Dihn, T. Jonsson, K. Irgum, Water uptakeon polar stationary phases under conditions for hydrophilic interaction chromatography and its relation to solute retention, J. Chromatogr. A 1320 (2013) 33–47. [27] K. Broeckhoven, D. Cabooter, G. Desmet, Kinetic performance of fully and superficially porous partilces with sizes ranging beween 2.7 m and 5 m: intrinsic evaluatio and applicationto a pharmaceutical test compound, J. Pharma. Anal. 3 (2013) 313–323. [28] F. Gritti, G. Guiochon, Unusual behavior of the height equivalent to a theoretical plate of a new poroshell stationary phase at high temperatures, J. Chromatogr. A 1169 (2007) 125–138. [29] F. Gritti, G. Guiochon, Mass transfer mechanism in hydrophilic interaction chromatography, J. Chromatogr. A 1302 (2013) 55–64. [30] F. Gritti, G. Guiochon, Impact of the nature and composition of the mobile phase on the mass transfer mechanism in chiral reversed phase liquid chromatography. Application to the minimization of the solvent cost in chiral separations, J. Chromatogr. A 1327 (2014) 57–65.
Please cite this article in press as: M.D. Dolzan, et al., Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.09.010
