Journal of Chromatography A, 1360 (2014) 275–287

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Evaluation of stationary phases packed with superficially porous particles for the analysis of pharmaceutical compounds using supercritical fluid chromatography Alexandre Grand-Guillaume Perrenoud a , William P. Farrell b,∗ , Christine M. Aurigemma b , Nicole C. Aurigemma b , Szabolcs Fekete a , Davy Guillarme a a b

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland Pfizer, Inc., Worldwide Medicinal Chemistry, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA

a r t i c l e

i n f o

Article history: Received 9 April 2014 Received in revised form 23 July 2014 Accepted 24 July 2014 Available online 1 August 2014 Keywords: Superficially porous particles Core shell 2-Ethyl pyridine Diol 4-Ethyl pyridine HILIC

a b s t r a c t Superficially porous particles (SPP), or core shell particles, which consist of a non-porous silica core surrounded by a thin shell of porous silica, have gained popularity as a solid support for chromatography over the last decade. In the present study, five unbonded silica, one diol, and two ethylpyridine (2ethyl and 4-ethyl) SPP columns were evaluated under SFC conditions using two mixtures, one with 17 drug-like compounds and the other one with 7 drug-like basic compounds. Three of the SPP phases, SunShellTM 2-ethylpyridine (2-EP), PoroshellTM HILIC, and Ascentis® Express HILIC, exhibited superior performances relative to the others (reduced theoretical plate height (hmin ) values of 1.9–2.5 for neutral compounds). When accounting for both achievable plate count and permeability of the support using kinetic plot evaluation, the CortecsTM HILIC 1.6 ␮m and Ascentis® Express HILIC 2.7 ␮m phases were found to be the best choices among tested SPPs to reach efficiencies up to 30,000 plates in the minimum amount of time. For desired efficiencies ranging from 30,000 to 60,000 plates, the SunShellTM 2-EP 2.6 ␮m column clearly outperformed all other SPPs. With the addition of a mobile phase additive such as 10 mM ammonium formate, which was required to elute the basic components with sharp peaks, the PoroshellTM HILIC, SunShellTM Diol and SunShellTM 2-EP phases represent the most orthogonal SPP columns with the highest peak capacities. This study demonstrates the obvious benefits of using columns packed with SPP on current SFC instrumentation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Resurgence in the use of supercritical fluid chromatography (SFC) for the analysis of pharmaceutical compounds was observed following the acetonitrile shortage of 2008. In addition to the obvious cost effectiveness of using non-acetonitrile based mobile phases, SFC benefits in part from the unique physico-chemical properties of the mobile phase such as lower viscosity and higher diffusivity when compared to liquids at a comparable density. SFC also exhibits high kinetic performance (peak capacities and efficiencies) [1–3] with lower column pressure drops, when compared to the use of hydro-organic based mobile phase under similar flow and temperature conditions [4]. From a chromatographic point of view, this enables high-resolution separations on longer columns or

∗ Corresponding author. Tel.: +1 858 638 3658; fax: +1 877 481 9809. E-mail address: bill.farrell@pfizer.com (W.P. Farrell). http://dx.doi.org/10.1016/j.chroma.2014.07.078 0021-9673/© 2014 Elsevier B.V. All rights reserved.

ultra-fast separations using ballistic mobile phase gradients without a significant loss of chromatographic efficiency. Compressed carbon dioxide (CO2 ) mixed with an organic modifier (typically an alcohol) is the most common mobile phase for SFC and offers a broad range of conditions suitable for separating a wide variety of polar and non-polar compounds [5–13]. SFC is considered to be thermodynamically comparable to liquid chromatography (LC), offering complementary selectivity based on stationary phase chemistry, mobile phase (organic solvents and additives), pressure, and column temperature. Similar to LC applications, SFC has been successfully implemented for high throughput separation and purification of combinatorial libraries and chiral compounds primarily using packed columns and is often combined with mass spectrometry [14–17]. Inspired by ultra-high performance LC (UHPLC) technology with overall reduced system dead volumes, increased pressure ranges, and highly accurate pumping modules, new SFC instrumentation has evolved to make SFC a highly competitive technique for

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analytical scale separations. These SFC instruments have further exploited the benefits of supercritical fluids for chromatography through redesigned and accurate backpressure regulators (BPR) and an overall reduction in system dead volumes. This new generation of analytical SFC instrumentation (e.g. Waters Acquity UPC2 , Agilent 1260 Infinity Hybrid SFC/UHPLC) has demonstrated full compatibility with columns packed with sub-2 ␮m fully porous particles (FPP) and comparable performance to UHPLC [4,18]. At the same time, these studies also highlighted the limitations of current instrumentation for use with sub-2 ␮m particles for high resolution analysis (N > 60,000) where column pressures can exceed 400 bar. In this context, the use of sub-3 ␮m superficially porous particles (SPP) could constitute an elegant alternative. In contrast to conventional FPPs, SPP consist of solid core silica microspheres surrounded by a thin, porous outer shell. In LC, it has been experimentally demonstrated that chromatographic efficiency of columns packed with SPP could be increased by approximately 30–50% when compared to columns packed with FPP and having the same dimensions [19–21], while the pressure drops remain similar between both particles types. The enhanced performance of SPP material can be attributed to significant improvements of eddy dispersion (A term of the van Deemter equation), moderate decrease of longitudinal diffusion (B term of the van Deemter equation), and limited improvement of mass transfer resistance (C term of the van Deemter equation) [22,23]. Under SFC conditions, SPPs were found to offer a significant decrease in analysis time with better efficiency compared to columns packed with fully porous 3 ␮m particles, at pressure drops of only ∼100 bar at optimal linear velocity [24]. In another study, significantly improved performance of SPPs under SFC conditions was demonstrated in a systematic kinetic study of ten C18 SPP columns [25]. The potential of those C18 SPPs for high resolution separation of triglycerides contained in vegetable oils was highlighted using 450 mm length SPP columns [26]. A comparison of SPP and FPP performance using isopycnic methodology and kinetic plot representation was also performed for columns bonded with C18 moiety [27]. To date, detailed evaluations of SPP columns under SFC conditions have primarily focused on non-polar stationary phases, and no systematic studies of polar SPP phases using UHPSFC instrumentation have been reported. This paper provides a systematic evaluation of kinetic performances of seven polar columns packed with sub-3 ␮m SPP exhibiting 4 different surface chemistries (unbonded silica, diol, 2-ethylpyridine and 4-ethylpyridine (4-EP)), as well as one column packed with polar, 1.6 ␮m SPP stationary phase material. Stationary phase chemistry plays the most important role for retention and selectivity using SFC conditions [28]. However, retention mechanisms in SFC are far more complex than in RPLC and remain difficult to predict. Therefore, column screening is usually an inevitable first step for method development for both chiral [29,30] and achiral separations [31,32]. Since there are a large number of available chemistries that can be used in SFC, several models have been proposed to rationalize the selection of stationary phases and to point out the chemistry that should offer the most diverse retention and selectivity [33,34]. SPP columns are slowly being integrated into those models as the diversity of chemistry expands [35,36]. This paper illustrates the difference in selectivity on a small set of polar SPP phases.

2. Experimental 2.1. Reagents and columns Pressurized liquid CO2 , 3.0 grade, (99.9%) was purchased from PanGas (Dagmerstellen, Switzerland). LC-MS grade solvents such as methanol (MeOH), isopropanol (iPrOH) and heptane were purchased from VWR (Radnor, PA, USA). The following

chemicals were used in this study: ammonium formate (MS grade), benzocaine, butylparaben, duloxetine, papaverine, nortriptyline, resorcinol, phloroglucinol, and noscapine were purchased from Sigma-Aldrich (Buchs, Switzerland); alprazolam, and midazolam were purchased from Lipomed AG (Arlesheim, Switzerland); flurbiprofen, naproxen, ketoprofen, caffeine, warfarin, thymine, etophylline, uracil, acetaminophen, ibuprofen, sulfamethoxazole, sulfadimethoxine, sulfamethazine, sulfaquinoxaline, sulfamethizole, hydrocortisone and prednisolone were purchased from Sigma–Aldrich (St. Louis, MO, USA). The CortecsTM HILIC (100 × 3.0 mm, 1.6 ␮m) column was purchased from Waters Corporation (Milford, MA, USA). The following columns share the same geometry, 150 × 3.0 mm, and were all purchased as indicated: PoroshellTM HILIC (2.7 ␮m) from Agilent Technologies, Inc. (Wilmington, DE, USA); Ascentis® Express HILIC (2.7 ␮m) from Supelco/Sigma-Aldrich (Bellefonte, PA, USA); KinetexTM HILIC (2.6 ␮m) from Phenomenex (Torrance, CA, USA); SunShellTM Silica (2.6 ␮m), SunShellTM 2-ethylpyridine (2.6 ␮m), SunShellTM 4ethylpyridine (2.6 ␮m), and SunShellTM Diol (2.6 ␮m), which were manufactured by ChromaNik Technologies, Inc. (Osaka, Japan), were purchased from Nacalai USA, Inc. (San Diego, CA, USA). The SunShell Diol and 4-EP SPP phases are considered prototype columns. The HILIC columns used in this study are unbonded (bare) silica. 2.2. Instrumentation 2.2.1. UHPSFC system Analyses were performed on an Acquity UPC2 system (Waters Corp., Milford, MA, USA) consisting of a binary solvent manager, a sample manager equipped with a 10 ␮L loop for partial loop injection, a UV/PDA detector fitted with an 8 ␮L flow cell, a thermostatic column manager, and a convergence manager with a two-step (passive and active) backpressure regulator. The transfer capillaries that connect the injector and column inlet (preheater included) and the column outlet and UV detector were not modified. The total length was equal to 600 mm with an I.D. of 0.175 mm. Extra-column volume was measured at 59 ␮L with a gradient delay volume of 440 ␮L, and the average extra-column variance was found to be approximately 85 ␮L2 for this system. To avoid some possible issues related to mobile phase compressibility, the extra-column volume was measured under LC conditions [4]. 2.2.2. Software Instrument control, data acquisition and data handling of the UHPSFC system were performed with Waters EmpowerTM software (Version 3.0). The physico-chemical properties of the pharmaceutical compounds used in this study were predicted using ACD/ADME Suite software (Version 5, Advanced Chemistry Development, Inc., Toronto, ON, Canada). Kinetic performance and selectivity calculations were performed using MS Excel 2007 Software (Microsoft Corporation, Redmond, WA, USA) and OriginPro 8 (OriginLab Corporation, Northampton, MA, USA). 2.3. Procedure and methodology 2.3.1. Evaluation of column performance The complex dependence of the supercritical fluid mobile phase properties and the chromatographic parameters on the SFC operating conditions complicates the evaluation of kinetic performance in SFC versus LC [37]. Mobile phase density was identified as the key parameter to accurately assess kinetic performance. Changes in density inherent to variations in pressure and temperature significantly affect analyte retention, solubility and diffusion coefficient (DM ), as well as mobile phase viscosity and column efficiency [38]. Under isothermal mobile phase conditions, changes in mobile

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phase density are closely related to pressure variations. Therefore, parameters affecting total pressure and column pressure drop, such as flow rates and/or small particles diameters and backpressure, induce density variations that can be significant depending on the working zone of temperature and pressure, as well as on the mobile phase composition as described by Tarafder and Guiochon [39]. Because of these complex behaviors, the reliability of kinetic performance data for SFC conditions is questionable. To address this, a specific methodology called the isopycnic approach is generally preferred to the conventional van Deemter strategy to accurately evaluate kinetic performance. Under isopycnic conditions, the average mobile phase pressure (and thus average density) is kept nearly constant between the different flow rates by adjusting the outlet pressure [40]. This ensures that all experiments are performed in the same average density environment and provide more reliable efficiency results. However, this method is clearly less representative of the routine use of SFC and is also more time-consuming. The main disadvantage of the conventional van Deemter method is the possible variation of retention factor as a function of mobile phase density and analyte solubility at elevated pressures. As the average pressure and density increase with increasing flow rates, there is a subsequent increase in compound solubility and a decrease in analyte retention [37,38]. However, the variations in retention factor (k) could be considered acceptable especially when working in the supercritical B zone (high backpressure and reasonable temperature) defined by Tarafder and Guiochon [39], with a representative amount of MeOH in CO2 (2–40% (v/v)). The overall mobile phase becomes less compressible with the addition of MeOH and, therefore, the impact of pressure changes become less important. It was recently shown that relative changes in solute retention vary by only 15–20% for flow rates between 0.3 and 3.5 mL/min [18]. The reliability of the results achieved with the conventional van Deemter approach and extrapolated kinetic plot representation was verified and efficiencies with less than 10% relative error were observed for column lengths up to 400–500 mm [18]. Since the dead time does not increase proportionally to the column length due to compressibility effects, the reliability of kinetic plot methodology may be questioned since the van Deemter data is often extrapolated to shorter or longer columns. However, when working within the 5,000–60,000 plate count range, the results were found to be reliable [41]. Therefore, on the basis of these attributes and the overall practicality of collecting data, the conventional methodology was used in the present study. The performance of new SPP columns was evaluated under SFC conditions using butylparaben and prednisolone standards (50 ␮g/mL in 30:70 (v/v) iPrOH/heptane), at injection volumes of 0.7 and 1.0 ␮L for 100 mm and 150 mm columns, respectively. For each column, the methanol composition was adjusted to maintain a k value between 4 and 6.2 for butylparaben (ranging from 2 to 5% MeOH in the mobile phase) and between 5.5 and 9 for prednisolone (ranging from 9 to 18% MeOH) on all eight SPP columns across the entire pressure range studied. Initially, the efficiencies of the eight SPP columns were studied and compared by constructing their respective plate height versus linear velocity (H–u0 ) plots. By employing 3.0 mm I.D. columns exclusively, system dispersion was minimized and the contribution of the SFC system to band broadening was determined to be negligible, provided that the retention remains sufficiently high (k ≥ 6) [18]. Therefore, no additional corrections for extra-column band broadening were applied. The backpressure at the column outlet was set to 150 bar and temperature was maintained at 40 ◦ C. Fifteen different flow rates between 0.3 and 3.5 mL/min were utilized to construct H–u0 plots for each column. This range was used as not to exceed the specified maximum system pressure of 400 bar, resulting in a maximum pressure drop of 250 bar. Chromatograms

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were recorded at 237 nm and 254 nm for prednisolone and butylparaben, respectively. The smooth lines were constructed using the van Deemter equation, a least square regression procedure and the solver function macro contained in Excel. The column void time (t0 ) was experimentally measured for each column/flow rate combination using the “minor UV disturbance method” (system peak), and corrected for system transit time. Column permeability (KV ) was also experimentally determined, using the following relationship [42]: KV =

u0 ·  · L P

(1)

where  is the mobile phase viscosity (at a given column head pressure, including the BPR), L is the column length and P is the experimentally observed column pressure drop measured at the column inlet. P was corrected for the native system pressure drop determined without a column in place. Estimation of supercritical CO2 viscosity was made on the correlations proposed by Ouyang [43] and applied to different CO2 /methanol mixtures using a second order correlation between viscosity and MeOH concentration, based on previously published experimentally-determined data [44]. By combining these two correlations, the viscosity of mixtures of supercritical CO2 and MeOH for a given pressure and temperature can easily be predicted. Predicted viscosity values were experimentally validated for several different conditions and values. The kinetic plot methodology is a facile way to visualize the maximum achievable plate number per analysis time, or the minimum analysis time to achieve a desired theoretical plate number, while operating the column at its optimum performance. The kinetic plot methodology is also a useful tool for evaluating column efficiency within the limits of column mechanical stability or system pressure capability. Plots of minimal achievable analysis time, expressed as column dead time (t0 ) versus required plate numbers (N), were constructed using the following equations: t0 =

Pmax max

N=

Pmax max



KV



u20

K  V u0 H

(2)

(3)

where Pmax and max are related to the maximum system pressure capability of the system (400 bar, including 150 bar BPR). In these representations, achievable analysis times for the different columns and mobile phase temperatures can easily be compared and discussed when the maximum performance of the instrument is utilized. Overall column performance can also be compared by using separation impedance (E), which considers plate count, analysis time, and column permeability. Separation impedance is based on the formula derived by Bristow and Knox [45]: E=

H2 t0 P = 2 KV N 

(4)

2.3.2. Evaluation of selectivity and peak capacity of superficially porous columns The selectivity and peak capacity of eight SPP columns were assessed using a mixture of 17 compounds including flurbiprofen, naproxen, ketoprofen, caffeine, warfarin, thymine, etophylline, uracil, acetaminophen, ibuprofen, sulfamethoxazole, sulfadimethoxine, sulfamethazine, sulfaquinoxaline, sulfamethizole, hydrocortisone and prednisolone dissolved in a binary sample solvent mixture of 30:70 (v/v) iPrOH/heptane at different concentrations. CO2 /MeOH mobile phase conditions were adjusted to maintain the same gradient program (5–20% gradient of MeOH in CO2 for all columns except the SunShell 4-EP, for which a gradient from 5 to 25% of MeOH in CO2 was used) and steepness irrespective

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Table 1 Mobile phase gradient conditions for selectivity and peak capacity evaluation. Column

Geometry

Flow rate (mL/min)

Gradient program

Run time (min)

Ascentis HILIC Poroshell HILIC Kinetex HILIC Cortecs HILIC SunShell silica SunShell diol SunShell 2EP SunShell 4EP

150 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm 100 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm

2.50 2.50 2.50 2.00 2.50 2.50 2.50 2.50

5 to 20% MeOH 5 to 20% MeOH 5 to 20% MeOH 5 to 20% MeOH 5 to 20% MeOH 5 to 25% MeOH 5 to 25% MeOH 5 to 25% MeOH

4.00 4.00 4.00 3.33 4.00 5.33 5.33 5.33

of the column length and flow rate (Table 1). For this purpose, the gradient time (directly related to the gradient slope and modifier concentration range) was scaled in direct proportion to the column dead time (which depends on column dimensions, and mobile phase flow rate), to maintain the same product of gradient slope by column dead time. Injection volumes were also scaled to column volumes, 0.7 and 1.0 ␮L injected on 100 and 150 mm long columns, respectively. Mobile phase flow rate was set at 2.0 mL/min when the CortecsTM HILIC column was used and at 2.5 mL/min for the separation performed on the seven remaining stationary phases. Column temperature, backpressure and detection wavelength were set at 40 ◦ C, 150 bar and 220 nm, respectively. Peak capacity (nc ) values were calculated using equation 5: nc = 1 +

tgrad 1.699 × w50%

(5)

where tgrad is the gradient time and w50% is the peak width at half height. 2.3.3. Basic compound analyses on superficially porous columns Peak shape, selectivity and retention of basic compounds on SPP material were assessed using a simple but representative mixture of basic pharmaceutical drugs. The compounds were selected to cover a fairly large range of pKa values (determined in water) and were sub-classified into 3 groups: weakly basic (pKa < 6) including benzocaine (pKa = 2.4) and alprazolam (pKa = 5.7); moderately basic (pKa between 6 and 8) including noscapine (pKa = 6.3), papaverine (pKa = 6.3) and midazolam (pKa = 6.5); and strongly basic (pKa > 8) including nortriptyline (pKa = 9.9) and duloxetine (pKa = 9.6). The mixture was prepared in 30:70 (v/v) iPrOH/heptane, with a compound concentration ranging from 10 to 100 ␮g/mL. Separation was performed in gradient mode on the eight SPP columns using the same gradient program and steepness, injection volumes and mobile phase flow rates as those described in the previous section. A second study was conducted with 10 mM ammonium formate in methanol as the organic modifier. For all columns and conditions, oven temperature, BPR, and UV detection wavelength were set to, 40 ◦ C, 150 bar and 235 nm, respectively. 3. Results and discussion 3.1. Comparison of the plate heights obtained with SPP materials The kinetic properties of the eight columns studied, which were packed with SPP particles (2.7, 2.6 and 1.6 ␮m), were first evaluated based on the H–u0 plots obtained with two different test analytes and summarized in Table 2. Figs. 1 and 2 show the corresponding van Deemter plots obtained by injecting butylparaben and prednisolone, respectively. Fig. 1A and B illustrate the van Deemter curves obtained on bare silica stationary phases, while Fig. 2A and B correspond to alternative chemistries such as diol and ethylpyridine (EP) bonded phases. For butylparaben, the lowest plate height value (Hmin = 3.7 ␮m, corresponding to hmin of 2.3) was observed with the 1.6 ␮m

CortecsTM HILIC column (Fig. 1A). With the other silica phases (2.6 and 2.7 ␮m particles), similar plate height values were demonstrated, with Hmin between 5.9 and 6.3 ␮m, corresponding to hmin of 2.2 to 2.4. Similar H–u0 curve shapes were also observed. Among the bonded phases, the 2-EP had the lowest Hmin of 5.0 ␮m (corresponding to hmin of 1.9), while Hmin for the diol and 4EP phases was 7.9 ␮m (corresponding to hmin of 3) (Fig. 2A). All of the columns packed with 2.6–2.7 ␮m particles showed very similar H–u0 curve shapes and comparable B- and C-terms, with the exception of the 1.6 ␮m CortecsTM HILIC, which is in agreement with theoretical expectations. Particle size does indeed have a strong impact on both the eddy dispersion (A-term) and the trans-particle mass transfer resistance (C-term). It should, however, be noted that with the 1.6 ␮m particles, the optimal linear velocity and thus the lowest possible H value could not be reached before exceeding the upper pressure limit of the instrument (380 bar was reached at u0 = 11 mm/s at T = 40 ◦ C). In this case, the major contributions to band broadening were eddy dispersion (A-term dominated region of the van Deemter curve) and longitudinal diffusion (B-term dominated region of the van Deemter curve). Due to the low permeability of this particular column (KV = 3.8 × 10−11 cm2 ), the 1.6 ␮m SPP material suffered from the limitations of our current instrumentation with respect to upper pressure capability (when analyzing small molecules with high diffusivity). The same phenomenon was recently reported in reversed phase mode when working with columns packed with 1.3–1.6 ␮m superficially porous particles [46,47]. Despite the fact that the maximum performance could not be attained, column efficiencies of approximately 270,000 plates/meter could be achieved with 1.6 ␮m SPP material, which is quite impressive in SFC. Figs. 1B and 2B illustrate the H–u0 curves obtained with prednisolone. The impact of diffusion rate (i.e. due to modifications of mobile phase viscosity and molecular weights of the model compounds) on the shape of the plate height curve is well illustrated. The B-term is reduced, and the C-term is higher with prednisolone when compared to butylparaben (the molecular weight of prednisolone is 1.8-fold larger than butylparaben). However, the curves show unexpected deviations that cannot be attributed only to molecular weight differences. For instance in Fig. 1B, the minimum plate heights varied from Hmin = 5 ␮m (hmin of 3.1) (CortecsTM HILIC) to 11.2 ␮m (hmin of 4.2) (Kinetex HILIC). The obvious difference between the chemical structures of prednisolone and butylparaben is the number of hydroxyl groups (e.g. 3 H-bond donor groups for prednisolone versus only one for butylparaben), which results in disparate Hbond interactions between the solutes and the silica stationary phase. (Note: the average strength of one intermolecular H-bond is approximately 5 kcal/mol). However, because H-bond interactions are cumulative, the adsorption of prednisolone at the surface of the stationary phase will be much more pronounced than that of butylparaben. In other words, the interaction of prednisolone with the stationary phase is kinetically less favorable than the interaction of butylparaben. Among the five silica

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Table 2 Core–shell columns features and achieved kinetic performance. Name

Column

Ascentis HILIC Poroshell HILIC Kinetex HILIC Cortecs HILIC SunShell silica SunShell diol SunShell 2EP SunShell 4EP

Butylparaben dp

KV

MeOH

Hmin

hmin

MeOH

Hmin

hmin

150 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm 100 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm 150 × 3.0 mm

2.7 ␮m 2.7 ␮m 2.6 ␮m 1.6 ␮m 2.6 ␮m 2.6 ␮m 2.6 ␮m 2.6 ␮m

9.1E−11 cm2 6.2E−11 cm2 6.6E−11 cm2 3.8E−11 cm2 8.1E−11 cm2 8.1E−11 cm2 8.2E−11 cm2 8.2E−11 cm2

2% 2% 2% 2% 2% 3% 5% 5%

5.9 ␮m 5.9 ␮m 6.3 ␮m 3.7 ␮m 6.3 ␮m 7.6 ␮m 5.0 ␮m 7.9 ␮m

2.2 2.2 2.4 2.3 2.4 2.9 1.9 3.0

9% 10% 11% 11% 11% 12% 11% 18%

6.7 ␮m 6.2 ␮m 11.2 ␮m 5.0 ␮m 8.8 ␮m 8.1 ␮m 5.5 ␮m 6.8 ␮m

2.5 2.3 4.3 3.1 3.4 3.1 2.1 2.6

phases, the hydrogen bond affinity (acidity) of the stationary phase varies, as it depends on the ratio of single, geminal, and vicinal silanols at the surface of the stationary phase. It is not possible to precisely estimate the surface characteristics of each tested phase; however, these interactions can certainly explain why the kinetic performances achieved on the SunShell and Kinetex HILIC silica phases were vastly different for butylparaben and prednisolone. To verify that the strength of H-bond interaction can impact the kinetic performance in SFC, two additional compounds, namely dihydroxybenzene (resorcinol) and trihydroxybenzene (phloroglucinol), having a similar size to butylparaben, but a different H-bond donor capability (one, two and three

H-bond donor groups for butylparaben, resorcinol and phloroglucinol, respectively) were injected on the Kinetex HILIC and Poroshell HILIC phases. On the Poroshell HILIC, the Hmin values were comparable irrespective of the investigated compound and were situated between 5.58 and 6.48 ␮m. These results are in agreement with the data generated with butylparaben and prednisolone. On the contrary, the Hmin values observed on the Kinetex HILIC were very different and strongly related to the H-bond interactions. The Hmin values varied from 6.12 ␮m (butylparaben, one H-bond donor group), to 7.40 ␮m (resorcinol, 2 H-bond donor groups), to 11.21 ␮m (prednisolone, 3 H-bond donor groups), and 16.71 ␮m (phloroglucinol, 3 H-bond donor groups). This additional data

20.0

20.0

18.0

16.0

14.0

14.0

12.0

12.0

H (µm)

H (µm)

18.0

A.

16.0

10.0 8.0

8.0 6.0

4.0

4.0

2.0

2.0 2.0

4.0

6.0

8.0

u (mm/s)

10.0

12.0

14.0

B.

10.0

6.0

0.0 0.0

Prednisolone

Geometry

0.0 0.0

16.0

2.0

4.0

6.0

8.0

u (mm/s)

10.0

12.0

14.0

16.0

Fig. 1. Van Deemter plots for butylparaben (A) and prednisolone (B) were obtained for the 5 bare silica stationary phases; Poroshell HILIC (red squares), Ascentis Express HILIC (purple diamonds), Kinetex HILIC (light green triangles), SunShell Silica (deep blue circles) and Cortecs HILIC (light blue barred Xs). SFC conditions for A consisted of a fixed mobile phase composition of 2% MeOH in CO2 (retention factors variations between 4 and 6 depending on the column and on the flow rate). B utilized a variable and column-dependent isocratic mobile phase composition where sufficient retention was achieved with k values between 6 and 8; 9% MeOH in CO2 for Ascentis Express HILIC, 10% for Poroshell HILIC and 11% for Kinetex HILIC, SunShell Silica and Cortecs HILIC columns. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

20.0

20.0

18.0

16.0

14.0

14.0

12.0

12.0

H (µm)

H (µm)

18.0

A.

16.0

10.0 8.0

10.0 8.0

6.0

6.0

4.0

4.0

2.0

2.0

0.0 0.0

2.0

4.0

6.0

8.0

u (mm/s)

10.0

12.0

14.0

16.0

B.

0.0 0.0

2.0

4.0

6.0

8.0

u (mm/s)

10.0

12.0

14.0

16.0

Fig. 2. Van Deemter plots for butylparaben (2A) and prednisolone (2B) were obtained for the SunShell Diol (deep green crosses), SunShell 2-EP (orange Xs) and SunShell 4-EP (black dashed line) columns. These compounds eluted with two variable but isocratic mobile phase compositions to ensure sufficient retention factors for both individual molecules (e.g. between 6 and 8, depending on column and flow rate). Isocratic SFC conditions were as follows: 3% MeOH in CO2 (2A) and 12% (2B) for SunShell Diol; 5% (2A) and 11% (2B) for SunShell 2-EP; and 5% (2A) and 18% (2B) for SunShell 4-EP. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

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1 x 10-3

t0 = 100 min

t0/N (min)

1 x 10-4

t0 = 10 min 1 x 10-5

t0 = 0.1 min 1 x 10-6 10000

100000

t0 = 1 min

1000000

Plate number Fig. 3. t0 /N vs. N-type kinetic plot representation extrapolated to 400 bar from the data measured using butylparaben as a model compound on 4 different core–shell columns; Poroshell HILIC (red squares), Ascentis Express HILIC (purple diamonds), Cortecs HILIC (light blue barred Xs) and SunShell 2-EP (orange crosses). (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

confirms that the strength of interaction (H-bond in this case) may in some cases impact kinetic performance in SFC. Aside from variations in Hmin values, a change in mass transfer rate for prednisolone versus butylparaben was also observed on the 4-EP phase, manifesting in a higher C-term. This increased curvature in the region dominated by the C-term observed with prednisolone is further exacerbated by the MeOH mobile phase proportion required for elution, which generates a non-negligible reduction of diffusion coefficients. Another possible explanation for the observed differences in Hmin values is related to the pore size distribution of the SPP

phases considered in this study. It is possible that the discrepancies observed for the larger and more rigid prednisolone may be related to its difficulty in entering small pores without trouble. However, a precise distribution of pore size is not available for all these columns. Finally, optimal linear velocity can be attained with prednisolone, even when using the 1.6 ␮m CortecsTM HILIC material. Clearly, this behavior is related to the lower diffusion coefficients of prednisolone relative to butylparaben and suggests that the pressure limit of the system is particularly critical for small molecule solutes (MW < 200 g/mol).

Fig. 4. t0 /N vs. N-type kinetic plot representation extrapolated to 400 bar from the data measured using prednisolone as a model compound on 4 different core–shell columns (Column designation symbols and colors are identical to those described in Fig. 3).

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When measuring the H–u0 data, the column pressure drop and t0 values were also recorded, allowing for the calculation of separation impedance values. The minimum separation impedance (E) with the superficially porous materials ranged between 1,000 and 2,000, which was significantly better than what could be achieved with fully porous particles. For example, E values between 2,500 and 5,100 were reported using fully porous packings in SFC [18]. The lower separation impedance of SPP materials may enable faster separations than columns packed with fully porous particles. While accounting for mechanical stability (maximal operating pressure) of the columns and the system pressure threshold, the limits of kinetic performance can be calculated using kinetic plot methodology. In the present study, it was observed that changes in solute retention factor under SFC conditions over the whole range of tested flow rates were of the same order of magnitude found under UHPLC conditions, but contributing factors for both techniques were different. Typically, the solute retention factor decreases by 15–20% while the pressure is increased from 100 to 1,000 bar (by increasing the flow rate) under UHPLC conditions, due primarily to the mobile phase compressibility and frictional heating effects [48]. Despite the inherent system pressure limitation of 400 bar (including the back pressure regulation) with the Acquity UPC2 system used in this study, SFC flow rates of 3–4 mL/min were feasible with the 3.0 mm I.D. columns due to the low viscosity of the mobile phase. During this kinetic evaluation, the decrease in solute retention factor ranged from 15 and 25% over the entire flow rate range due to mobile phase density variation, but also due to temperature effects (decompression and adiabatic cooling, frictional heating related to the use of small particles at high flow rate) which is quite similar to the retention characteristics observed in UHPLC [18]. The plots in Fig. 3 represent the theoretical plate–time values (t0 /N) and minimal achievable analysis time (t0 ) for butylparaben on four SPP columns at maximum system performance. For low plate numbers (N < 30,000), which often corresponds to the typical plate number range in current SFC practices, the 1.6 ␮m CortecsTM HILIC and the 2.7 ␮m silica columns offer practically the same separation speed, when operating the columns at their kinetic performance thresholds. For example, the column dead time corresponding to an efficiency of 25,000 plates was equal to 0.12 min on the CortecsTM HILIC 1.6 ␮m and Ascentis Express HILIC 2.7 ␮m columns, while the Poroshell HILIC 2.7 ␮m column came close at 0.15 min. The SunShell 2-EP 2.6 ␮m phase provides somewhat longer analysis times within the same plate number region, but is clearly the best choice for high resolution separations (N > 60,000 plates). Due to the low permeability of the CortecsTM HILIC 1.6 ␮m material, this column is not suitable for high resolution separations. In order to reach the N > 60,000 plate level, a minimum of 600–800 bar would be required. A similar trend was observed with prednisolone (Fig. 4). When N < 30,000, all four columns provided similar analysis times. The best choice for N > 70,000 is the SunShell 2-EP 2.6 ␮m phase. Again, the 1.6 ␮m CortecsTM HILIC was not well suited for high resolution separations even with the larger analyte due to limitations in operating pressure. Based on kinetic plot comparisons, the four SPP materials used to construct Figs. 3 and 4 offer similar plate-time values in the case of fast separations (e.g. N < 30,000). Under typical SFC conditions (e.g. using 100–150 mm long columns), these SPP materials can be used without significant differences in expected analysis times, at least from a purely kinetic point of view. However, it is also important to keep in mind that retention and selectivity (thermodynamic parameters) may be altered between these different phases. Then, the speed of the separation may be reduced due to thermodynamic

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t0 = 10 min

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Plate number Fig. 5. t0 /N vs. N-type kinetic plot comparison of fully-porous and superficially porous materials extrapolated to 400 bar for butylparaben using Waters Acquity UPC2 BEH (hybrid silica) 1.7 ␮m (dark green triangles), 3.5 ␮m (red circles), and 5 ␮m (black squares); Ascentis Express HILIC 2.7 ␮m (purple diamonds); and Cortecs HILIC 1.6 ␮m (light blue barred Xs). (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

rather than kinetic parameters. For high resolution separations, our focus will be on the 2.6–2.7 ␮m particles, mainly due to the 400 bar system pressure limit. It is also of interest to highlight the potential as well as the limitations of fully porous and superficially porous materials. Fig. 5 illustrates plate-time values for butylparaben. Data previously published using columns packed with 1.7, 3.5 and 5 ␮m fully porous BEH (ethylene bridged hybrid) particles were also included in the figure [4,18]. Based on this comparison, the 2.7 ␮m superficially porous material outperforms the other columns in the plate count range of 20,000–120,000. For low plate numbers (e.g. N = 10,000), the 1.6 ␮m CortecsTM HILIC offers moderately faster analysis (∼10% faster), while for very high-efficiency separations (N > 120,000), the fully porous 3.5 ␮m packing demonstrated somewhat faster analysis due to its more favorable permeability. The superiority of 2.6–2.7 ␮m core–shell vs. 1.7 ␮m fully porous particles experimentally demonstrated here corroborates the theoretical predictions made by Gritti and Guiochon [49]. 3.3. Performance of columns packed with SPP with pharmaceutical compounds In addition to the kinetic performances achieved with butylparaben and prednisolone, the overall quality of the eight SPP columns was also assessed using a test mixture of 17 drugs which included steroids, non-steroidal anti-inflammatory drugs (NSAIDS), analgesics, and sulfonamide antibiotics, to name a few. The structures of these model compounds and the corresponding chromatograms achieved with SPP phases are provided in Fig. 6 and Fig. 7, respectively. Fig. 7A–D highlight the chromatograms obtained with bare silica phases of 2.6–2.7 ␮m SPP using the same generic gradient of 5–20% MeOH in 4.00 min. For these four stationary phases, the elution order remains relatively constant with only slight differences observed. The acidic compounds (peaks 1 to 4, corresponding to NSAIDs) were systematically eluted first, while the highest retention was observed for sulfonamide antibiotics (peaks 13 to 17). The elution order of the NSAIDs remained constant on these four bare silica phases. The most significant changes in selectivity were observed with the sulfonamide antibiotics. To better visualize the changes in

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Fig. 6. Structures of the 17 acidic and neutral compounds used for the selectivity evaluation.

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Fig. 7. Chromatograms of the 17-component mixture separation under gradient SFC conditions on all eight tested SPP columns; Ascentis Express HILIC (A., purple trace), Poroshell HILIC (B., red trace), Kinetex HILIC (C., light green trace), SunShell silica (D., deep blue trace), Cortecs HILIC (E. light blue trace), SunShell Diol (F., deep green trace), SunShell 2-EP (G., orange trace) and SunShell 4-EP (H., black trace). Numbers correspond to the compounds presented in Fig. 6 and analytical conditions are detailed in Section 2.3.3. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.) Table 3 Selectivity of the 8 core–shell stationary phase for the mixture of 17 compounds expressed according to the S values and apparent gradient efficiency expressed using peak capacity. S value Ascentis HILIC Poroshell HILIC Kinetex HILIC Cortecs HILIC SunShell silica SunShell diol SunShell 2EP SunShell 4EP Peak capacity

SunShell 4EP 43.9 56.2 56.2 59.7 50.3 95.4 19.9 104

SunShell 2EP 50.1 62.5 62.2 65.8 56.6 98.5

126

SunShell diol 92.3 91.3 91.4 90.9 91.7

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SunShell silica 8.5 8.2 7.5 12.5

128

Cortecs HILIC 20.8 5.2 5.3

113

Kinetex HILIC 15.9 2.8

117

Poroshell HILIC

Ascentis HILIC

16.6

138

130

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selectivity between the different phases, the corresponding S values were computed in Table 3, using equation (6): S = 100 ·



1 − r2

(6)

where r equals the Pearson correlation coefficient between two given conditions. S is consequently equal to 100 for two uncorrelated conditions (e.g. very distinct retentions for the 17 compounds) and equal to 0 for two identical separations [50]. As shown in Table 3, the S values ranged from 2.8 to 16.6 for these four SPP columns, which confirm their chemical similarity. From these values, it appears that the Poroshell HILIC and Kinetex HILIC were particularly close (S = 2.8). This is also illustrated in Fig. 7B and C, where only minor differences in selectivity were observed between peaks 12 and 15 (sulfamethoxazole and sulfamethazine). In contrast, the highest selectivity differences were found between the Ascentis Express HILIC and Kinetex HILIC (S = 15.9), as well as the Ascentis Express HILIC and the Poroshell HILIC (S = 16.6). Nevertheless, these S value differences were systematically low, suggesting that the retention mechanisms on these four silica phases were highly similar. These results support those previously shown by West et al. on a systematic study based on a solvation parameter model performed on 5 ␮m FPP and 2.6 ␮m SPP silica columns [36]. With the exception of selectivity and retention differences, the average peak capacities (nc ) were also calculated while accounting for average peak widths and gradient elution times. The nc values ranged from 117 to 138 for the four bare-silica phases. In agreement with the discussion in Section 3.1, the Kinetex HILIC offered the lowest peak capacity, while the Poroshell HILIC demonstrated the highest value (nc = 138) and was, therefore, preferentially selected as a reference bare silica SPP phase. The CortecsTM HILIC column, packed with 1.6 ␮m SPP particles, was also tested and the gradient was geometrically scaled (3.33 versus 4.00 min) in proportion to the column dead time while maintaining identical initial and final compositions of 5 and 20% MeOH, respectively. As shown in Fig. 7E, the early eluting peak for the acidic compound exhibited peak distortion (strong tailing), while the other peaks remained symmetrical and sharp. The strange behavior observed with these acidic drugs could be related either to the nature of the silica matrix itself or to a possible overloading of the stationary phase attributed to the reduced porous shell volume on this material compared to 2.6–2.7 ␮m SPP columns. The peak capacity of the CortecsTM HILIC was found to be 113, which is lower than the values achieved with the Ascentis Express HILIC (nc = 130), Poroshell HILIC (nc = 138) and SunShell Silica (nc = 128), but relatively close to the Kinetex HILIC (nc = 117). As discussed in Sections 3.1 and 3.2, a plate count improvement should have been observed with the CortecsTM HILIC when compared to the 2.6–2.7 ␮m SPP columns. However, since the column length was shortened to 100 mm and the gradient time was also shortened, it was logical that the peak capacity was also reduced. In addition, the flow rate employed on the CortecsTM HILIC was lowered to 2.00 mL/min, which further contributed to the reduced peak capacity. As expected, the elution order was not vastly different between the CortecsTM HILIC and the other bare silica SPP phases. Indeed, the S value between the CortecsTM HILIC column and the four other bare silica phases ranged from 5.2 to 20.8. The most significant difference was found between the Ascentis Express HILIC and the CortecsTM HILIC (S = 20.8). The low S values confirm that all tested bare silica (HILIC) SPP phases were chemically similar. The experiments were repeated on the SunShell Diol, 2-EP and 4-EP SPP phases, and the chromatograms were reported in Fig. 7F, G and H, respectively. The gradient steepness used for the unbonded silica columns was maintained, but due to higher peak retention with the bonded columns, the gradient program was extended from 5 to 25% MeOH in 5.33 min. As shown in Fig. 7F, G and H, the

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elution orders were quite different from those achieved on the bare silica phases, and the calculated S-values varied from 43.9 to 92.3. This behavior is in agreement with our expectations due to differences in the retention mechanisms of the SunShell SPP columns that result from the chemical modifications of these bonded phases. Surprisingly, the most orthogonal stationary phase was the SunShell Diol, with significantly high S-values of 90.9–92.3, when compared to the bare silica phase. The results support previously published data obtained for FPP diol columns that result in orthogonal selectivity compared to bare silica [36]. Changes in selectivity were observed between the SunShell Diol and its ethylpyridine counterparts, where S-values for the SunShell 2-EP and 4-EP phases were even higher (95.4 and 98.5, respectively). The SunShell 2-EP and 4EP were also relatively orthogonal compared to bare silica phases (S-values between 43.9 and 65.8), but to a lesser extent than the Diol SPP column. Finally, selectivity differences between the 2-EP and 4-EP were relatively small (S equal to 19.9). It is important to note that the SunShell 4-EP phase consistently demonstrated the most retentivity, except for peak 6 (caffeine). The Diol and 2-EP moieties also offer good chromatographic behaviors for all the tested compounds, which is in complete agreement with data published by De la Puente et al. for FPP stationary phases [31]. Finally, peak capacities of the three bonded SPP phases were found to be 104 for the 4-EP, 106 for the Diol, and 126 for the 2-EP. This column ranking is consistent with the results of the kinetic performance evaluation presented in section 3.1, where the SunShell 2-EP clearly outperformed the two other bonded phases. If initial column screening under SFC conditions requires limiting to 2 or 3 stationary phases, the Poroshell HILIC, SunShell Diol and the SunShell 2-EP phases are the most orthogonal and offer the highest peak capacities as demonstrated in this study.

3.4. Performance of columns packed with SPP with ionizable drugs 3.4.1. In absence of mobile phase additives The 17-component mixture used for the evaluations in Section 3.3 consisted primarily of acidic and neutral compounds. Since basic substances are often much more challenging to analyze in SFC conditions, we have included a mixture of seven basic drugs as described in Section 2.3.3 for evaluating the SPP phases. The structures of these drugs are given in Fig. 8 and corresponding chromatograms achieved on the eight columns packed with 1.6–2.7 ␮m SPP are provided in Figs. 9 and 10. The chromatograms reported in Fig. 9 were obtained without the use of additives in the mobile phase. Under these SFC conditions, only five peaks could be eluted from the bare silica SPP phases (Fig. 9A–E). The interactions between the two strongly basic drugs, nortriptyline and duloxetine, and the stationary phases were quite pronounced, due to significant ionic interactions between the positively charged secondary amino group of these drugs and the negatively charged silanols (which are present in abundance on these non-endcapped, unbonded phases). This observation is in agreement with data previously published, where a non-negligible number of basic substances were not eluted from fully porous bare silica phases [51]. Since there are fewer silanols on the Diol, 2-EP and 4-EP materials, these two basic compounds are eluted from these bonded phases with a reasonable proportion of methanol (up to 20%) as shown in Fig. 9F, G and H. On the five bare silica SPP phases, peaks II (noscapine) and III (midazolam) also display pronounced tailing and significant band broadening, respectively. Finally, the weak bases (peaks I and V) were eluted from the columns as sharp peaks. This behavior supports the conclusion that bare silica SPP phases are not well suited for SFC analysis of basic drugs in the absence of additives.

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Fig. 8. Structures of the seven basic model drugs. According to their pKa values (in water), the compounds were distributed into 3 sub-groups: low-range basic (pKa < 6; compounds I and V), middle-range basic (pKa between 6 and 8; compounds II, III and IV) and higher-range basic (pKa > 8, compounds VI and VII).

The 2-EP stationary phase is regarded as a reference stationary phase for the analysis of basic compounds in the absence of mobile phase additives [31]. However, it has recently been demonstrated that not all commercially available 2-EP phases are equivalent [51]. In this study, only one prototype 2-EP phase (SPP) was tested. As shown in Fig. 9G, all the basic drugs eluted from this column with suitable retention, but significant tailing was observed for midazolam, nortriptyline and duloxetine. The 4-EP exhibited relatively the same peak shape behavior (Fig. 9H), but with higher retention under similar conditions. This indicates that the SunShell 2-EP and 4-EP SPP phases are also poorly suited for SFC analysis of strongly basic substances without additives.

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3.4.2. In the presence of mobile phase additives Utilizing additives in the SFC mobile phase tends to improve both peak shapes and separations of many problematic compounds [1,5,52–55]. In the last few years, it has been demonstrated that the addition of ammonium hydroxide was beneficial for

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In contrast to the 2-EP and 4-EP, the performance of SunShell Diol in the absence of additives was found to be superior to the other SPP phases. Indeed, the seven basic drugs were all eluted from the column with reasonable peak shapes. The two strongly basic compounds display symmetrical bands, but with correspondingly broader peaks.

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Fig. 10. Chromatograms obtained for the separation of the seven basic drugs mixture in gradient mode using 10 mM ammonium formate as mobile phase additive. (Column designations colors are identical to those described in Fig. 7).

improving the peak shapes of basic drugs, by disrupting the interactions between silanols and basic solutes under SFC conditions [6,51,56]. Therefore, the use of a relatively strong base will likely modify the surface chemistry of the silica, which usually results in improved peak shapes. Taylor et al. have demonstrated that the use of ammonium formate additives was also a good way to improve peak shapes in SFC and could potentially be a better solution than ammonium hydroxide [57]. Indeed, formate ions are able to undergo ion-pairing with basic analytes, while ammonium ions tend to mask the surface silanols. It was demonstrated that when using ammonium acetate, a higher ionic strength was generally beneficial for peak shape of compounds that exhibited late elution [53]. This is logical since the ion pairing behavior is directly related to the nature of the salt (pKa ) but also to its ionic strength. However, the MS sensitivity is reduced at higher concentrations. Because we typically use MS detection, we decided to add a concentration of 10 mM ammonium formate to the organic (methanol) part of the mobile phase. This evaluation was repeated on the eight SPP columns, and the corresponding chromatograms are presented in Fig. 10. All basic substances were now eluted from the SPP columns during SFC gradients (Fig. 10) regardless of phase chemistry (e.g. bare silica, Diol, 2-EP and 4-EP). In addition, the peaks were all symmetrical, except with the 2-EP, where tailing was observed for peaks III, VI and VII (Fig. 10G) and for peaks VI and VII on the 4-EP (Fig. 10H). On the SunShell Diol, the two peaks corresponding to strongly basic compounds were narrower compared to other stationary phases and their peak widths were then comparable to those of the 5 other basic substances. These chromatograms exemplify the benefits of adding 10 mM ammonium formate to the SFC mobile phase for the analysis of basic substances. For this mixture of basic drugs, the elution order and overall selectivity remained nearly identical on the bare silica columns (Fig. 10A–E), with only the Ascentis Express HILIC demonstrating less overall retention than the other SPP phases. On the SunShell Diol and 4-EP, the

selectivity was slightly different compared to bare silica phases, with an elution order reversal seen only for peaks III and IV (midazolam and papaverine). Lastly, the SunShell 2-EP phase demonstrated the largest difference in terms of selectivity and retention, where the elution of the seven basic drugs was compressed and elution order of peaks II and III (noscapine and midazolam) was completely reversed. 4. Conclusion The chromatographic potential of various SPP columns packed with 1.6, 2.6, and 2.7 ␮m material was evaluated exclusively under SFC conditions. Only polar stationary phases were tested, including five bare-silica, one diol and 2 ethylpyridine (2-EP and 4-EP). Similar to that observed in liquid chromatography, 2.6–2.7 ␮m SPP phases provide excellent kinetic performance in SFC at a lower pressure than columns packed with sub-2 ␮m particles. From a kinetic point of view, van Deemter curves and kinetic plot representations were constructed for all tested phases using two model analytes, butylparaben and prednisolone. First of all, the observed kinetic performance varied significantly among several columns, particularly for the Kinetex HILIC (but less so for the SunShell Silica and CortecsTM HILIC columns) simply by changing the nature of the test compound. This behavior was explained by notable differences in the strengths of possible interactions between solutes and the stationary phase while considering structural differences between butylparaben and prednisolone. In terms of kinetic performance, the lowest hmin values were consistently observed with the SunShell 2-EP, Poroshell HILIC and Ascentis Express HILIC. Corresponding hmin values ranged from 1.9–2.2 and 2.1–2.5 for butylparaben and prednisolone, respectively. When factoring in the plate count and permeability of the support (kinetic plot representation), the CortecsTM HILIC 1.6 ␮m and Ascentis Express HILIC 2.7 ␮m outperformed the other phases for N below 30,000, while the SunShell 2-EP 2.6 ␮m was superior above 60,000

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plates, with a Pmax of 400 bar. However, by extending the instrumentation pressure threshold from 400 to 600 bar, it is clear that the CortecsTM HILIC 1.6 ␮m column will provide a greater plate count than the other SPP phases tested. Finally, the great interest of core–shell particles in SFC was confirmed when comparing their potential and limitations versus fully porous particles (1.7, 3.5 and 5 ␮m). The 2.7 ␮m SPP material appears ideally suitable in the range of 20,000 < N < 120,000. For low plate numbers (e.g. N = 10,000), the 1.6 ␮m CortecsTM HILIC offers moderately faster (approximately 10%) analysis, while for very high resolution separations (N > 120,000), the fully porous 3.5 ␮m packing offers somewhat faster analysis due to increased permeability. To evaluate the behavior of these SPP phases with a more representative set of pharmaceutical compounds, 24 small drug-like compounds were analyzed under generic gradient SFC conditions on the eight different SPP materials. For the 17-component mixture of neutral and acidic drugs, the achieved peak capacities were in agreement with the observations made on van Deemter curves. Based on this study, it is recommended to employ the Poroshell HILIC, SunShell Diol and SunShell 2-EP phases for an initial screening purpose under SFC conditions, as they are the most orthogonal and offer highest peak capacity. However, when analyzing basic drugs (particularly with pKa > 8), addition of 10 mM ammonium formate as a mobile phase additive appears necessary to elute compounds from the columns and maintain sharp peaks, excluding the Diol, 2-EP and 4-EP phases. The results obtained on the Diol stationary phase support the great interest for this stationary phase chemistry emphasized by De la Puente et al. [31,58]. In conclusion, SPP materials look extremely promising for SFC applications despite the limited diversity in available polar chemistries and have the potential to further expand the use of SFC. Acknowledgments The authors wish to thank Toshiyuki Ono (Nacalai USA) and Norikazu Nagae (ChromaNik Technologies, Inc.) for the synthesis and manufacture of the SunShell columns used in this study. Furthermore, the authors wish to thank Terry Berger for his discussion on his work with superficially porous particles. References [1] C. Brunelli, Y. Zhao, M.-H. Brown, P. Sandra, Development of a supercritical fluid chromatography high-resolution separation method suitable for pharmaceuticals using cyanopropyl silica, J. Chromatogr. A 1185 (2008) 263–272. [2] C.M. Harris, The SFC comeback. Pharmaceuticals give supercritical fluid chromatography a fighting chance, Anal. Chem. 74 (2002) 87A–91A. [3] T.A. Berger, Packed Column SFC, The Royal Society of Chemistry, Cambridge, 1995. [4] A. Grand-Guillaume Perrenoud, J.-L. Veuthey, D. Guillarme, Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performace liquid chromatography for the analysis of pharmaceutical compounds, J. Chromatogr. A 1266 (2012) 158–167. [5] C. Brunelli, Y. Zhao, M.-H. Brown, P. Sandra, Pharmaceutical analysis by supercritical fluid chromatography: Optimization of the mobile phase composition on a 2-ethylpyridine column, J. Sep. Sci. 31 (2008) 1299–1306. [6] M. Ventura, B. Murphy, W. Goetzinger, Ammonia as a preferred additive in chiral and achiral applications of supercritical fluid chromatography for small, drug-like molecules, J. Chromatogr. A 1220 (2012) 147–155. [7] A. Periat, A. Grand-Guillaume Perrenoud, D. Guillarme, Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and MS compatibility, J. Sep. Sci. 36 (2013) 3141–3151. [8] T.A. Berger, B.K. Berger, Separation of Natural Food Pigments in Saponified and Un-Saponified Paprika Oleoresin by Ultra High Performance Supercritical Fluid Chromatography (UHPSFC), Chromatographia 76 (2013) 591–601. [9] L.T. Taylor, Separation of ionic analytes via supercritical fluid chromatography, LC-GC North Am. (2009) 62–68. [10] M.A. Patel, F. Riley, M. Ashraf-Khorassani, L.T. Taylor, Supercritical Fluid Chromatograpy resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation, J. Chromatogr. A 1233 (2012) 85–90.

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Evaluation of stationary phases packed with superficially porous particles for the analysis of pharmaceutical compounds using supercritical fluid chromatography.

Superficially porous particles (SPP), or core shell particles, which consist of a non-porous silica core surrounded by a thin shell of porous silica, ...
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