Extension of the carotenoid test to superficially porous C18 bonded phases, aromatic ligand types and new classical C18 bonded phases

Journal of Chromatography A, 1266 (2012) 34–42

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Extension of the carotenoid test to superficially porous C18 bonded phases, aromatic ligand types and new classical C18 bonded phases夽 E. Lesellier ∗ Institut de Chimie Organique et Analytique (ICOA), Université d’Orléans, CNRS UMR 6005, B.P. 6759, rue de Chartres, 45067 Orléans cedex 2, France

a r t i c l e

i n f o

Article history: Received 11 July 2012 Received in revised form 6 September 2012 Accepted 10 September 2012 Available online 12 October 2012 Keywords: Carotenoid test Column classification Superficially porous particles SFC Aromatic ligands New silica technology

a b s t r a c t The recent introduction of new stationary phases for liquid chromatography based on superficially porous particles, called core–shell or fused-core, dramatically improved the separation performances through very high efficiency, due mainly to reduced eddy diffusion. However, few studies have evaluated the retention and selectivity of C18 phases based on such particles, despite some retention order change reported in literature between some of these phases. The carotenoid test has been developed a few years ago in the goal to compare the chromatographic properties of C18 bonded phases. Based on the analysis of carotenoid pigments by using Supercritical Fluid Chromatography (SFC), it allows, with a single analysis, to measure three main properties of reversed phase chromatography stationary phases: hydrophobicity, polar surface activity and shape selectivity. Previous studies showed the effect of the endcapping treatment, the bonding density, the pore size, and the type of bonding (monomeric vs. polymeric) on these studied properties, and described the classification map used for a direct column comparison. It was applied to ten ODS superficially porous stationary phases, showing varied chromatographic behaviors amongst these phases. As expected, due to the lower specific surface area, these superficially porous phases are less hydrophobic than the fully porous one. In regards of the polar surface activity (residual silanols) and to the shape selectivity, some of these superficially porous phases display close chromatographic properties (Poroshell 120, Halo C18, Ascentis Express, Accucore C18, Nucleoshell C18 on one side and Aeris Wide pore, Aeris peptide and Kinetex XDB on the other side), whereas others, Kinetex C18 and Halo peptide ES C18 display more specific ones. Besides, they can be compared to classical fully porous phases, in the goal to improve method transfer from fully to superficially porous particles. By the way, the paper also report the extension of the test to other ligands such as naphtyl, cholester, phenyl-hexyl, or to the new ODS bonded phases, such as charge surface hybrid phases, High Strength Silica, and Hybrid ones, and also presents results for identical brands using different particle size, such as Luna and Synergi phases. Phenyl-hexyl and napthyl ligands show rather close properties, low hydrophobicity, high polar surface activity and specific shape selectivity, whereas, at the opposite, the cholester phase display a polymeric behavior and a high hydrophobicity. Finally, additional classical (fully porous particles) C18 bonded phases are also reported to complete the data set presented in previous papers. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The technology of silica particles has strongly been enhanced for the last 30 years, mainly to ensure higher resistance of the particles to pressure or extreme pH values. The analysis of basic compounds is one of the great challenges encountered in HPLC, explaining most particle development. First, type A silica was used, but severe peak tailing was reported for basic compounds with this particle type, because of the presence of isolated acidic silanol groups. The

夽 Presented at the 38th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Anaheim, CA, USA, 16–21 June 2012. ∗ Tel.: +33 1 69336131; fax: +33 1 69336048. E-mail address: [email protected] 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.09.068

decrease of the metal content of silica, in association with dehydration of silica followed by re-hydroxylation allowed to decrease the acidity of silanol groups onto the silica surface, leading to a type B silica better suited to the analysis of basic compounds [1,2]. Finally, type C silica was developed on the basis of silicone hydride groups instead of silanol ones [3,4]. A huge variety of bonding technologies was also applied in the goal to limit interactions between compounds and residual silanols, such as vertical and horizontal polymerization [5–9], polymer coating [10–14], the use of lateral bulky chains (stable bond phases) [15–17] or propylene bridge for bidentate phases [18,19]. Some years ago, the use of hybrid silica including an organic part into silica, or multilayer organic–inorganic was also supposed to reduce the amount of residual silanol groups and favor pH resistance [20–23]. The introduction of polar embedded phases, by using

E. Lesellier / J. Chromatogr. A 1266 (2012) 34–42

amide, carbamate, urea, sulfonamide or ether part into the alkyl bonded chain, was also performed to overcome the silanophilic interactions [24–28]. The polar group inserted in the bonded chain interacts with water molecules of the mobile phase to make an immobilized layer of water above the silica surface [27]. This water film should repulse the organic compounds reducing interactions of the compounds with residual silanols. Recently, ODS bonded phase called charge surface hybrid (CSH) was introduced to improve peak symmetry in low ionic strength mobile phase due to a first coating with charged compounds [29]. Concerning silica design, three major innovations were done in regard of classical fully porous particles of 5 or 3 microns generally used: monolith, sub-2 microns and superficially porous particles. The goal was to improve the chromatographic efficiency and reduce the analysis duration, although different ways were used to achieve it. The high permeability of monolith enables high flow rates and short analysis time [30], whereas the use of sub2 microns particles with short column length using high pressure drop in ultra-high-pressure liquid chromatography leads to significant chromatographic improvements [31]. Satisfactory separation enhancements were also achieved with lower pressure drop by the use of superficially porous particles, also called core–shell or fused-core particles [32–36]. In parallel, during these last 30 years, varied chromatographic tests have been developed to study ODS phase properties [37–70]. Most of these tests were performed in high performance liquid chromatography (HPLC), using different probes and analytical conditions: organic solvent nature (methanol or acetonitrile), pH value, temperature [71]. Some of these tests display results as simple two dimensional maps allowing a direct comparison of the bonded phases [55–59], others require the use of a calculated comparative factor due to the greater number of parameters studied [60–70], others require chemometric data treatment to define clusters of phases having close properties [38–41,47–55]. Among these tests, two are largely applied on numerous brands and type of ligands, from C8 to pentafluorophenyl ones: the SRM 870 [60] and the Hydrophobic Substraction Model [61–70]. Another test was developed by using supercritical fluid chromatography (SFC) and carotenoid pigments [72,78]. With one single analysis, it assesses the polar surface activity, through the all trans-beta-carotene/zeaxanthin separation factor, the shape selectivity, through the 13-cis/all trans-beta-carotene separation factor, and the phase hydrophobicity through the retention factor of all trans-beta-carotene. The difficulty of the separation of carotene isomers, the use of varied bonded stationary phases (C30 and other) in HPLC to achieve this type of separation [79–82], and the relationships between this separation, called shape selectivity, and the TBN/BaP test was addressed by several papers [75,81–83]. The major difference between the HPLC and SFC tests is obviously the mobile phase composition, with or without water. The absence of water in SFC induces that silanol groups cannot be covered by water and are probably more available for interactions with polar groups of the probe used, i.e. zeaxanthin. Consequently, for bonded phases that are fully endcapped, zeaxanthin is eluted very fast of the column and the separation factor between all trans-beta-carotene and zeaxanthin is high, whereas for phases with residual silanol groups, the retention of zeaxanthin is larger, thereby reducing the studied separation factor. In some cases, mainly for the polar embedded phases, zeaxanthin may even be more retained than all trans-beta-carotene [75]. In previous studies, we observed that the effect of the endcapping treatment was related to the decrease in the polar surface activity. Besides, vertical polymeric stationary phases showed a higher shape selectivity than horizontally polymerized or monomeric ones [6,8,9,75,81,82].

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In this paper, 10 ODS phases based on fused-core particles are studied by using the carotenoid test. Extension of the test to aromatic phases (naphtyl, phenylhexyl) and a cholester phase is also studied, and the location of recent C18 phases on the classification map is reported. 2. Materials and methods Chromatographic conditions are reported elsewhere [72–78]. A mixture of beta carotene isomers, produced by the addition of iodine, and zeaxanthin dissolved in MeOH/CH2 Cl2 is injected (5 ␮l) on each column, with a mobile phase composes of CO2/MeOH 85/15 (v/v), a temperature of 25 ◦ C, a back pressure equal to 15 MPa, a flow rate of 3 ml/min, and a UV detection at 440 nm. The columns tested are listed in Table 1. The ten superficially porous particles were: Kinetex C18 (150 × 4.6; 2.6 ␮m) (Phenomenex) (#168), Kinetex XB X18 (150 × 4.6, 2.6 ␮m) (#201) (Phenomenex), Halo C18 (150 × 4.6; 2.7 ␮m) (#162) (AMT), Halo Peptide ES (150 × 4.6; 2.7 ␮m) (#204) (AMT), Nucleoshell C18 (150 × 4.6; 2.7 ␮m) (#205) (MachereyNagel), Poroshell 120 EC C18 (150 × 4.6; 2.7 ␮m) (#199) (Agilent), Accucore C18 (150 × 4.6; 2.6 ␮m) (#210) (Thermo Fisher Scientific), Aeris Peptide (150 × 4.6; 3.6 ␮m) (#206) (Phenomenex); Aeris WP (150 × 4.6; 3.5 ␮m) (#207) (Phenomenex), Ascentis Express C18 (150 × 4.6; 2.6 ␮m) (#161) (Supelco/Sigma–Aldrich). The aromatic and cholesteric ones were: Cosmosil Cholester (250 × 4.6; 5 ␮m) (#158) (Nacalai), Cosmosil pi-Naphtyl (250 × 4.6; 5 ␮m) (#180) (Nacalai), Nucleosil Sphinx (250 × 4.6; 5 ␮m) (#158) (MachereyNagel), Luna phenyl-hexyl (250 × 4.6; 5 ␮m) (#159) (Phenomenex), Zorbax Phenyl-hexyl (250 × 4.6; 5 ␮m) (#165) (Agilent), Gemini Phenyl-hexyl (250 × 4.6; 5 ␮m) (#166) (Phenomenex), XSelect CSH Phenyl-hexyl (250 × 4.6; 5 ␮m) (#203) (Waters). Other regular C18 phases were described in Table 1. 3. Results and discussions 3.1. Superficially porous particles Among numerous studied stationary phases, Fig. 1 shows the location of the ten superficially porous particles ODS phases. Five phases display close chromatographic behavior, Accucore, Ascentis Express, Halo C18, Nucleoshell C18 and Poroshell 120 C18 ES. All these phases are monomeric with a low polar surface activity. Hydrophobicity increases following this order Accucore < Nucleoshell < Ascentis express < Halo C18 < Poroshell. The high hydrophobicity of Poroshell phase was also reported in HPLC with ACN/water mobile phase [84]. Whatever the phase, hydrophobicity is rather lower than the one of most of the fully porous particles, because the solid core reduces the pore volume in regards of the one of the fully porous particles. Obviously, the specific surface area and the total carbon loading of superficially porous particle is reduced, explaining the lower hydrophobicity Besides, the bonding density, expressed in ␮mole/m2 , does not depends on the particle type (fully or superficially porous). One phase displays a particular chromatographic behavior compared to the five previous ones: the Kinetex C18. The lower shape selectivity indicates a reduced bonding density, and despite an endcapping treatment, the polar surface activity is higher than the one of the five previous phases. Hydrophobicity of Kinetex C18 is also strongly reduced. Several recent studies carried out in HPLC show different selectivities between Kinetex C18 and Ascentis Express, Poroshell 120 C18 EC, Halo C18 on amine containing agricultural references [84] and pharmaceutical compounds [85,86], with reversal of elution orders between codeine and acebutol [85] or degradation products of ethinyl-estradiol [86].

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E. Lesellier / J. Chromatogr. A 1266 (2012) 34–42

Table 1 Column names and properties. Columns

Manufacturer

No

Group

SSA m2 /g

CC %

Pore Ø Å

Specificity

Acclaim C18 Accucore C18 Ace 5 C18 Ace 5 C18 HL Ace 5 C18 AR Adsorbosil Adsorbosphere HS Adsorbosphere XL AERIS Peptide AERIS Wide Pore Alltima C18 Alltima HP C18 Alltima HP C18 HL Alphabond Apex C18 Ascentis C18 Ascentis Express C18 Atlantis dC18 Atlantis T3 Baker C18 NP Baker C18 WP Betabasic Bondasorb Brava BDS C18 C18 micro-bondapak Capcell pak C18 Capcell pak UG 80 Capcell pak C18 MGII Chromegabond C22 Chromolith C18 Clipeus C18 Cogent C18 Colosphere C18 Cosmosil C18 AR II Cosmosil C18 MS II Cosmosil p-Naphtyl Cosmosil Cholester Delta-Pak C18 Denali Develosil C18 Discovery C18 Discovery HS C18 Econosil Econosphere Equisil BDS Equisil Gold C18 Exelsphere 120 C 18 H Exelsphere ODS 2 120 Exsil ODS Gammabond C18 Gemini C18 Gemini NX C18 Gemini Phenylhexyl Genesis C18 Grace Smart RP 18 HAIsil C18 HAIsil HL C18 HALO C18 120 HALO Peptide ES C18 HSS C18 HSS SB HSS T3 Hydrosphere C18 Hypersil 100 C18 Hypersil BDS Hypersil Elite Hypersil Gold Hypersil Green-PAH Hypersil HyPurity Hypersil ODS Hypersil PAH Inertsil ODS 2 Inertsil ODS 3 Inertsil ODS4 J’Sphere 80H J’Sphere 80L

Thermo Thermo ACT ACT ACT Discovery Discovery Discovery Phenomenex Phenomenex Discovery Discovery Discovery Discovery Jones Supelco Supelco Waters Waters Baker Baker Thermo SFCC Discovery Waters Shiseido Shiseido Shiseido ES industries VWR HIGGINS ACT Colochrom Nacalai Nacalai Nacalai Nacalai Waters Discovery Develosil Supelco Supelco Discovery Discovery Dr Maisch Dr Maisch Colochrom Colochrom SGE ES industries Phenomenex Phenomenex Phenomenex Jones Discovery Higgins Higgins Mac-Mod Mac-Mod Waters Waters Waters YMC Thermo Thermo Thermo Thermo Thermo Thermo Thermo Thermo GL Science GL Science GL Science YMC YMC

115 210 128 152 197 28 55 82 206 207 85 124 123 12 46 134 161 120 153 110 105 113 25 78 13 58 186 187 30 79 47 129 67 122 121 180 158 53 164 45 91 126 29 9 176 177 21 59 75 5 127 156 166 54 139 41 98 162 204 189 190 191 4 49 90 96 125 35 92 48 32 95 43 167 143 141

11 11 11 11 10 8 9 10 1 1 11 11 11 3 9 11 11 1 1 7 6 11 8 10 3 9 9 9 8 10 9 10 10 7 9 1 6 9 11 9 11 11 8 3 9 8 4 9 10 1 9 9 1 9 3 9 11 11 1 11 6 1 1 9 11 11 9 5 11 9 5 11 9 9 11 1

300 130 300 400 300 450 350 200

18 9 15.5 20 15.5 15 21 11

120 80 100

Superficially porous

310 200 450 300 170 450 150 330 300 170

16.2 12 24 10 10 25

100 60 60 90 100 200 100 190 100 125 100 100 90 100 100 120 300 150

200

12 14 17.2 7.3 13

185 330 300 340 260 350 300 350 350

8.5 10 15 18 15 22 17 18 16

145 125 120 80 100

300 300 300 300 300 320 350 200 300 450 200 180 180 300 300 220

17 16 11 20 17 20 20 12.5 20 15 10 11

120 120 120 120 100 120 100 180 120 60 80 120 120

375 375 390 300 220 190 300 150 80 230 230 230 340 300 170 250 220 170 200 170 170 320 450 450 500 500

14 14 12 18 8 12 18 8

15 17 12

15 8 11 12 16 11.1 15 10 13.5 13 9.5 13.5 18 15 11 22 9

Phenyl embedded Monofunctional Monofunctional Superficially porous Superficially porous

Monofunctional

Difunctional Trifunctional

Y Y Y Y Y

D Y Y Y Y

Y Y

Y

Monofunctional Coated polymer

Monofunctional 130 120 100

Endcapping

Y Y

N Y

Monofunctional Type C silica Polyfunctional Naphtylethyl group Cholester group Coated polymer

Y Y

Y Y Y Y

80 110 105 120 120 100 100 90 160 100 100 100 120 100 130 115 175 120 190 120 150 100 100 80 80

coated polymer hybrid silica hybrid silica Phenyl-hexyl

Monofunctional Monofunctional Superficially porous Superficially porous Trifunctional ligand Trifunctional ligand Trifunctional ligand

Polyfunctional Monofunctional Polyfunctional Monofunctional

High bonding dens. Low bonding density

Y Y Y Y Y Y Y N Y N Y

Y Y Y Y Y Y Y Y Y Y Y Y

E. Lesellier / J. Chromatogr. A 1266 (2012) 34–42

37

Table 1 (Continued) Columns

Manufacturer

No

Group

SSA m2 /g

CC %

Pore Ø Å

Specificity

Endcapping

J’Sphere 80 M Jupiter C18 300 Kinetex C18 Kinetex XB-C18 Kromasil C18 Lichrosorb RP 18 Lichrospher 100 RP 18 Lichrospher 100 RP 18e Lichrospher LC-PAH Luna C18(2) 5 mm Luna C18(2) 2.5 mm Luna Phenylhexyl Normasphere ODS 2 Nova-Pak C18 Nucleodur 100 C18 ec Nucleodur Gravity C18 Hypersil HyPurity Nucleodur ISIS C18 5 mm Nucleodur ISIS C18 1.9 mm Nucleodur Sphinx Nucleodur C18 Htec Nucleoshell C18 Nucleosil 100 C18 Nucleosil 100 C18 HD Nucleosil 100 C18 PAH Nucleosil 300 C18 Nucleosil 5 C18 AB Nucleosil 50 C18 Nucleosil 50 C18 ec Omnisphere Partisil ODS 1 Partisil ODS 2 Partisil ODS 3 Pathfinder EP PE CR C18 Platinum C18 Poroshell 120 EC-C18 Prodigy ODS2 Prodigy ODS3 Prosphere C18 300 Protecol HQ 105 C18 Protecol C18 GP125 Protecol C18 PC105 Protecol HPH125 Purospher 100 RP 18 Purospher 100 RP 18e Purospher star RP18e Pursuit C18 Pursuit XRs C18 Reprosil 100 C18 Reprosil pur ODS3 Reprosil 80 ODS2 Reprosil pur basic C18 Reprosil pur basic C18HD Reprosil saphir Reprospher C18 DE RES-ELUT 5C18 Resolve C18 Restek Allure C18 Restek Ultra C18 Satisfaction RP 18 AB Separon C18 Separon C18 ec SGE-250 GL4 P-C18 SMT C18 Spheri-5 ODS Spherisorb ODS 1 Spherisorb ODS 2 Spherisorb ODSB Stability ODS 2 Strategy C18-2 Strategy C18-2 2.2 mm Strategy C18-3 Sunfire C18 Supelcosil LC-18 Supelcosil LC-18 DB Supelcosil LC-18S

YMC Phenomenex Phenomenex Phenomenex EKA Nobel VWR VWR VWR VWR Phenomenex Phenomenex Phenomenex Colochrom Waters Macherey Macherey

142 157 168 201 100 10 74 88 34 52 196 159 70 84 117 118

1 7 8 1 11 3 10 11 5 9 9 1 10 10 10 11

500 170 200 200 350 300 350 350 200 440 400 400 450 120 340 340

14 13.3 12

80 300 100 100 100 100 100 100 150 100 100 100

Medium bond. dens. Polyfunctional Superficially porous Superficially porous Monofunctional

Y

Macherey Macherey Macherey Macherey Macherey Macherey Macherey Macherey Macherey Macherey Macherey Macherey Agilent Whatmann Whatmann Whatmann Shimadzu Perkin Discovery Agilent Phenomenex Phenomenex Discovery SGE SGE SGE SGE VWR VWR VWR Agilent Agilent Dr Maisch Dr Maisch Dr Maisch Dr Maisch Dr Maisch Dr Maisch Dr Maisch Varian Waters Restek Restek Cluzeau Tessek Tessek SGE SMT Brownlee Waters Waters Waters Cluzeau Interchim Interchim Interchim Waters Supelco Supelco Supelco

131 194 140 198 205 37 97 33 83 103 69 73 102 6 31 8 130 40 24 199 136 137 106 182 183 184 185 72 86 114 119 135 169 170 171 172 173 174 175 11 39 61 99 62 26 38 2 68 80 36 76 66 81 132 194 133 154 44 56 50

7 7 1 9 11 8 11 5 10 7 10 10 11 2 5 3 4 9 8 9 9 9 6 4 9 9 11 10 11 11 9 9 9 9 10 4 11 11 9 3 8 9 11 9 8 8 1 10 10 5 10 10 10 11 11 9 9 9 9 9

340 340 340

20 20 15 18 7.5 14 20

130 350 350 350 100 350 450 450 350 350 350 350 300 200 120 310 450 100

21.4 18 18 21 20 19 17.5 17.5 21 7 17.5 18

6.5 25 14 14.5 20 4.7 17.3 10.7

6 18.5 15.5 9

Y Y N N Y N Y

Phenyl-hexyl

Monofunctional Monofunctional

Y Y Y Y

110 110 110 110 90 100 100 100 300 100 50 50 110 85 85 85 100

Cross linked

Y

C18/C3-phenyl

Y Y Y Y Y N Y Y N Y Y Y N Y

100 120 150 100 300

Monofunctional

80 110 110

Superficially porous Monofunctional Monofunctional Polyfunctional Cross linked Monofunctional Monofunctional

Coated polymer

Monofunctional Monofunctional Polyfunctional

Y Y Y Y Y

120

350 350 350 180 440 280 300 220 450 450

18 21 18 20 25 15 17 12 17 25

120 120 120 120 200 100 100 120 80 100 100

200

10 27 20 17

90 90 60 100 120

320

Monofunctional N Y Y

Y Y Y Y Y

N N Monofunctional

Coated polymer 340 180 220 220 220 320 425 425 425 340 170 170 170

24 14 7 12 12 15 19 19 21 16 11 11 11

Y N Y

100 80 80 80 100 100 100 100 100 120 120 120

Monofunctional Monofunctional Monofunctional

N Y Y Y N Y Y Y Y Y

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E. Lesellier / J. Chromatogr. A 1266 (2012) 34–42

Table 1 (Continued) Columns

Manufacturer

No

Group

SSA m2 /g

CC %

Pore Ø Å

Supelcosil LC-18 T Superspher 100 RP 18 Superspher 100 RP 18e Symmetry C18 Synchropak C18 Synergi Fusion Synergi Fusion 2.5 mm Synergi Hydro Synergi Hydro 2.5 ␮m Synergi Max RP Synergi Max RP 2.5 ␮m Targa C18 TSKgel ODS 80TS TSKgel ODS 100V TSKgel ODS 100Z TSKgel ODS 120A TSKgel ODS 120 T TSKgel ODS 80TM Ultrasphere ODS Ultrasphere XL ODS Unisphere C18 Uptisphere HDO Uptisphere HSC Uptisphere NEC Uptisphere ODB Uptisphere TF Vision C18 Classic Vision C18 HL Vydac 201 HS Vydac 201 TP 300 Vydac 202 TP 300 Vydac 218 MR 300 Vydac 218 TP 300 Vydac 238 TP 300 Wakosil II C18 AR Wakosil II C18 HG Wakosil II C18 RS X Bridge C18 Xselect CHS C18 Xselect CHS PheHex XTerra MS C18 YMC Pack ODS A 120 YMC Pack ODS A 200 YMC Pack ODS A 300 YMC Pack ODS-AQ YMC Pack Pro C18 RS YMC Pack Pro C18 YMC Triart C18 Zorbax 300 SB C18 Zorbax Eclipse Plus Zorbas Eclipse Plus PAH Zorbax Eclipse XDB Zorbax Extend Zorbax E + Phenylhexyl Zorbax RX-C18 Zorbax SB C18

Supelco VWR VWR Waters Eichrom Phenomenex Phenomenex Phenomenex Phenomenex Phenomenex Phenomenex Higgins Tosoh Tosoh Tosoh Tosoh Tosoh Tosoh Beckmann Beckmann Biotage Interchim Interchim Interchim Interchim Interchim Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery SGE SGE SGE Waters Waters Waters Waters YMC YMC YMC YMC YMC YMC YMC Agilent Agilent Agilent Agilent Agilent Agilent Agilent Agilent

93 71 94 87 16 179 193 178 192 160 191 18 111 148 149 112 77 15 60 65 1 20 64 27 51 116 208 209 23 109 104 108 107 14 150 151 22 155 202 203 42 144 145 146 19 147 57 200 3 138 163 63 101 165 89 17

11 10 11 11 4 1 1 8 9 4 4 4 9 1 9 6 10 8 9 9 1 4 9 8 9 7 8 9 8 6 6 6 6 3 7 9 4 9 9 1 9 9 9 11 4 11 9 9 1 9 10 9 11 1 11 4

170 350 350 330 300 475 400 475 400 475 400 330

12.3 18 22 19.4

120 100 100 100

12 12 17 17 19 19 16 15 15 20 22 22 15 12 12

80 100 80 100 80 100 120 80 100 100 120 120 80 80

Four other phases, Aeris Peptide, Aeris WP, Halo Peptide ES-C18 and Kinetex XB C18 display very different shape selectivity, showed by the retention inversion of the beta-carotene isomers. These phases are rather devoted to the analysis of peptides and large biomolecules [87,88]. Among the phases having a close chromatographic behavior to these, are C18 phases with very low bonding density (J’Sphere L80), or C18 phases with multiple anchors onto silica (Atlantis phases), or C18 phases with bulky side chains (Zorbax SB). The two Aeris phases and the Kinetex XB C18 display the same bonding ligand, including isobutyl side chains, such as the Zorbax SB, which could induce a lower bonding density, explaining the identical shape selectivity of these phases. In regards of their close location in Fig. 1, Aeris Peptide and Kinetex XB C18 seem to differ only by the particle size, 3.6 and 2.6 ␮m. Of course, due to the greater pore size, and a probably smaller porous layer, the

450 450 200 200 250 50 320 310 320 320 310 200 220 450 90 90 90 90 90 300 300 350 185 185 185 175 330 175 100 300 510 340 45 160 160 180 185 160 180 180

18 20 16 17 6 11 13.5 8

8 20 15 17 18 15 14 15.5 17 12 7 14.6 22 17 20 2.8 8 14 10.3 12.1 9 12 10

Specificity

N Y Y

C12 C12 Monofunctional Monofunctional Difunctional Difunctional

Monofunctional

220 120

Monofunctional

120 120

Monofunctional Monofunctional

100 120 90 300 300 300 300 300 120 120 120 185 135 135 125 120 200 300 120 80 120 120 300 95 95 80 80 95 80 120

Endcapping

Y Y Y Y difunc Y monfunc N Y Y Y Y

Monofunctional Polyfunctional Polyfunctional Polyfunctional Monofunctional Trifunctional Monofunctional Monofunctional Hybrid silica Charged silica Phenyl-hexyl Hybrid silica

Hybrid silica DiBuC18

DiMeC18 Bidentate Pheny hexyl DiMeC18 DiBuC18

Y Y N Y Y Y Y

Y Y Y Y

Y Y Y Y Y Y Y Y N Y Y Y D Y N N

hydrophobicity of Aeris WP is strongly reduced, but this does not affect shape selectivity. In the goal to transfer a separative method from a classical stationary phase to a superficially porous one, one can compare the location of some phases provided by the same manufacturer. Poroshell 120 C18 ES is very close to Zorbax Eclipse XDB (#63) or Zorbax Eclipse plus (#138), Nucleoshell C18 is close to Nucleodur Gravity C18 (#118), while Accucore C18 is close to Hypersil BDS (#90) and Hypersil Elite (#96). 3.2. Aromatic phases The test was extended to various aromatic and cholesteryl ligands and: cholesteryl (#158); phenylhexyl (#159, #165, #166, #203); naphtylethyl (#180); and propylphenyl/C18 (#140) (Fig. 1).

E. Lesellier / J. Chromatogr. A 1266 (2012) 34–42

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Fig. 1. Classification diagram, polar surface activity (separation factor of all trans beta carotene and zeaxanthin) vs shape selectivity (separation factor of all trans and 13 mono-cis beta carotene) for fused core ODS phases (full red points), aromatic and cholesteric ligands (full yellow points). The point size is related to the phase hydrophobicity, i.e. the retention factor of all trans beta carotene. For column identification see Table 1 and Section 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

The cholester phase (Nacalai) is one of the more hydrophobic phases tested (large size of the yellow data point), and displays a high shape selectivity close to polymeric C18 stationary phases. This behavior is not related to a large surface coverage because of the high polar surface activity (low separation factor between all trans ␤-carotene and zeaxanthin) which indicates the presence of residual silanol groups on the silica surface. This phase type is well known to provide high separation of isomers for vitamins, antibiotics [89] or for steroid separation and even for enantiomers [90]. The four phenylhexyl phases (#159, 165, 166, 203) display similar chromatographic behavior; they are located in group 1, meaning a retention order inversion of the 13-cis and all trans ␤-carotene isomers, compared to most C18 phases. The reason of this inversion is unknown, but indicates that phases of group 1 are able to provide different separations of compounds on the basis of shape recognition. Their polar surface activity is high showing that these phases can also interact with polar parts of compounds to achieve separations. Because of the points proximity, Zorbax (#165) and Luna phenylhexyl ((#159) phases seem to be fully exchangeable. XSelect CSH phenyl hexyl (#203) displays a lower hydrophobicity, probably due to the hybrid silica of the particle, reducing the bonding sites. The naphtyl phase (Nacalai) (#180) has a close behavior,

with a more pronounced retention order inversion. This phase is near the C18 Unisphere phase, showing that different ligands can sometimes provide similar chromatographic behavior. Besides the Unisphere particle was alumina, and not silica. 3.3. Additional classical C18 phases (fully porous particles) The following discussion is focused on new C18 phases which were not presented in previous papers. The presentation of results is made by ranking the columns in eleven groups having different polar surface activity and shape selectivity. For groups 1, 2, 3, 4, 5, 6 and 7 the limits are established by previous studies based on well-known phases [24,49–54]. For groups 8–11, which include monomeric bonded phases, the limits are established as follow: the middle of the range of the measured values for shape selectivity, i.e. at 1.1 between 1 and 1.2, and at 5 for the alltrans-beta-carotene/zeaxanthin selectivity, because most of the non-endcapped phases display a value below 5 for this parameter, and because the effect of endcapping on this selectivity is well described. However, some phases included in groups 8, 9, 10 and 11 are very close to some phases from other groups. For instance, the no 15 (TSK ODS 80TM), 167 (Inertsil ODS4), 176 (Equisil BDS), 177 (Equisil Gold C18), and YMC Triart (#200) which are located

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either in the bottom of group 9 or in the top of group 8, would probably display the same selectivity for most separations due to their proximity. In group 1 (Fig. 2), one can find two J’Sphere 80 A˚ L (#141) and M (#142) whose surface coverage are rather lower than most of the other studied phases (1.0 and 1.6 m2 /g). Neue showed that a straight line could be drawn between the data of the three J’Sphere 80 L, M and H (#143) by plotting the silanol activity vs. hydrophobicity [55]. These packings were based on the same silica with an increased bonding density, explaining both the decrease in the silanol activity and the increase in selectivity. In a previous paper [78], it was also demonstrated that a straight line could be drawn for these three packing on the classification map obtained by plotting the polar surface activity vs. the shape selectivity. This behavior underlines the relevance of the studied parameters by the cartenoid test. Three Waters phases are also located in this group: Atlantis dC18 (#120), Atlantis T3 (#153) and HSS T3 (#188). The reactive silane was either a dichlorosilane or a trichlorosilane, inducing more than one link between the bonded chain and the silica surface. Those three phases display very close chromatographic behavior, the polar surface activity of the HSS phase being a little bit reduced in regards of the one on Atlantis silica. The two Synergi Fusion, 4 and 2.5 ␮m (#179 and #193) are also located in this group, as well as the TSKgel ODS 100 V(#148). In group 4, containing monomeric bonded phases, are located the Synergi Max RP 4 ␮m (#160), and 2.5 ␮m ((#191) with a C12 bonded chain, and the Protecol XQ105 (#182). As explained previously, the C12 chain (Synergi Max RP) is unable to separate the cis/trans ␤-carotene isomers due to the short chain length. The particle size reduction for Synergi Max RP ˚ and a speis related to a pore size increase (from 80 to 100 A) cific surface area reduction (from 475 to 400 m2 /g) leading to a reduced hydrophobicity and a lower polar surface activity from 4 to 2.5 ␮m. The Protecol phase displays a great hydrophobicity, and is very close to Uptisphere HDO (#20) and YMC Pack Pro AQ (#19). In group 6, containing polymeric stationary phases, one can find the Jupiter C18 (#157), the Isis 2.2 ␮m (#194) and HSS SB (#190). This recent phase is not end-capped and made with a trifunctional silane. This explains why it is located in group 6 including polymeric C18 phases, which present similar chromatographic properties. Among the phases produced by Waters, which are mainly located in group 9 and 11, this HSS SB is the first one having a polymeric chromatographic behavior, improving the range of choices from this manufacturer in terms of separation selectivity. The same remark can be done for the Phenomenex phases with the Jupiter C18 which displays a polymeric behavior. Isis 2.2 ␮m (#194) is very close to the Isis 5 ␮m (#131) showing that for this phase, the particle size reduction does not modify the chromatographic properties of the phase. In group 8, monomeric phases with low bonding density and high polar surface activity, are the Equisil Gold (#177), the Vision C18 Classic (#208) and the Synergi Hydro RP (#178). This last phase is comparable to the “famous” Resolve C18 (#39) in terms of polar surface activity, i.e. in terms of separation selectivity based on polar interactions. Vision C18 Classic is exactly located at the same place as the Platinum C18. We noticed that for Uptisphere NEC (nonendcapped) located in the same group (#27), the peak symmetry of basic compounds was satisfactory, despite the rather strong polar surface activity (unpublished results). One should be aware that the varied silanol groups (germinal, vicinal, isolated) on silica surface do not all have same properties (acidity), and do not always induce peak tailing. Recently, Engelhardt et al. showed that the reduction of the amount of silanol groups on hybrid silica leads to a higher percentage of isolated acidic ones [66].

In group 9, having the same type of ligand and bonding density as group 8 with a low polar surface activity, are located Capcell Pak UG80 (#186), Capcell Pak C18 MGII (#187) Equisil BDS (#176), Inertsil ODS4 (#167), Protecol C18 GP125 (#183), Protecol C18 PC105 (#184), ReproSil-Pur ODS3 (#170), Reprospher C18 DE (#175), ReproSil 100 C18 (#169), Sunfire C18 (#154), TSKgel ODS 100Z (#149), YMC ODSA 120 (#144), YMC ODSA 200 (#145), Luna C18 (2) 2.5 ␮m (#196), Wakosil II C18 HG (#151), XBridge C18 (#155), Gemini NX (#156), HSS C18 (#189), Strategy-2 (2.2 ␮), Nucleodur C18 HTec (#198), YMC Triart C18 (#200), XSelect CHS C18 ((#202) and Vision C18 HL (#209). The two new generations of hybrid silica (XBridge and Gemini NX) are located in the same group: they display low hydrophobicity due to this hybrid character which reduces the surface silanol groups, i.e. the bonding density and the carbon content. YMC Triart C18 (#200) is also in this group, but hydrophobicity and polar surface activity is closest to the first hybrid generation (XTerra and Gemini). Inertsil ODS4 (#167) is close to Inertsil ODS3 (#43), with a lower hydrophobicity, due to its lower carbon content (11% against 15%), with an identical surface area (400 m2 /g). These two phases show satisfactory peak symmetry for dextrometorphan in ACN/phosphate buffer pH 7 [48]. We can remark that YMC Triart C18 and Inertsil ODS4 display identical chromatographic behavior, based on the carotenoid test. The two Capcell Pak are very close to the oldest Capcell (#58), with a higher hydrophobicity. The ReproSil Pur ODS3 (#170), which is supposed to be an alternative to YMC ODSA 120 (#144), displays the same shape selectivity, a slightly higher hydrophobicity and a slightly lower polar surface activity. One can remark that the Uptisphere ODB (#51), which is just located between these two phases, has the same pore size and specific surface area as the two previous ones. The two Luna C18 (2) 2.5 and 5 ␮m (#52) are close because the silica properties (pore size and surface area) are almost identical. The Nucleodur C18 HTec (#198) displays a classical behavior for a monomeric phase, and could be exchanged with Luna C18 (2) (#52). One can notice that their pore diameter and carbon content are similar. Another interesting fact is that this manufacturer did not have a column located in this group until this Nucleodur C18 HTec. It means that the variety of Macherey–Nagel phases is improved with this new one. The last XSelect CSH C18 (#202) is very close to XTerra MS C18 (#42), both being based on a hybrid silica. The new “ionic” coverage applied to XSelect does not seem to modify the properties studied through the carotenoid test. However, a recent study underlined some similarities of the XSelect CSH C18 and a polar embedded C18 phase [29]. In group 10, in which stationary phases display a high bonding density and a high polar surface activity, one finds few newly tested phases, Synergi Hydro RP 2.5 ␮m (#192), ReproSil 80 ODS2 (#171), Zorbax Eclipse Plus PAH (#163) and ACE C18 AR (#197). Due to the very high polar surface activity (inversion of the retention order between zeaxanthin and all trans-␤-carotene), the Zorbax Eclipse Plus PAH can be considered as a polymeric one, despite the rather low value of 13-cis/all trans beta-carotene selectivity. The intermediate position of this phase in regards of the two polymeric groups (groups 6 and 5) could be due to the small pore ˚ such as phases of group 5, and low carbon content (9%) size (95 A), close to the one of columns in group 6. The ReproSil 80 ODS2 (#171) is supposed (from the manufacturer) to be close to Spherisorb ODS2 (#76), and their chromatographic properties are almost identical. The shape selectivity and hydrophobicity of Synergi Hydro RP 2.5 ␮m are shifted in regards of the 4 ␮m phase for identical reasons that for Synergi Fusion, i.e. a change in the pore size and the surface area in regards of the particle size. The new ACE C18 AR (#197) is a mixed phase with phenyl groups and alkyl chains. It could be compared

E. Lesellier / J. Chromatogr. A 1266 (2012) 34–42

41

Fig. 2. Classification diagram for new studied regular C18 phases (colored full points). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

to the Sphinx Nucleodur RP (#140), which seems be to have similar chemistry. However, the location of the two phases is rather different, the Sphinx belonging to group 1 due to the inversion of the cis/trans beta carotene retention order, as most of the aromatic studied phases display. Group 11 contains phases displaying high shape selectivity and low polar surface activity. The new tested phases are ACE C18 HL (#152), J’Sphere 80H (#143), Protecol HPH125 (#185), ReproSil Pur Basic C18 HD (#173), ReproSil Saphir (#174), YMC ODSA 300 (#146), YMC Pack Pro C18 RS (#147), and HSS C18 (#189). Most of these phases have a high carbon content explaining their chromatographic properties. The increase in carbon content of ReproSil Pur Basic C18 HD in regards of ReproSil Pur Basic C18 (#172) in group 4, induces a very large shift in shape selectivity and polar surface activity. The pore size change for YMC ODSA 300 A˚ (#146) induces a shape selectivity increase, and, as expected due to the surface area diminution, a decrease in hydrophobicity, in regards of 120 and 200 A˚ YMC ODSA phases. The HSS C18 (#189) displays a very low polar surface activity whereas its hydrophobicity is rather low, in regards of the surrounding columns in this area: Kromasil (#100), HAIsil HL C18 (#98), Restek Ultra (#99), Nucleodur Gravity (#118), Alltima HP HL (#123), Discovery HS C18 (#126), ACE 5 C18 HL (#152) and Denali C18 120 (#164), which are columns with high carbon content (around 20%). This behavior can be explained by the smaller specific surface area of HSS (230 m2 /g)

and the smaller carbon content (15%), leading to a weaker retention, whereas the bonding density is close.

4. Conclusion This study shows different chromatographic properties for fused core particles in terms of hydrophobicity, polar surface activity and shape selectivity. It means that, despite their common high efficiency, the choice of these phases should take into account their chemical differences. As reported in the literature [85–88], these differences in interactions can lead to changes in elution order for varied couples of compounds. Phenylhexyl phases, often supposed to be complementary to C18 or C8 ones, display a high polar surface activity and a specific behavior in regards of the shape selectivity. They are very close to each other. The naphtyl phase is located in the same group as the phenylhexyl phases, and could also be used as an alternative in RP-LC. The effect of particle size variation (Synergi, Strategy, Luna C18(2)) has generally no effect on selectivity, except when the surface area also varies, as for the Strategy columns. Minor changes (less than 20%) due to particle size modification were reported elsewhere [91] for some brands. The change in the pore size (Aeris

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