Poly(4-vinylpyridine) based novel stationary phase investigated under supercritical fluid chromatography conditions

A novel poly(4-vinylpyridine) based stationary phase was investigated for its performance under supercritical fluid chromatography (SFC) mode. Due to its unique structure, this stationary phase has high molecular planarity recognition ability for aromatic samples possessing the same number of aromatic rings and -electrons.

Journal of Chromatography A, 1572 (2018) 119–127 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Poly(4-vinylpyridine) based novel stationary phase investigated under supercritical fluid chromatography conditions Kanji Nagai ∗ , Tohru Shibata, Satoshi Shinkura, Atsushi Ohnishi DAICEL Corporation, CPI Company, Life Science Development Center, Innovation Park, 1239, Shinzaike, Aboshi-ku, Himeji, Hyogo, 671-1283, Japan a r t i c l e i n f o Article history: Received 31 May 2018 Received in revised form August 2018 Accepted 16 August 2018 Available online 23 August 2018 Keywords: Supercritical fluid chromatography Stationary phase Ligand Selector Polymer Poly(4-vinylpyridine) a b s t r a c t A novel poly(4-vinylpyridine) based stationary phase was investigated for its performance under supercritical fluid chromatography (SFC) mode Due to its unique structure, this stationary phase has high molecular planarity recognition ability for aromatic samples possessing the same number of aromatic rings and ␲-electrons Taking advantage of the planarity recognition ability observed, separations of structurally similar polycyclic aromatic hydrocarbons and steroids were achieved This novel stationary phase afforded good peak symmetry for both acidic and basic active pharmaceutical ingredients even when excluding the use of additives such as acids, bases, and salts These findings may be attributed to the polymeric pyridyl groups covalently-attached on silica gel, which will effectively shield the undesirable interaction between residual silanol groups on the surface and the analytes Moreover, the properties of pyridyl group on the selector can be reversibly tuned to cationic pyridinium form by eluting trifluoroacetic acid containing modifier Column robustness toward cycle durability testing was also confirmed © 2018 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Introduction Supercritical fluid chromatography (SFC) is increasing inuse in the analytical and preparative separation field [1] SFC uses supercritical or subcritical mobile phases consisting of pressurized carbon dioxide (CO2 ), usually mixed with a miscible organic solvent (e.g an alcohol) This technology has major advantages over more conventional liquid chromatography (HPLC) or gas chromatography (GC), because it has a low viscosity allowing high diffusivities and limited pressure drop Therefore, high flow rates can be applied without losing efficiency [2–8] In addition, the “green” aspect is a significant motivation for SFC because CO2 is a nontoxic recycled material and generates no waste disposal issues The high-throughput potential together with ecological advantages contribute to making SFC attractive technology for a wide range of applications, not only for chiral [9–15], but also in the achiral field [16–30] The retention and separation mechanisms in SFC are likely to depend on a combination of both mobile phase and stationary phase (SP)[5] A variety of SPs are currently available for use in SFC mode Most of these phases have been developed in and transferred from the commercially available portfolios of HPLC SPs (e.g ∗ Corresponding author E-mail address: kn nagai@jp.daicel.com (K Nagai) reverse phase, normal phase, and/or HILIC) In parallel, there are some activities to develop novel SPs specifically designed for SFC use [31] One of the most recognized SP dedicated to achiral SFC separation is 2-ethylpyridine (2-EP) bonded silica phase This 2-EP SP affords good peak shapes especially for basic compounds, without any additive in the mobile phase [32] Other novel SPs for SFC have been developed by academic and industry groups [33–38] Most of the SPs used for achiral SFC separations are composed of low-molecular-weight selectors covalently bonded onto a solid support, usually silica gel Polymer type selectors would be expected to interact with analytes by utilizing multiple and cooperative mechanisms and in addition possess high durability However, only a very limited number of examples have been introduced that utilize polymer-based ligands for achiral SFC separation [36] Based on the experience of our research team in the polymeric field, we recently developed a novel poly(butylene terephthalate) based column, which exhibited unique molecular recognition ability together with high robustness in cycle durability tests [38] We considered that these features may be attributed to the associated macromolecular effect and as a result we decided to develop various polymer-based SPs and to evaluate their performance For the design of the novel polymer stationary phase series, we attempted to prepare several polymers based on the ethylpyridine moiety, mainly as the commercial phases containing such synthon are considered as benchmarks for many researchers In https://doi.org/10.1016/j.chroma.2018.08.038 0021-9673/© 2018 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) 120 K Nagai et al / J Chromatogr A 1572 (2018) 119–127 2.2 Instrumentation and chromatographic conditions Fig Structure of poly(4-vinylpyridine) or P4VP-based selector of the column DCpak P4VP this perspective, moving from a monomeric to a polymeric selector, we were expecting that the molecular recognition ability may be improved by multiple concerted interactions between the more abundant polymeric pyridyl ligand interactions with the analyte sample Similar to the recently reported poly(butylene terephthalate) selector, these polymer type selectors were anticipated to display a high durability as the polymer layer on silica gel should effectively shield any undesirable chemical interaction The HPLC separation behavior of pyridine containing polymer SP has been studied by Ihara [39–41] This SP showed good selectivity particularly for planar and disk-like aromatic molecules in reverse and normal phase HPLC modes The investigations of the polymeric phase in HPLC mode was the objective of this work In an earlier study, we focused on novel vinylpyridine polymers and related vinyl heteroaromatic polymers, and evaluated their performance in SFC mode We tested various poly(vinylpyridine isomers), including poly(2-vinylpyridine), poly(3-vinylpyridine), and poly(4-vinylpyridine), together with poly(vinylimidazole) [42,43] These SPs afforded distinctive molecular recognition abilities, particularly for structurally-similar isomeric samples Among them, poly(4-vinylpyridine) (P4VP) SP was found to provide better molecular shape recognition performance (Fig 1) The present article focuses on this P4VP column and describes its separation behavior by using various samples Based on these results, its characteristics and suitable chromatographic conditions are discussed Materials and methods 2.1 Chemicals The modifier used in this study was Japanese Industrial Standard special grade methanol (MeOH) obtained from Nacalai Tesque Inc (Kyoto, Japan) Carbon dioxide of industrial grade (over 99.5%) was purchased from Tatsumi Industry Co., Ltd (Hyogo, Japan) Ammonium formate was obtained from Wako Pure Chemical Industries (Osaka, Japan) o-Terphenyl, triphenylene, anthracene, phenanthrene, pyrene, chrysene, perylene, theobromine, trans-cinnamic acid, 3phenylphenol, adenine, and diethylamine were purchased from Tokyo Chemical Industry Co (Tokyo, Japan) 1,3,5-Tritert-butylbenzene, 2-acetylanthracene, 9-acetylanthracene, 3-acetylphenanthrene, 9-acetylphenanthrene, paraxanthine, fenoprophen, ketoprofen, naproxen, alprenolol, propranolol, atenolol, pindolol, and cyanocobalamin were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA) Trifluoroacetic acid (TFA), naphthacene and dexamethasone were purchased from Nacalai Tesque Inc (Kyoto, Japan) 2-Propanol (IPA), prednisone, estrone, prednisolone, estradiol, estriol, caffeine, theophylline, nicotinamide, and pyridoxine were purchased from Wako Pure Chemical Industries (Osaka, Japan) N-hexane (nHex) was purchased from Kanto Chemical Co (Tokyo, Japan) DCpak P4VP column (initially launched as DCpak SFC-B), sized 150 mm × 4.6 mm (i.d.), was supplied by DAICEL Corporation (Tokyo, Japan) This selector is composed of immobilized P4VP on ␮m silica particle (N.B also available on ␮m) A Silica 2-ethylpyridine (2-EP) column of ␮m particle, sized 150 mm × 4.6 mm (i.d.), was purchased from Waters Corporation (Milford, MA, USA) The SFC instrument used in this study is NexeraUC supplied by Shimadzu Corporation (Kyoto, Japan) equipped with a CO2 pump, a modifier pump, a vacuum degasser, a column oven, a multiple wavelength UV detector, and automated back pressure regulator (ABPR) Lab Solutions software (V 5.89) was used for system control and data acquisition Chromatographic conditions, such as modifier, column temperature, ABPR pressure, total flow rate, detection wavelength, sample concentration, and injection volume were described in each figure, respectively 2.3 Data analysis Relative retention factor (k) and separation factor (˛) were calculated with the equations below k= (V/V )–1, ˛=k2 /k1 (i) (ii) where V is the elution volume of an analyte and V0 is the column void volume V0 was estimated by injecting 1,3,5-tri-tertbutylbenzene as a non-retained marker k1 and k2 in Eq (ii) are the retention factors of the first and second eluted peaks, respectively Results and discussion 3.1 Planarity recognition of aromatics Considering the structure features of the poly(4-vinylpyridine) SP, it was expected to interact with planar aromatic samples as a result of the multiple aromatic pyridyl units covalently attached on silica gel Non-planar o-terphenyl (1) and planar triphenylene (2) with the same number of aromatic rings and ␲-electrons will provide detailed perception of the planarity recognition ability, because they have been considered as indicator for molecular planarity recognition in HPLC [44,45] and SFC [46] Fig 2A shows the SFC chromatogram of and by using P4VP, when its performance was compared with commercially available 2-EP SP under isocratic conditions The retention time of non-planar was almost identical for the new selector and 2-EP, while that of planar sample significantly increased for P4VP The separation factor for P4VP selector between and (˛: k2 /k1 ) reached 30.6, whereas that obtained by 2-EP is 4.4 This result indicates that ␲-electron rich planar could strongly interact with vinylpyridine polymer selector via ␲–␲ interaction Taking advantage of this high planarity recognition ability, commercially available polycyclic aromatic hydrocarbons (PAHs) were analyzed Fig shows the SFC chromatogram of eight PAHs (2–9) under gradient condition Eight peaks were well separated on the P4VP column Of particular note is that anthracene (4) and phenanthrene (5) have the same molecular weight and similar molecular size and polarity Therefore, these two compounds cannot be distinguished by MS detector, which mean that the only method to separate and must be by column separation This new selector achieved a baseline resolution for and On the other hand, when these compounds were analyzed by 2-EP SP under the same condition, and co-eluted The slight adjustment of gradient conditions was necessary to separate and in isocratic mode, immediately after eluting and 5, a linear gradient program started K Nagai et al / J Chromatogr A 1572 (2018) 119–127 121 Fig SFC chromatograms of o-terphenyl and triphenylene on (A) DCpak P4VP and (B) 2-EP SPs Modifier, MeOH (isocratic condition, 3%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 254 nm; sample concentration, 0.3 mg/mL in nHex/IPA = 9/1; injection volume, ␮L The longer aspect ratio of the PAH analyzed resulted in a shorter retention time Wise et al proposed length-to-breadth (L/B) ratio for describing two dimensional aspect ratio of PAH, and the smaller L/B ratio indicates the disk-like molecule [47] Indeed, L/B ratio of naphthacene (7), chrysene (8), and triphenylene (2) which have the same number of aromatic rings and ␲-electrons is 1.89, 1.72, and 1.12, respectively The elution order of 7, 8, and is < < 2, which is the inverse of L/B ratio These tendencies are almost identical for similar stationary phase used in HPLC mode [41] Similar to the separation of non-substituted PAH separation, regioselective acetylated anthracene (10, 12) and phenanthrene (11, 13) were also examined under isocratic conditions Although these samples have almost the same molecular size and polarity, the P4VP selector can recognize the slight structural differences and the four peaks were well resolved as shown in Fig From these results, the new column shows excellent planarity recognition and molecular shape recognition of various aromatic isomers and PAHs Combining P4VP column with supercritical fluid extraction (SFE) and subsequent SFC technique would enable us to analyze the residual PAH in soil and atmosphere etc [21] 3.2 Separation of samples with different structural features Inspired by the interesting planarity and molecular shape recognition, other type of planar sample families e.g steroids mixtures (14–19) were analyzed under isocratic conditions Fig shows the SFC chromatogram of six structurally related steroids In particular, prednisone (14), prednisolone (16), and dexamethasone (18) Fig SFC chromatogram of eight polycyclic aromatic hydrocarbons on (A) DCpak P4VP and (B) 2-EP SPs Inset shows magnified chromatogram Modifier, MeOH (gradient condition); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 254 nm; sample concentration, 0.1 mg/mL (except 9), 0.3 mg/mL (9) in nHex/IPA = 9/1; injection volume, ␮L The gradient started with 3% of MeOH, after hold at 3% of MeOH, linear gradient ramped up to 38% of MeOH over 14 min, followed by hold at 38% of MeOH, then returned to 3% of MeOH over min, followed by hold at 3% of MeOH have almost same skeleton with some slight differences in the substituents, and therefore they are difficult to separate When investigating these samples by using 2-EP SP under the same conditions, they were co-eluting We note that the conditions applied were only to compare the selectivity in exactly identical conditions A better separation might be possible for 2-EP by using different gradient condition The direct separation of such steroid mixtures in SFC mode would have significance in steroid profiling, as potential biomarkers[48] or also in anti-doping control The analysis of caffeine (20) and its demethylated derivatives, theophylline (21), theobromine (22) and paraxanthine (23) was also investigated Fig shows the SFC chromatogram of the mixture Good peak resolution with symmetrical peaks was observed for these polar analytes as well as for less-polar PAH derivatives and steroids The longer retention of 21–23 than 20 may be attributed to the hydrogen bonding interactions between the demethylated proton of the analytes and the proton acceptor behavior of P4VP selector 122 K Nagai et al / J Chromatogr A 1572 (2018) 119–127 Fig SFC chromatogram of acetylated anthracene and phenanthrene on DCpak P4VP SP Modifier, MeOH (isocratic condition, 3%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 254 nm; sample concentration, 0.15 mg/mL in nHex/IPA = 9/1; injection volume, ␮L This novel SP contains the basic poly(4-vinylpyridine) moiety and one may guess that acidic samples would be strongly retained and/or would show tailing peaks on it In order to confirm this point, we tested propionic acid nonsteroidal anti-inflammatory drugs (NSAIDs), fenoprofen (24), ketoprofen (25), and naproxen (26) Fig shows the SFC chromatogram of three NSAIDs under isocratic conditions without any additives, which gave three resolved peaks Surprisingly, their peak shapes were relatively symmetric, with peak symmetry factors (Ps) for 24 of1.11, 1.15 for 25, and 1.19 for 26 These results indicate that the pyridine polymer ligand must efficiently shield the undesirable interactions between analytes and residual silanol groups on SP Fig SFC chromatogram of six steroids on (A) DCpak P4VP and (B) 2-EP SPs Modifier, MeOH (isocratic condition, 30%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 225 nm; sample concentration, 0.33 mg/mL in nHex/IPA = 1/1; injection volume, ␮L 3.3 Effect of additives In the SFC field, it is common to use additives as a third component in the mobile phase, such as acids, bases and salts They improve the peak shapes and/or increase the solubility in the mobile phase especially for polar analytes [5,31,49] Basic additives are often used for basic samples; acidic additives for acids, but other combinations are also possible The current trend for both acidic and basic sample analysis is to use volatile salts, such as ammonium formate and ammonium acetate These additives are often used when separating APIs in SFC in analytical as well as preparative applications, because many APIs bear polar and/or ionizable groups which can easily interact with the residual silanol groups, and often result in deficient peak shapes (leading, tailing, and asymmetric peaks) [31] However, if a SP could afford good peak symmetry without any additive, it would be considered advantageous The absence of additives would be a positive feature for preparative applications (no salts to be recuperated together with the product), but also for analytical UV detection (the high UV absorption of Fig SFC chromatogram of caffeine, theophylline, theobromine, and paraxanthine on DCpak P4VP SP Modifier, MeOH (isocratic condition, 5%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 225 nm; sample concentration, 0.2 mg/mL in MeOH/IPA = 1/1; injection volume, ␮L K Nagai et al / J Chromatogr A 1572 (2018) 119–127 123 any additive In general, for such basic samples, their peaks were broadened without any additives However, when an appropriate additive (e.g 20 mM of ammonium formate) was used, the peak shapes were substantially improved (Fig 8B) As expected, the P4VP column can produce relatively symmetrical peaks even in the absence of any additive (Fig 8C) As for 2-EP, after the addition of salts, peaks were further sharpened (Fig 8D) The fact that the P4VP column could gain sharp peaks without any additives for these basic APIs may be attributed to the polymeric ligand effect The covalently bonded P4VP chains may spread on the porous silica gel surface and will contribute to reduce the undesirable interactions between the residual silanol groups and the basic analytes 3.4 Effect of conditioning with different additive Fig SFC chromatogram of nonsteroidal anti-inflammatory drugs on DCpak P4VP SP Modifier, MeOH (isocratic condition, 10%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 210 nm; sample concentration, 0.2 mg/mL in IPA; injection volume, ␮L ammonium formate or ammonium acetate especially in gradient conditions sometimes leads to unstable baselines) Accordingly, we analyzed four ␤-adrenergic blocking agents (␤-blockers) (27–30) in the presence and absence of ammonium formate, to compare the chromatographic performance of the two SPs Fig 8A shows the SFC chromatogram of four ␤-blockers under gradient conditions obtained by using the 2-EP column without As the new selector consists of basic poly(4-vinylpyridine) moieties, it can be envisaged to form a cationic pyridinium form by reaction with strong acids such as trifluoroacetic acid (TFA) and be converted to a quaternized amphiphilic salt form by reaction with the corresponding alkyl halide Recently, Ihara and Takafuji reported amphiphilic poly(N-alkylpyridinium salt) based HPLC SPs through quaternization reactions Their separation mode can be easily tuned by changing the N-alkyl side chain length [50,51] The protonated pyridinium salt effect was investigated for the P4VP phase by passing through TFA-containing modifier We selected neutral (2, 13), acidic (31, 32) and basic samples (21, 34) for this test under isocratic conditions Fig SFC chromatograms of ␤-blockers (A, C) in the absence or (B, D) presence of ammonium formate on (A, B) 2-EP and (C, D) DCpak P4VP SPs Modifier, MeOH (gradient condition); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, (A, C) 220 or (B, D) 280 nm; sample concentration, 0.1 mg/mL in MeOH; injection volume, ␮L The gradient started with 10% of modifier, after hold at 10% of modifier, linear gradient ramped up to 35% of modifier over 10 min, followed by hold at 35% of modifier, then returned to 10% of modifier over min, followed by hold at 10% of modifier 124 K Nagai et al / J Chromatogr A 1572 (2018) 119–127 Fig (A–E) SFC chromatograms and (F) retention factor dependent of neutral, acidic, and basic samples on DCpak P4VP SP by using various modifier under isocratic condition (10%) Before each analysis, the testing modifier was eluted for more than 30 for equilibration Each modifier was, (A) MeOH, (B) MeOH/TFA = 100/1, (C) MeOH, (D) MeOH/DEA = 100/1, and (E) MeOH For detail, please see text Temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 254 nm; sample concentration, 0.13 mg/mL in nHex/IPA = 1/1; injection volume, ␮L Fig 9A shows the SFC chromatogram of the six sample mixture with MeOH as modifier These samples were eluted as relatively symmetric peaks After passing a TFA-containing modifier (MeOH/TFA = 100/1, (v/v)) for more than 30 min., the same sample mixture was injected Fig 9B shows the corresponding chromatogram and Fig 9F the retention factor (k) dependence on modifier composition Their elution order dramatically changed Acidic samples (31, 32) eluted faster than in the initial analysis, whereas basic theophylline (21) hardly changed its retention time and more basic adenine (34) eluted significantly later Surprisingly, neutral samples (2, 13) also eluted faster than in the original analysis However, by using MeOH again as a modifier during 30 min., retention time of all analytes were likely to recover the original profile (Fig 9C) We then used a diethylamine (DEA)-containing MeOH (MeOH/DEA = 100/1, (v/v)) as a modifier, and the same experiment was conducted However, the elution order hardly changed (Fig 9D) After passing MeOH as a modifier again, the elution almost reverted to that of the first injection (Fig 9E) Based on these results, we proposed the following retention mechanism As discussed in the previous section, the pyridyl groups on the polymer side chains effectively mask the silanol groups on silica gel surface, which lead to a shielding of undesirable interactions between silanols and analytes When only MeOH was used as modifier, the acidic samples (e.g 31), can interact with the SP via acid-base interaction, while such an interaction between SP and basic analytes (e.g 34) should not be expected (Fig 10A) For this reason, the elution of 34 might be faster than the one of 31 When a TFA-containing MeOH was used as a modifier, the pyridyl groups on the side chain are protonated (Fig 10B) Contrary to Fig 10A, the protonated SP and 34 can interact We note that the pyridyl groups in the SP are more prone to protonate than 34 because pyridine is more basic than 34 based on the pKa values of the corresponding conjugate acids [52,53] Hence, the elution of basic 34 was slower than acidic 31 The reason why the retention time of neutral samples decreased under acidic conditions is still unclear We envisage that the electron density of the pyridyl ring in the SP might be decreased by protonation, or MeOH may solvate the protonated side chains, which will interfere with the SP and neutral analyte interaction In such a case, it may affect the retention behavior of neutral samples in acidic conditions After gradually passing MeOH as modifier again, the protonated SP side chains gradually deprotonated to be in their original state Therefore, the initial retention behavior gradually recovered DEA seems to have less effect on the retention of these samples As demonstrated here, the elution order of this selector can be reversely tuned through acid mediated pyridinium salt formation K Nagai et al / J Chromatogr A 1572 (2018) 119–127 125 Fig 10 Postulated retention mechanism of DCpak P4VP in neutral conditions (A) and acidic conditions (B) Fig 11 Cycle dependent SFC chromatograms of water soluble vitamins on (A) 2-EP and (B) DCpak P4VP SPs Modifier; MeOH (isocratic condition, 25%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, mL/min; UV detection, 230 nm; sample concentration, 0.2 mg/mL in MeOH; injection volume,2 ␮L 3.5 Cycle durability tests As mentioned in the introduction, polymer-type selectors were expected to show good durability as we estimate that undesirable attack may be interfered with the polymer ligand layer on the SP surface Recently, we confirmed the column robustness of poly(butylene terephthalate) selector by a range of cycle durability testing [38] In order to investigate the durability of a new P4VP column, three water soluble vitamins (WSVs), nicotinamide (vitamin B3 ), pyridoxine (vitamin B6 ), and cyanocobalamin (vitamin B12 ) were selected Fig 11A shows the cycle-dependent SFC chromatograms of three WSVs by using 2-EP column under isocratic conditions For the first injection, three peaks were well separated and a characteristic long retention was observed for vitamin B12 However, as the cycle passed, the retention time gradually decreased for vitamin B3 and vitamin B6 , and sharply decreased for vitamin B12 Fig 11A also shows the chromatograms after 20, 41, 60, and 80 cycles The retention time continuously decreased and that of vitamin B12 reduced to less than half of the original time Fairchild et al proposed that 126 K Nagai et al / J Chromatogr A 1572 (2018) 119–127 Fig 12 Cycle versus retention factor (k) of (A) vitamin B3 , (B) vitamin B6 , and (C) vitamin B12 by using DCpak P4VP and 2-EP columns Experimental condition is same as Fig 11 silyl ether formation by a condensation reaction between silanols and MeOH used as a modifier is a major contribution to retention variation over time in SFC mode [54] We investigated the same cycle test for the P4VP case (Fig 11B) In contrast to 2-EP case, the retention time of these samples did not change after 20, 40, 60, and 80 cycles Fig 12 shows cycle versus their retention factors (k) We note that the systematic cycle investigation here reported was run over 80 sequential cycles in brand-new column, however the P4VP columns used in this study to generate all experimental data reported were periodically tested with the standard samples and confirmed the durability and the stability over several months Conclusion A novel P4VP based column was designed and its performance was evaluated under SFC conditions This SP showed unique molecular shape recognition for planar molecules such as structurally related polycyclic aromatic hydrocarbons and steroid mixtures The new SP afforded symmetric peaks for active pharmaceutical ingredient analysis even in the absence of any additives, e.g acids, bases, or salts, probably due to the effective shield of residual silanols by the polymeric pyridine selector The surface chemical properties of the new column can be easily converted to cationic pyridinium form by eluting TFA containing modifiers, which dramatically change elution order of acidic, basic, and even neutral analytes This significant elution order change can be recovered to the original state by passing through DEA containing modifier Additionally, the column performance did not change as a result of cycle durability testing of water soluble vitamins The present study together with that of another our polymeric SP [38,43,55] 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Shinkura, A Ohnishi, Poly(butylene terephthalate) based novel achiral stationary phase investigated under supercritical fluid chromatography conditions, J Chromatogr A 1549 (2018) 85–92, http://dx... Bertin, P Hennig, E Lesellier, Synthesis and characterization of novel polymer -based pyridine stationary phases for supercritical fluid chromatography, Submitted (n.d.) K Kimata, K Iwaguchi, S Onishi,... analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography, J Chromatogr A 1362 (2014) 270–277,