Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

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Journal of Chromatography A, 1175 (2007) 7–15

Study of a monolithic silica capillary column coated with poly (octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds Oscar N´un˜ ez ∗ , Tohru Ikegami, Kosuke Miyamoto, Nobuo Tanaka Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Goisho-Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan Received 29 August 2007; received in revised form 25 September 2007; accepted 27 September 2007 Available online 10 October 2007

Abstract The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of ␤- and ␥-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography. © 2007 Elsevier B.V. All rights reserved. Keywords: Monolithic silica capillary columns; Reversed-phase liquid chromatography; Chemical polymerization; LC

1. Introduction Monolithic columns have emerged as an alternative to traditional packed-bed columns for high efficiency separations in high-performance liquid chromatography (HPLC) due to their small-sized skeletons and wide through-pores [1–4]. In a previous work, a hybrid monolithic silica capillary column for micro-HPLC was reported, based on silica prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS), and chemically modified for reversed-phase liquid chromatography by free radical polymerization of octadecyl methacrylate using ␣,␣ -azobis-isobutyronitrile (AIBN) as an initiator [5]. By performing the polymerization at high

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Corresponding author. Tel.: +81 75 724 7809; fax: +81 75 724 7710. E-mail addresses: [email protected] (O. N´un˜ ez), [email protected] (N. Tanaka). 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.09.071

monomer and initiator concentrations columns with high retention (kamylbenzene up to 4.2 in 80% methanol mobile phases) and high efficiency (Huracil 10 ␮m) were obtained. So far, the performance of these columns was mainly tested by using alkylbenzenes and a few other compounds as solutes in terms of permeability, retention properties and chromatographic efficiency. The high efficiency and resolution of monolithic silica capillary columns made them ideal for proteomic and metabolomic studies and some applications have been reported in the last years [6–11]. These properties combined with the high permeability and low pressure drop allow to use these columns in two-dimensional chromatography to separate complex mixtures [12,13]. The performance of a monolithic silica-C18 capillary column for polar compounds is then of particular interest because there are many polar compounds in the metabolites present in a cell. Moreover, the separation of very complex mixtures such as metabolome by using long monolithic silica

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capillary columns seems to be highly advantageous compared to conventional particle-packed columns [14]. In the present work, the performance for some polar and non-polar compounds of a hybrid type monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for reversed-phase liquid chromatography was studied. The results were compared to those obtained with columns modified using octadecylsilyl-(N,N-diethylamino)silane (ODS columns). Different families of compounds including benzene and naphthalene derivatives, PAHs, steroids, alkyl phthalates, and tocopherols were used as test samples. The chromatographic performance of these compounds was evaluated using both methanol and acetonitrile mobile phases. 2. Experimental 2.1. Chemicals All the reagents used were of analytical grade. Tetramethoxysilane (TMOS), methyltrimethoxysilane (MTMS) and 3-methacryloxypropyltrimethoxysilane (MOP) were purchased from ShinEtsu Silicon Chemicals (Tokyo, Japan); acetonitrile, urea, and pyridine from Wako (Osaka, Japan); poly(ethylene glycol) (PEG, Mr = 10,000) from Sigma–Aldrich (Steinheim, Germany); octadecyl methacrylate from TCI (Tokyo, Japan); and methanol, hexane, toluene, acetic acid (1 M), diethylamine and ␣,␣ -azobis-isobutyronitrile (AIBN) from Nacalai Tesque (Kyoto, Japan). Methanol, acetonitrile and toluene were distilled before used. Water was purified by using a Milli-Q Gradient A10 system from Millipore. All chemical standards were purchased from Nacalai, TCI, Sigma–Aldrich and Merck (Darmstadt, Germany). The following sample mixtures were used in this study: benzene and naphthalene derivatives (1, benzene; 2, toluene; 3, phenol; 4, o-cresol; 5, p-cresol; 6, 2-naphthol; 7, benzonitrile; 8, nitrobenzene; 9, methyl benzoate; 10, methyl phenylacetate; 11, methyl phenyl ketone; 12, ethyl phenyl ketone; 13, phenyl propyl ketone; 14, p-nitrotoluene; 15, o-nitrophenol; 16, m-nitrophenol; 17, p-nitrophenol; 18, p-fluorophenol; 19, p-chlorophenol; 20, p-bromophenol; 21, p-iodophenol; 22, odinitrobenzene; 23, p-dinitrobenzene; 24, p-nitrobenzyl alcohol; 25, p-cyanophenol; 26, methyl p-hydroxybenzoate; 27, ethyl phydroxybenzoate; 28, propyl p-hydroxybenzoate; 29, dimethyl phthalate; 30, diethyl phthalate; 31, dimethyl terephthalate; 32, 1,5-dinitronaphthalene; 33, 1,8-dinitronaphthalene; 34, 2.4dinitrochlorobenzene; 35, 2-amino-4-nitrophenol), polycyclic aromatic hydrocarbons (PAHs) (1, napthalene; 2, acenaphtylene; 3, fluorene; 4, acenaphthene; 5, phenanthrene; 6, anthracene; 7, fluoranthene; 8, pyrene; 9, chrysene; 10, benz(a)anthracene; 11, benzo(b)fluoranthene; 12, benzo(k)fluoranthene; 13, benzo(a)pyrene; 14, dibenz(a,h)anthracene; 15, indeno(1,2,3cd)pyrene; 16, benzo(g,h,i)perylene), steroids (1, cortisone; 2, prednisolone; 3, corticosterone; 4, testosterone; 5, 17-␤estradiol; 6, 17-␣-estradiol; 7, estrone; 8, progesterone), alkyl phthalates (1, dimethyl phthalate; 2, dimethyl terephthalate; 3, diethyl phthalate; 4, di-n-butyl phthalate; 5, benzyl butyl phthalate), tocopherol homologues (1, dl-␦-tocopherol; 2,

dl-␤-tocopherol; 3, dl-␥-tocopherol; 4, dl-␣-tocopherol), and xylenols (1, 3,4-xylenol; 2, 3,5-xylenol; 3, 2,4-xylenol; 4, 2,6-xylenol). 2.2. Instrumentation Column chromatographic evaluation was performed using a split-injection HPLC system which consists of a pump (LC10AD, Shimadzu, Kyoto, Japan), a MU-701 UV–vis detector (GL Science Inc., Tokyo, Japan), and an injection valve (model 7125, Rheodyne, CA, USA) fitted with a T-union which serves as a splitter, with one end connected to the capillary column and the other end to a flow restrictor that is a stainless steel tubing (0.1 mm I.D., 1/16 in. O.D.). Measurements were carried out in triplicate by keeping all system including the pump, the injector and the detector at 30 ◦ C in an air-circulating oven. Chromatographic data acquisition and processing were performed with an EZChrom Elite Client/Server software version 2.8.3. 2.3. Preparation and chemical modification of columns All monolithic silica capillary columns (hybrid type) were prepared from a mixture of TMOS and MTMS in 200 ␮m I.D. fused-silica capillaries following a previously reported method [15,16]. ODM columns were obtained by free radical polymerization of octadecyl methacrylate following the previously described procedure [5]. Briefly, columns were washed with methanol for 24 h, then MOP bonding was performed by rinsing the columns with a MOP:pyridine 1:1 solution for 48 h at 80 ◦ C, followed by methanol washes. The MOP-bonded columns were then rinsed with toluene for 3 h and the polymerization reaction solution, a mixture of 250 ␮L octadecyl methacrylate monomer and 250 ␮L toluene solution of AIBN (38.6 mg/ml), was charged into the columns and allowed to react at 80 ◦ C for 3 h. Finally, columns were washed with toluene and methanol. ODS columns were modified using octadecylsilyl-(N,N-diethylamino)silane (ODS-DEA). The bonding reaction was carried out as previously described [14], by continuously feeding the columns with 20% ODS-DEA (v/v) solution in toluene at 0.8 MPa and allowing it to react for 3 h at 60 ◦ C, followed by washes of toluene and THF. This procedure was repeated twice. 3. Results and discussion 3.1. Benzene and naphthalene derivatives In order to study and compare the performance of columns for a variety of compounds under the same separation conditions, a mixture of 35 benzene and naphthalene derivatives was analyzed with an ODS column and the proposed ODM monolithic silica capillary column. The plots of log k values for these 35 benzene and naphthalene derivatives on an ODM column against those on an ODS column are given in Fig. 1. In order to simplify this figure nitro compounds and phenols are given in a different plot than the other compounds. The ODS stationary phase provides a retention factor range for these compounds from 0.08 to 1.52 (retention factors

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Fig. 1. Plots of log k values for benzene and naphthalene derivatives on an ODM column against those on an ODS column. Mobile phase: (a) a methanol:water:formic acid 60:40:0.1 (v/v/v) solution, (b) an acetonitrile:water:formic acid 70:30:0.1 (v/v/v) solution.

of 2-amino-4-nitrophenol and toluene, respectively) with the methanol mobile phase. However, under the same conditions the column coated with poly(octadecyl methacrylate) allow to increase the retention factor range by a factor of 3.5, with values from 0.19 to 5.18 (retention factors of 2-amino4-nitrophenol and 1,5-dinitronaphthalene, respectively). Both columns showed symmetrical peaks and similar efficiency. In contrast, with acetonitrile mobile phase similar retention factor range was achieved with both columns (k values between 0.07–1.0 and 0.1–0.8 for ODM and ODS, respectively). Some differences in selectivity were also observed between both columns. For instance, toluene and 1,5-dinitronaphthalene are the more retained compounds on both the ODS and the ODM columns with the methanol mobile phases although the retention times are greater on the latter column. With the acetonitrile mobile phase, the retention times are similar with both columns. The preference for the polymer coated column is notable for nitro compounds, such as nitrophenols, dinitrobenzenes and dinitronaphthalenes, and for phenols, especially acidic ones, which show higher retentions on the ODM column compared to the ODS column. This could be related to the acidic character of these compounds (␲-acids or Bronsted acids) which can better interact with the ester groups of the poly(octadecyl methacrylate)

(electron donor or proton acceptor). Other compounds such as phthalates and hydroxybenzoates seem to show a similar behavior with both columns. With acetonitrile mobile phase, although all compounds are slightly more retained on ODS column, there is not much difference between acidic compounds and the other benzene and naphthalene derivatives (Fig. 1b). In order to increase retention and separation factors based on the contribution of polar interactions the chromatographic separation of seven of the 32 naphthalene and benzene derivatives (methyl phenylacetate, p-nitrotoluene, p-fluorophenol, p-dinitrobenzene, methyl p-hydroxybenzoate, diethyl phthalate and 1,8-dinitronaphthalene) was studied with both columns at a lower methanol content in the mobile phase. Fig. 2 shows the chromatograms of this mixture using methanol:water:formic acid (50:50:0.1) (v/v/v). Retention is slightly higher with 60% methanol mobile phase and a higher differentiation for this family of compounds according to polar interactions is observed. As can be seen in the figure, important changes in selectivity were obtained. For instance, on the ODM stationary phase pfluorophenol is more retained than methyl p-hydroxybenzoate, p-dinitrobenzene is more retained than methyl phenylacetate, and both p-nitrotoluene and 1,8-dinitronaphthalene are more retained than diethyl phthalate, while this behavior changes

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polymerization reaction mixture containing only 100 ␮L of octadecyl methacrylate monomer (which corresponds to an ODM column with the lowest phase loading, expected to be similar to that of an ODS column from the comparison of retention factors of alkylbenzenes [5]). As can be seen, some differences in selectivity with the other two columns (Fig. 2a) were obtained that could be attributed to the differences on the amount of C18 phase. For instance, the retention of p-fluorophenol and p-dinitrobenzene increases with the amount of C18 stationary phase bonded to the silica, while the retention of the last three compounds is very similar on all ODM columns, independently of the amount of stationary phase. These results allow proposing the polymer-bonded ODM column as an alternative to conventional silica ODS columns, and their different selectivity with methanol mobile phase can produce promising results in future 2D-HPLC studies with both C18 stationary phases. 3.2. PAHs

Fig. 2. Separation of a mixture of seven benzene and naphthalene derivatives on (a) ODM and ODS columns and (b) ODM column prepared from 100 ␮L octadecyl methacrylate monomer, using a methanol:water:formic acid 50:50:0.1 (v/v/v) solution as mobile phase. Uracil was used as tM standard. Detection was performed at 210 nm and 30 ◦ C. See Section 2 for peak identification.

on the ODS stationary phase. So, in general the ODM column using methanol mobile phases can show the selectivity of an ODS column using THF mobile phases [17]. Phenols and nitro compounds that are acids or ␲-acids seems to better undergo interactions with the poly(octadecyl methacrylate) polymer (electron donor) as in the case of THF with ODS, while hydrogen-bond acceptors (bases) compounds such as esters, ethers and ketones showed less interaction with the ODM column. Differences in phase loading between ODM and ODS columns can also account for significant changes in shape selectivity. In our previous work we observed that as the monomer concentration on the polymerization reaction mixture increases higher retention factors were observed for alkylbenzenes because of the higher amount of C18 stationary phase bonded to the silica [5]. Fig. 2b shows the chromatogram obtained for a mixture of seven selected benzene and naphthalene derivatives using an ODM column prepared from a

The 16 priority pollutant PAHs legislated by the Environmental Protection Agency (EPA) were also used to compare the performance of ODM and ODS columns. Fig. 3 shows the plot of log k values for PAHs on an ODM column against those on an ODS column using an acetonitrile:water 70:30 (v/v) solution as mobile phase. As can be seen, PAHs are always more retained on the polymer coated column than on ODS, probably due to the increase of ␲–␲ interactions with the polymeric stationary phase because of the aromatic character of these compounds. For instance, a threefold increase in the retention range for PAHs was observed with the ODM column, with k values from 0.79 to 14.1 (retention factors of naphthalene and benzo(g,h,i)perylene, respectively), allowing a higher differentiation for compounds with similar planar structures than the ODS column. Fig. 4 shows

Fig. 3. Plots of log k values for PAHs on ODM column against those on ODS column using acetonitrile:water 70:30 (v/v) as mobile phase. See Section 2 for peak identification. The chemical structure of some PAHs is also included.

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retention is observed. However, a more deep study would be necessary in order to verify this behavior. 3.3. Steroids and alkyl phthalates

Fig. 4. Separation of the 16 EPA priority pollutants PAHs with ODM and ODS columns using an acetonitrile:water 70:30 (v/v) solution as mobile phase. Thiourea was used as tM standard. Detection performed at 254 nm and 30 ◦ C. See Section 2 for peak identification.

the chromatograms obtained with both ODM and ODS columns using 70% acetonitrile mobile phase. Symmetrical peaks and similar column efficiency were obtained with both C18 stationary phases. Some differences in selectivity and the elution order were observed between ODM and ODS columns. For instance, fluorene and acenaphthene (peaks 3 and 4, respectively) cannot be separated with the ODS column while better separation can be achieved with the ODM column. The behavior of this ODM stationary phase for PAHs seems to be different from that of other polymeric C18 columns where retention increases with the increase of the length-to-breadth (L/B) ratio of the compound [18]. In this case it seems that as more compacted is the structure of the compound (lower L/B ratio) higher is the retention observed with the polymer coated column. For this reason, acenapthene (peak 4, see structure in Fig. 3) which has a more compacted structure than fluorene (peak 3) is more retained on the ODM column allowing the separation of both compounds. This behavior can also be observed for other PAHs. For instance, benzo(g,h,i)perylene (peak 16, with an L/B ratio of 1.16 [19]) is more retained than indeno(1,2,3-cd)pyrene (peak 15, with an L/B ratio of 1.44 [19]), allowing also the separation on the ODM column while no separation at all was observed with the ODS. Up to this point, we can conclude that the ODM polymeric coated column has a preference for aromatic compounds, with rigid and planar structures. When working with acetonitrile/water mobile phase it seems that retention increases with the decrease in L/B ratio (in contrast to the behavior of other structural stationary phases, probably due to the contribution of methyl groups on the hybrid monolith), so as more compacted is the structure of the PAH (see some structures in Fig. 3) higher

The chromatographic performance of ODM columns versus that of ODS columns for some polar but relatively hydrophobic compounds such as steroids and alkyl phthalates was also studied. Fig. 5 shows the chromatograms obtained for a mixture of eight steroids using a methanol:water 40:60 (v/v) solution (Fig. 5a) and an acetonitrile:water 40:60 (v/v) solution (Fig. 5b) as mobile phases. In general, both columns showed good performance for the separation of steroids, with good peak symmetry and efficiency, with some differences in selectivity. Retention is higher in methanol than in acetonitrile, probably due to the loss of dispersion interactions in the acetonitrile mobile phase. In general, all compounds have more preference for the ODS column than for the ODM, less for 17-␤-estradiol, 17-␣-estradiol and estrone (peaks 5, 6 and 7, respectively) using the methanol mobile phase that present a slightly higher retention on ODM. This is probably due to the higher aromatic character of these compounds (they have an aromatic ring on their structures) which increases the interactions (␲–␲ or dipole–␲) with the polymer coated stationary phase, effect which is more noticeable with methanol mobile phases. ODS columns produce a better separation for cortisone and prednisolone steroids (peaks 1 and 2, respectively) than ODM columns with both methanol and acetonitrile mobile phases. In contrast, ODM can better separate testosterone, 17-␤-estradiol, 17-␣-estradiol and estrone (peaks 4, 5, 6 and 7, respectively) steroids than ODS column, also with both mobile phases evaluated. Some differences in selectivity and in the elution order were also obtained. For instance, the change in the elution order of cortisone and prednisolone (peaks 1 and 2, respectively) on ODS column when changing from acetonitrile to methanol mobile phase could be due to the increase of dispersion interactions on prednisolone that has a hydroxyl group in its structure instead of the CO group in cortisone. It seems that aromatic nature increase retention on ODM compared to ODS, at least with acetonitrile mobile phase (Fig. 5a). This could be observed with peaks 5, 6 and 7 which are more retained than testosterone (peak 4 and not aromatic) on ODM compared to ODS. When going to methanol, this behavior seems also to be valid in general, although estrone steroid (peak 7) is less retained compared to 17-␤-estradiol, and the explanation could be similar to that of peaks 1 and 2, as the only difference in the structure of these two steroids is that 17-␤-estradiol has an hydroxyl group while estrone has a CO group in the same position. Fig. 6 shows the chromatograms obtained for a mixture of five alkyl phthalates using a methanol:water 50:50 (v/v) solution (Fig. 6a) and an acetonitrile:water 50:50 (v/v) solution (Fig. 6b) as mobile phases. In general, similar retention was observed for alkyl phthalates with both C18 stationary phases although they are slightly more retained on ODS when acetonitrile mobile phase was used. However, although similar retention is obtained, the pair of compounds di-n-butyl phthalate and benzyl butyl phthalate (peaks 4 and 5, respectively) show a baseline separa-

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Fig. 5. Separation of a mixture of eight steroids on ODM and ODS columns using (a) a acetonitrile:water 40:60 (v/v) solution and (b) a methanol:water 40:60 (v/v) solution as mobile phases. Uracil was used as tM standard. Detection was performed at 220 nm and 30 ◦ C. See Section 2 for peak identification.

Fig. 6. Separation of a mixture of five alkyl phthalates on ODM and ODS columns using (a) a acetonitrile:water 50:50 (v/v) solution and (b) a methanol:water 50:50 (v/v) solution as mobile phases. Uracil was used as tM standard. Detection was performed at 254 nm and 30 ◦ C. See Section 2 for peak identification.

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tion with the ODM column when acetonitrile was used as mobile phase (Fig. 6a), probably due to the higher aromatic character of benzyl butyl phthalate which increase interactions with the polymer coated column. When using methanol as mobile phase (Fig. 6b) higher retention with acetonitrile was observed for all compounds (because of the increase in dispersion interactions), and benzyl butyl phthalate shows a higher preference for the ODM phase. However, the separation between dimethyl tereph-

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thalate and diethyl phthalate (peaks 2 and 3, respectively) was lost on ODM column, and worse peak symmetry was observed for the more retained compounds in both C18 stationary phases. 3.4. Tocopherol homologues The separation of some tocopherols, term used to describe all tocol derivatives included in Vitamin E, has been reported to

Fig. 7. (a) Chemical structures of tocopherol homologues. (b) Separation of tocopherols with ODM and ODS columns using a methanol:water 95:5 (v/v) solution as mobile phase. Thiourea was used as tM standard. Detection performed at 295 nm and 30 ◦ C. (c) Separation of tocopherols using a long ODS monolithic silica capillary column (440 cm). Other conditions as in (b). See Section 2 for peak identification.

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be difficult. In fact, it is hard to separate ␤- and ␥-tocopherol isomers (see chemical structures in Fig. 7a) by reversed-phase liquid chromatography using conventional C18 stationary phases [20–24], while their separation can be achieved with other stationary phases such as octadecylpolyvinyl alcohol [20] and C30 [24], or in normal-phase chromatography [24,25]. Fig. 7b compares the separation of these compounds using an ODM polymer coated column with that obtained on ODS column. Good peak symmetry and similar column efficiency was observed with both columns. As described in the literature for some C18 stationary phases, ␤- and ␥-tocopherol isomers were not separated using the ODS column but an acceptable separation can be obtained with the ODM stationary phase using a methanol:water 95:5 (v/v) solution as mobile phase. It should be noted that baseline separation can be achieved when decreasing to 90% the amount of methanol on the mobile phase. Conventional ODS columns neither produce the separation of ␤- and ␥-tocopherol isomers nor using a long monolithic silica capillary column as can be seen in Fig. 7c where the separation of tocopherol homologues with a 440 cm ODS column (which can generate 400,000 theoretical plates) is shown. In general, these compounds have a higher preference for the ODM stationary phase than for the ODS, using both acetonitrile and methanol mobile phases, probably due to the aromatic ring on the structure which increased ␲–␲ interactions with the polymer coated column. The higher rigidity of the ODM stationary phase compared to that of ODS helped on the structure recognition of ␤- and ␥-tocopherol isomers, thus allowing their separation. The ␥-tocopherol isomer has the two methyl groups

closer to the hydroxyl group (see structure in Fig. 7a) creating a more compacted compound than the ␤-tocopherol isomer where methyl groups in the aromatic ring are more separated. As in the case of PAHs (Section 3.2), as more compacted is the structure of a compound better interaction with the ODM polymer coated column is produced, which could explain the separation of ␤- and ␥-tocopherol isomers on this stationary phase. To try to confirm this behavior a mixture of xylenols was also used as a test sample with both ODM and ODS columns, and the chromatograms obtained using a methanol:water 60:40 (v:v) solution as mobile phase are shown in Fig. 8. As can be seen, the poly(octadecyl methacrylate) ODM phase allowed the separation of 2,4- and 2,6-xylenol isomers that cannot be separated under the same conditions on the ODS column. As in the case of tocopherols, the proximity of both methyl groups to the hydroxyl group in 2,6-xylenol isomer could probably generate a more compacted structure than in 2,4-xylenol isomer, and consequently 2,6-xylenol has a higher retention on the more rigid ODM stationary phase allowing the separation of both isomers. 4. Conclusions The monolithic silica capillary column coated with poly(octadecyl methacrylate), ODM column, showed a very good performance for polar and non-polar compounds in reversed-phase liquid chromatography when compared to conventional ODS columns. The separation of some compounds which cannot be separated under the same conditions with ODS columns, such as ␤- and ␥-tocopherol, and some PAHs, steroids and alkyl phthalates was reported, while keeping similar column efficiency. Thus, ODM columns allowed a higher differentiation for compounds with similar planar structures than ODS columns, probably because of the higher amount of C18 stationary phase bonded to the silica. As the aromatic character, acidic character and steric congestion increase, so does the retention on the ODM column. ODM column with methanol mobile phase also provides similar selectivity to ODS column with THF mobile phases. In general, the good performance of ODM columns, their higher retention and the differences in selectivity against ODS columns, makes them a good alternative to conventional C18 stationary phases, and allowed to consider them for future 2D-HPLC applications. Acknowledgements The authors gratefully acknowledge the postdoctoral fellowship awarded to Oscar N´un˜ ez and the Grant-in-Aid for Scientific Research No. P05394 and 17350036 funded by Japan Society for the Promotion of Science. References

Fig. 8. Separation of xylenols with ODM and ODS columns using a methanol:water 60:40 (v/v) solution as mobile phase. Uracil was used as tM standard. Detection was performed at 254 nm and at room temperature. See Section 2 for peak identification.

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