Evaluation of functionalized mesoporous silicas for reverse phase high performance liquid chromatography: An application for the separation of steroids

Microchemical Journal 114 (2014) 53–58

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Evaluation of functionalized mesoporous silicas for reverse phase high performance liquid chromatography: An application for the separation of steroids Judith Gañán, Sonia Morante-Zarcero, Damián Pérez-Quintanilla, Isabel Sierra ⁎ Departamento de Química Inorgánica y Analítica, E.S.C.E.T, Universidad Rey Juan Carlos, C/Tulipán s/n, 28933 Móstoles, Madrid, Spain

a r t i c l e

i n f o

Article history: Received 3 July 2013 Received in revised form 4 December 2013 Accepted 4 December 2013 Available online 16 December 2013 Keywords: Functionalized mesoporous silica Reversed-phase column High performance liquid chromatography Steroids

a b s t r a c t Conventional mesoporous silica (SM) and ethylene-bridged periodic mesoporous organosilica (PMO) were synthesized and modified with mono-functional octadecylsilane (C18). The materials were comprehensively characterized, before and after surface modification. Small angle X-ray diffraction, N2 adsorption–desorption measurement, scaning electron microscopy and transmission electron microscopy showed that all the prepared materials had non-symmetrical 3-D wormhole-like mesostructure and spherical morphology. Fourier transform infrared spectroscopy, 29Si MAS NMR and 13C MAS NMR spectroscopy, elemental and thermogravimetric analysis demonstrate the successful funcionalization of the materials with 0.242–0.388 and 0.204–0.402 mmol of C18 per g of SM-C18 and PMO-C18 silicas, respectively. HPLC columns packed with the prepared materials were tested by using a RP test mixture. From chromatographic parameters it was found that the separation power primarily depends on surface coverage. PMO-C18-2 and SM-C18-3 silicas were tested as a potential stationary phase for the effective separation of a mixture of five steroids. Separation for the five hormones was observed with the PMO-C18-2 column, which can be ascribed to the ethane groups in the mesoporous framework of this material. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Since the discovery of surfactant-template synthesis of ordered mesoporous silicas, the research on the preparation and characterization of these novel materials has expanded very fast. They have attractive properties such as high specific surface area, large mesopore volume, adjustable pore diameter and narrow pore size distribution. In addition, another advantageous property of these materials is the ease with which they can be functionalized in order to obtain hybrid mesoporous silicas [1]. Organic groups inside the silicate framework can also be introduced by using bisilylating organic precursors that leads to organic/inorganic mesoporous silicas (the so called periodic Abbreviations: ACN, acetonitrile; BET, Brunauer–Emmett–Teller; BJH, Barret–Joyner– Halenda; BTME, 1,2-bis(trimehoxysilyl)ethane; CTAB, cetyltrimethylammonium bromide; C8, octylsilane; C18, octadecylsilane; 13C MAS NMR, 13C magic angle spinning nuclear magnetic resonance; DAD, diode array detector; DES, diethylstilbestrol; dp, particle diameter; E2, 17β-estradiol; EE2, ethynilestradiol; F, flow rate; FTIR, Fourier transform infrared spectroscopy; K, permeability; k, retention factor; Lc, column length; Lo, functionalization degree; MeOH, methanol; N, number of theoretical plates; P, progesterone; PMO, periodic mesoporous organosilica; Rs, resolution; SEM, scaning electron microscopy; SM, conventional mesoporous silica; SP, stationary phase; 29Si MAS NMR, 29Si magic angle spinning nuclear magnetic resonance; T, testosterone; TEM, transmission electron microscopy; TEOS, tetraethylorthosilicate; TGA, thermogravimetric analysis; tR, retention time; t0, dead time; u, linear velocity; wh, width at the half of peak height; XRD, X-ray diffraction; α, separation factor; ΔP, back pressure. ⁎ Corresponding author. Tel.: +34 914887018; fax: +34 914888143. E-mail address: [email protected] (I. Sierra). 0026-265X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.microc.2013.12.003

mesoporous organosilicas). Compared with conventional mesoporous silicas, the periodic mesoporous organosilicas exhibit excellent hydrothermal, chemical and mechanical stability. In addition, the existence of the organic fragments and their neighboring silanol groups offers the possibility of synthesizing well-defined bifunctional materials. Cooperative synergistic effects can enhance the potential applications of these materials, in particular in applications such as adsorption [2]. Silica particles are the most widely used stationary phases (SP) for HPLC. Commercial HPLC columns are usually packed with amorphous silicas that exhibit low surface areas and large pore size distribution. In order to improve the chromatographic performance of these columns, recently, the application of hybrid mesoporous silicas as SP in HPLC columns has attracted a great deal of attention. In this sense, the crucial parameters to optimize are surface area, particle morphology, particle size and pore size. The high surface area of mesoporous silicas is accompanied by a high amount of potential binding sites, because apart from attachment of the organic components at the outer particle surface, the large pore size gives access to the binding sites at the pore interior. As a result, these materials can give rise to a high retention of some selected analytes to enhance the HPLC separation efficiency of multi-components [3]. The control of pore size and morphology represents also important issues. Thus, columns with spherical shaped or spheroidal (almost spherical) mesoporous silica particles are superior for chromatographic applications, because spherical particles provide higher efficiency, better column stability and lower back-pressures compared to irregularly shaped particles [4]. Finally, the type and functionality of the silane

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J. Gañán et al. / Microchemical Journal 114 (2014) 53–58

employed for surface modification and the amount of surface coverage are very crucial to achieve good separations [5]. The most widely used method of HPLC is RP with alkyl-bonded stationary phases (mainly octadecylsilane, C18, and octylsilane, C8). In this chromatographic mode, the separation mechanism is dependent upon interactions between analytes to separate, the mobile phase (typically a polar phase) and the surface of the non-polar solid stationary phase. In this sense, during the last years, a variety of mesoporous silicas functionalized with alkylsilanes have been applied successfully in RP-HPLC mode [3,5–8]. Although benefits of mesoporus silicas as packing materials in HPLC columns are mentioned in almost every contribution making use of them, still very few materials have been evaluated for this purpose (mainly SBA-15 and MCM-41). In this sense, compared to SBA-15 and MCM-41 silicas which have a 1-D mesoporous structure [1], the intraparticle transport can be facilitated in other mesporous silicas with a 3-D interconected pore structure, so this may be in favor of fast separations [9]. In the present paper, we report the preparation and characterization of various spherical mesoporous silicas, with non-symmetrical 3-D wormhole-like mesostructure, which were modified by mono-functional C18 in order to obtain materials with different surface coverage. For comparative purposes conventional mesoporous silica and ethylene-bridged periodic mesoporous organosilica were prepared. These materials were comprehensively characterized, before and after surface modification. HPLC columns packed with the prepared materials were tested by using a RP test mixture. The effects of textural properties and surface coverage on the separation were investigated. Finally, some of the columns were used for the separation of five steroids. To the best of our knowledge, this is the first reported study for the use of hybrid mesoporous silicas as SP for RP-HPLC to separate hormones. 2. Materials and methods 2.1. Chemicals Tetraethylorthosilicate (TEOS) 98%, poly(ethylene glycol)block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic 123), cetyltrimethylammonium bromide (CTAB) 98%, octadecyldimethylchlorosilane, and 1,2-bis(trimehoxysilyl)ethane (BTME) 96% were purchased from Sigma-Aldrich (St. Louis, MO, USA). Organic solvents were purchased from SDS (Peypin, France). Hydrochloride acid 35% was purchased from Panreac (Barcelona, España). HPLC-grade solvents acetonitrile (ACN) and methanol (MeOH) were purchased from Sigma-Aldrich. Stock standard solutions of 500 mg/L were prepared by diluting adequate amounts of diethylstilbestrol (DES, 98%), ethynilestradiol (EE2, 98%), 17β-estradiol (E2, 98%), testosterone (T, 99%) and progesterone (P, 99%) from Sigma-Aldrich in methanol and stored at − 20 °C. Working solutions were prepared at 5 mg/L of each hormone by an appropriate dilution of each stock solution in methanol. All working solutions were filtered through a 0.45 μm pore size nylon filter membrane before analysis. Water (resistance 18.2 MΩ·cm) was obtained from a Millipore Milli-Q-System (Billerica, MA, USA). All other reagents were at least of analytical grade. 2.2. Synthesis of mesoporous silicas Conventional spherical mesoporous silica (denoted as SM) was prepared according to the method of Ma et al. [10]. Ethylene-bridged spherical periodic mesoporous organosilica (denoted as PMO) was prepared according to the method of Zhu et al. [11]. Octadecyldimethylchlorosilane was used to functionalize SM and PMO silicas by post-synthesis method as follows: 0.132, 0.263, 0.395 and 0.526 g octadecyldimethylchlorosilane were dissolved in 60 mL toluene. Then, 2.5 g SM or 2.5 g of PMO silicas (obtained after heating at 70 °C under vacuum for 12 h) was added to each solution and the mixture was stirred for 24 h at 80 °C. The resulting materials (denoted SM-C18-1 to SM-C18-4 and PMO-C18-1 to PMO-C18-4, respectively)

were recovered by filtration, washed with toluene (2 × 50 mL), ethanol (2 × 50 mL) and diethyl ether (2 × 50 mL), and dried at 90 °C for 24 h. 2.3. Characterization methods X-ray diffraction (XRD) patterns of the silicas were obtained on a Philips Diffractometer model PW3040/00 X'Pert MPD/MRD at 45 kV and 40 mA, using Cu-Kα radiation (λ = 1.5418 Å). Scanning electron micrograph was carried out on a XL30 ESEM Philips with an energy dispersive spectrometry system (EDS). Conventional transmission electron microscopy was carried out on a TECNAI 20 Philips, operating at 200 kV. N2 gas adsorption–desorption isotherms were obtained using a Micromeritics ASAP 2020 analyzer. The Brunauer–Emmett–Teller (BET) surface area was calculated from the adsorption data over the relative pressure range from 0.0 to 1.0. The pore size distribution was calculated using the Barret–Joyner–Halenda (BJH) method from the adsorption branches. Infrared spectra were recorded on an Avatar 380 FTIR spectrophotometer. Proton-decoupled 29Si MAS NMR spectra were recorded on a Varian-Infinity Plus 400 MHz Spectrometer operating at 79.44 MHz proton frequency. Cross polarization 13C MAS NMR spectra were recorded on a Varian-Infinity Plus 400 MHz Spectrometer operating at 100.52 MHz proton frequency. Elemental analysis (wt.% C) was performed with a LECO CHNS-932 analyzer. Thermal stability of the modified mesoporous silicas was studied using a Setsys 18 A thermogravimetric analyzer. 2.4. Chromatographic evaluation experiments Chromatographic columns (150 mm × 4.6 mm id) were packed with the prepared C18-modified mesoporous silicas by the slurry method with chloroform/2-propanol (80/20, v/v) as slurry solvent by Analisis Vinicos Company (Tomelloso, Spain). The stainless steel columns were packed by using an HPLC slurry packer under 483 bar (7000 psi) pressure with methanol as packing solvent. A commercially available Ultrabase C18 column (150 mm × 4.6 mm id, 5 μm, 110 Å, 310 m2/g) from Sugelabor (Barcelona, Spain) was used for comparative purposes. Chromatographic tests were performed on a Varian ProStar chromatographic system (Varian Ibérica, Madrid, Spain). The system consisted of a 230 ProStar ternary pump, a ProStar 410 autosampler with a six-port injection valve equipped with a 20 μL injection loop (Rheodyne), a photodiode array detector, DAD 335 ProStar UV–vis detector and a PC-based data acquisition system Varian Star Workstation. The chromatographic performance of the prepared columns was tested with a test mixture for RP-HPLC obtained from Análisis Vínicos which contains three compounds (2,4-dimethylphenol, toluene and naphthalene). They were separated on the prepared columns with an ACN/H2O mobile phase (70/30, v/v) at a flow of 1.0 mL/min (columns packed with SM materials) or 0.5 mL/min (columns packed with PMO materials) with the detector operating at 254 nm (column temperature 25 °C). The chromatographic performance of SM-C18-3 and PMO-C18-2 columns was also tested with a working solution mixture of DES, EE2, E2, T and P hormones (prepared as indicate in the 2.1 section). They were separated on the columns using an ACN/H2O (70/30, v/v) as mobile phase at 1.0 mL/min in a column packed with SM-C18-3 and at 0.7 mL/min in column packed with PMO-C18-2. Detection was performed at 249 nm for T and P and at 280 nm for DES, EE2 and E2. Column temperature was 25 °C. 3. Results and discussion 3.1. Mesoporous silica characterization 3.1.1. XRD patterns and N2 adsorption–desorption isotherms The XRD patterns of the obtained SM and PMO materials exhibit a single (100) diffraction peak at the low angle region (0.49 and 0.41°,

Adsorbed Volume (cm3/g) STP

Adsorbed Volume (cm3/g) STP

J. Gañán et al. / Microchemical Journal 114 (2014) 53–58

700 600

Adsorption Desorption

500 400 300

a)

200 100 0 0,0

0,2

0,4

0,6

0,8

1,0

600

400

Adsorbed Volume (cm3/g) STP

Adsorbed Volume (cm3/g) STP

700 Adsorption Desorption

500 400

c)

200 100 0 0,0

0,2

0,4

0,6

b)

300 200 100 0 0,0

0,2

0,4

0,6

0,8

1,0

Relative Pressure (P/P0)

800

300

Adsorption Desorption

500

Relative Pressure (P/P0)

600

55

0,8

1,0

700 600 500

Adsoprtion desorption

400 300

d)

200 100 0 0,0

Relative Pressure (P/P0)

0,2

0,4

0,6

0,8

1,0

Relative Pressure (P/P0)

Fig. 1. Nitrogen adsorption–desorption isotherms for (a) SM, (b) SM-C18-3,(c) PMO and (d) PMO-C18-2.

of independent pores. However, in PMO silicas an H2 type adsorption hysteresis was observed (desorption branch with two distinctive steps), that is explained as a consequence of the interconnectivity of pores (presence of “ink-bottle” pores) [2]. The textural properties for the unmodified and modified materials are summarized in Table 1. After functionalization a gradual decrease in the SBET and pore volume was observed in all materials. However, the BJH pore diameter was only reduced significantly in SM-C18-4 and PMO-C18-4. This fact indicates that whereas in SM-C18-1–SM-C18-3 and PMO-C18-1–PMO-C18-3 the C18 groups are found mainly on the silica surface, in SM-C18-4 and PMO-C18-4 silicas there is also a significant amount of C18 groups within the pores. For this reason, after functionalization an increase in the wall thickness takes place in these materials (from 27.4 to 40.0 Å in SM-C18-3 and from 29.9 to 43.5 Å in PMO-C18-4) that confirms the presence of a ligand inside of the SM and PMO mesopores. The narrow pore size distribution found for these silicas provides evidence for its uniform framework mesoporosity (see Appendix A, supporting information SM1).

respectively). In these materials the d100 spacing, assigned to the poreto-pore center correlation distance, was 89.7 and 106.6 Å, respectively. This pattern is typical of materials with uniform pores in the mesoporous range and non-symmetrical 3-D wormhole-like structure of the porous framework, with a pore structure lacking a long-range order. The C18-modified materials exhibit almost identical XRD pattern which clearly indicates that the basic SM and PMO pore structures remain unchanged after surface modification. N2 adsorption–desorption isotherms of the SM and PMO materials indicated that both samples exhibited type IV isotherms according to the I.U.P.A.C. classification, characteristic of a mesoporous structure, with well-defined steps associated with the filling of the mesopores due to capillary condensation (Fig. 1a and c). Surface modification leaves the overall appearance of these isotherms practically unchanged, indicating that the textural characteristics are not altered by surface modification with C18 organic moieties (Fig. 1b and d). As can be seen in Fig. 1, an important difference in the type of hysteresis loop between both materials was observed. As expected, in SM silicas the hysteresis loop was of type H1, characteristic Table 1 Characterization data for mesoporous silicas. Material

SBETa (m2/g)

Pore volume (cm3/g)

BJHb pore diameter (Å)

L0c(mmol/g)

Surface coverage (μmol/m2)

SM SM-C18-1 SM-C18-2 SM-C18-3 SM-C18-4 PMO PMO-C18-1 PMO-C18-2 PMO-C18-3 PMO-C18-4

740 714 635 595 526 1103 975 891 791 689

1.06 1.02 0.92 0.87 0.78 1.10 1.05 0.96 0.88 0.80

62.3 62.1 62.0 61.0 52.0 76.7 77.8 77.5 75.1 63.1

– 0.242 0.305 0.350 0.388 – 0.204 0.254 0.315 0.402

– 0.338 0.480 0.588 0.737 – 0.209 0.285 0.398 0.583

a b c

Brunauer–Emmett–Teller surface area. Barrett, Joyner and Halenda. Millimols of C18 per gram of mesoporous silica.

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J. Gañán et al. / Microchemical Journal 114 (2014) 53–58

3.1.2. SEM and TEM images Typical TEM images of SM and PMO (see Appendix A, supporting information) show irregularly aligned mesopores throughout the materials with relatively uniform pore sizes (wormhole-like pore arrangement). These results are in good agreement with the related XRD patterns and N2 adsorption–desorption isotherms. SEM pictures demonstrate the spherical morphology of the SM and PMO materials, with diameters ranging from 3 to 5.5 μm for SM and 1.5 to 4.5 μm for PMO (see Appendix A, supporting information SM2). In both preparations, the addition of ethanol as co-solvent plays an important role in the formation of silica spheres [9,10]. After functionalization, all prepared materials kept the same morphology, particle size and disordered wormhole-like structure as that of the as-synthesized silicas. 3.1.3. FTIR, 29Si MAS NMR and 13C MAS NMR spectra The FTIR spectra of SM and PMO materials, modified after reaction with octadecyldimethylchlorosilane, exhibited a main new peak near 2930 cm−1 due to the stretching vibration of the CH3 groups, confirming the functionalization of these materials. Furthermore, in the PMO spectra, the stretching vibration of –CH2–CH2– at 2900 and 2850 cm−1, respectively as well as the C–H deformation vibration at 1470 cm−1, clarified the existence of ethane groups in this material (see Appendix A, supporting information). 29Si MAS NMR spectrum of unmodified SM showed Q4 sites [Si(OSi)4], ascribed to surface silicon atoms with four siloxane bonds, as the most abundant sites at −110 ppm and two shoulders at −100 and −90 ppm ascribed to Q3 [Si(OH)1(OSi)3] and Q2 [Si(OH)2(OSi)2] sites, respectively. The spectra of the functionalized SM materials, showed a decrease in the intensity of the Q3 and Q2 signals and the apparition of a new signal at 10 ppm (M site corresponding to the silicon atom of the octadecyldimethylsilane), which verified the anchoring of the functional groups to the SM surface. On the other hand, 29Si MAS NMR spectrum for unmodified PMO silica exhibits a broad resonance at −60 ppm corresponding to the T2 [CSi(OH)(OSi)2] sites and two shoulders at −66 and −48 ppm, corresponding to the T3 [CSi(OSi)3] and T1 [CSi(OH)2(OSi)] sites, respectively. In addition, no signals related with Qn sites in the range from − 90 to −110 ppm were observed. After functionalization of PMO, the apparition of a new signal at 12 ppm (M site) corresponding to the silicon atom of the octadecyldimethylsilane verified the anchoring of the functional groups to the PMO surface (see Appendix A, supporting information SM3) 13C MAS NMR spectra in the solid state of the C18-modified SM clearly display peaks at 28, 21 and 14 ppm, corresponding to the carbon atoms on the octadecyl chain (−(CH2)16–, –CH3 and –CH2–, respectively). As expected, in PMO silicas similar signals were observed, but an intense additional new peak appeared at 5.5 ppm, assigned to the bridging ethane moiety (see Appendix A, supporting information). 3.1.4. Elemental and thermogravimetric analysis As expected, elemental analysis demonstrated that the C18 groups were successfully grafted in each material, and that the C18 group density increased with the increase of the octadecyldimethylchlorosilane concentration in the functionalization reaction (Table 1). The functionalization degree (Lo, mmol/g) obtained was similar or slightly higher than those reported previously for other mesoporous silicas modified with mono-functional octadecylsilane [9,12], leading a surface coverage between 0.34 and 0.74 μmol/m2 and between 0.21 and 0.58 μmol/m2, respectively. In the PMO silica the content of the ethane groups, calculated from the wt.% C before their functionalization, was 6.8 mmol/g, which is similar to the content in other ethylene-bridged PMOs [13]. TGA curves of the C18 modified silicas (see Appendix A, supporting information SM4) show that exothermic degradation processes occur between 200 and 550 °C, with weight loss between 6 and 10% in SM-C18 silicas and between 18 and 24% in PMO-C18 silicas. The thermal stability of these samples is in agreement with previous results given in the literature for other functionalized mesoporous silicas

[9], and the weight loss confirmed the Lo estimated by elemental analysis. 3.2. Chromatographic performance of the prepared HPLC columns 3.2.1. Separation of a reversed-phase test mixture As surface coverage is an important factor that affects the chromatographic performance of RP stationary phases, the functionalized mesoporous silicas prepared with different Lo were slurry-packed into HPLC columns for the separation of a test mixture of 2,4dimethylphenol, toluene and naphthalene. Analyses were carried out using an ACN/H2O (70/30, v/v) as mobile phase at a flow of 1.0 mL/min (columns packed with SM materials) or 0.5 mL/min (columns packed with PMO materials). The retention factor (k) for each analyte was measured according to the equation [14]:

k ¼ ðtR –t0 Þ=t0

ð1Þ

where t R is the retention time of the analyte; t 0 is the dead time, estimated by using the peak resulting from the change in refractive index from the injection solvent on the column. The t0 values were between 1.61 and 1.26 min in columns packed with C18 -modified SM and between 2.50 and 2.67 min in columns packed with C18-modified PMO. The k values calculated according Eq. (1) are summarized in Table 2. As expected, for RP chromatography, in all cases the more polar analyte (2,4-dimethylphenol) eluted faster than the less polar ones (toluene and naphthalene). In addition, from Table 2 we can see that longer retention times and better separation factor (α) were obtained using SM-C18-3 and SM-C18-4 columns. The different retention times can be explained by the different loads of the incorporated C18 groups in these four materials. Thus, the Lo in SM-C18-1 was 0.24 mmol/g based on elemental analysis, which was much lower than that of SM-C18-4 (0.39 mmol/g). However, in comparison with the commercial Ultrabase C18 column under the same separation conditions, lower k values were obtained on the home-made SM-C18-4 column for these compounds (k = 0.90, 2.17 and 2.78 in Ultrabase C18 and k = 0.75, 1.48 and 1.82 in SM-C18-4 for 2,4-dimethylphenol, toluene and naphthalene, respectively). These results were attributable to the lower hydrophobicity of the SM-C18-4 silica, due to their lower C18 surface coverage in comparison with the commercial column (9.3 and 18% C, respectively). In a recent paper, Ide et al. [12] demonstrated that the amount of C18 groups that can be grafted on the surface of ordered mesoporous silicas is much smaller than on the surface of the amorphous silicas. The main reason for this is that the grafting reaction is strongly diffusion limited, as the surface area of these materials is basically internal surface area. These results evidence that appropriate functionalization of mesoporous silicas, essential to their successful application as RP-HPLC packing, could be achieved by grafting smaller silanes [4]. Ethylene-bridged PMOs with different C18 coverage were also used to study the separation of the test mixture. As was shown in Table 2, the order of peaks in the PMO-C18 columns was the same as in the SM-C18 columns. However, as expected, the results obtained confirmed that despite the fact that Lo of PMO-C18 silicas was very similar than that of the SM-C18 silicas, the large amount of –CH2–CH2– groups (6.8 mmol/g) contributed to additional interactions and stronger retention of the analytes in these columns. Thus, a noticeable decrease in the α for 2,4-dimethylphenol and toluene and an increase in the α for toluene and naphthalene were observed in these columns. Therefore, compared to mono-functionalized SM-C18, the bifunctionalized PMO-C18 may be advantageous as a stationary phase in RP-HPLC because the ethane groups that are an integral part of their mesoporous framework can improve the chromatographic system potential for separating a mixture of different compounds.

J. Gañán et al. / Microchemical Journal 114 (2014) 53–58

57

Table 2 Retention factor and separation factor of columns packed with SM-C18 and PMO-C18 silicas for a reversed-phase test mixturea. Analyteb

SM-C18-1

1 2 3 Analyteb

SM-C18-2

k

αc1–2

0.311 0.509 0.509

–

k

α1–2

0.551 0.874 0.979

1.59

–

αd2–3

k

α1–2

0.732 1.020 1.214

1.39

1.14

PMO-C18-1

1 2 3

SM-C18-3

αd2–3

k

α1–2

0.765 1.249 1.450

1.63

1.12

α2–3

k

α1–2

0.808 1.141 1.372

1.41

1.19

PMO-C18-2

k

αc1–2

0.465 0.659 0.753

1.41

SM-C18-4

α2–3

α2–3

k

α1–2

0.754 1.478 1.827

1.68

1.16

α2–3

k

α1–2

1.184 1.848 2.308

1.56

1.20

PMO-C18-3

α2–3 1.17

PMO-C18-4 α2–3 1.25

a Analysis were carried out using acetonitrile/water (70/30, v/v) as mobile phase at a flow of 1.0 mL/min (columns packed with SM materials) or 0.5 mL/min (columns packed with PMO materials). – Not calculated. b 1 = 2,4-dimethylphenol, 2 = toluene; 3 = naphthalene. c α1–2 = Separation factor for 2,4-dimethylphenol and toluene (α1–2 = k2/k1). d α2–3 = Separation factor for toluene and naphthalene (α2–3 = k3/k2).

To measure an important chromatographic parameter such as column efficiency, expressed as the number of theoretical plates (N), the following equation was used [14]: 2

N ¼ 5:54 ðtR =wh Þ

ð2Þ

where wh is the width at the half of peak height. Table 3 shows the N values for SM-C18 and PMO-C18 columns calculated for the naphthalene peak in the test mixture according to Eq. (2). Taken into account that in the best case only 27,761 plates/m were observed (column SM-C18-3), it is obvious that this aspect needs further improvements by extensive size classification of the silica particles followed by fully optimized column packing processes. In this sense, more compact and uniform packing of sieved particles in chromatography columns seems to cause better separation efficiency [15]. Fig. 2 shows the chromatograms for the separation of the RP test mixture with the columns packed with SM-C18-3 and PMO-C18-2 materials at 0.4 and 0.6 mL/min, respectively. Well separated peaks for the three tested compounds were obtained in 5 and 8 min in the PMO-C18-2 and SM-C18-3 columns, respectively. However, as expected the peaks in both chromatograms appeared to be somewhat broad. 3.3. Chromatographic separation of steroid hormones with SM-C18-3 and PMO-C18-2 columns

important levels of these compounds in rivers, lakes, etc. can be a potential risk for the environment and in the future may be for humans, especially because synergistic and long-term effects are not fully understood. Fig. 3 shows typical chromatograms for the separation of a mixture of five steroids in SM-C18-3 and PMO-C18-2 columns using isocratic elution (ACN/H2O, 70/30, v/v) at 1 and 0.7 mL/min, respectively. In Fig. 3a, it can be seen that good separation was achieved for the steroids in 5.5 min in the SM-C18-3 column, except only for DES and EE2 that coeluted. On the other hand, all five steroids were separated in the PMO-C18-2 column (Fig. 3b) that can be ascribed to the presence of ethane groups in the mesoporous framework of this material. The retention of steroids decreased with increased concentration of ACN in the mobile phase under the investigated conditions, indicating that the hydrophobic interaction was predominant between the analytes and the stationary phase. The above results demonstrated the great potential of the studied silicas as column packing materials for RP-HPLC. 4. Concluding remarks Conventional mesoporous silica and ethylene-bridged periodic mesoporous organosilica with spherical morphology and non-symmetrical 3-D wormhole-like mesostructure have been successfully prepared and characterized. These silicas were functionalized with different amounts

3

Steroid hormones are a group of important emerging contaminants for their toxicity and persistence in the environment. The presence of Table 3 Number of theoretical plates of columns packed with SM-C18 and PMO-C18 silicas for a reversed-phase test mixture.a Analyteb

1 2 3 Analyteb

1 2 3

SM-C18-2 tR

wh

2.21 2.67 2.82

0.105 0.131 0.126

SM-C18-3 Nc

tR

wh

18,412

2.37 3.02 3.29

0.102 0.114 0.120

Nc

tR

wh

20,920

4.84 5.73 6.35

0.352 0.382 0.411

PMO-C18-2 tR

wh

4.37 5.11 5.60

0.235 0.264 0.235

SM-C18-4 N

tR

wh

N

27,762

2.40 3.10 3.39

0.105 0.131 0.157

17,024

PMO-C18-3

SM-C18-3

2

PMO-C18-2

Absorbance (AU)

1

3

1

2

PMO-C18-4 N

tR

wh

N

8787

5.46 7.12 8.27

0.241 0.323 0.352

20,287

0

a Analysis were carried out using acetonitrile/water (70/30, v/v) as mobile phase at a flow of 1.0 mL/min (columns packed with SM materials) or 0.5 mL/min (columns packed with PMO materials). b 1 = 2,4-dimethylphenol, 2 = toluene; 3 = naphthalene. c Number of plates per meter of column estimated from the naphthalene peak.

1

2

3

4

5

6

7

8

9

10

Time (min) Fig. 2. Chromatograms showing the separation of a reversed-phase test mixture of (1) 2,4-dimethylphenol, (2) toluene and (3) naphthalene Analysis were carried out using acetonitrile/water (70/30, v/v) as mobile phase at 25 °C, UV detection at 254 nm. Flow rate: 0.6 mL/min in PMO-C18-2 column and 0.4 mL/min in SM-C18-3 column.

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J. Gañán et al. / Microchemical Journal 114 (2014) 53–58

a)

Acknowledgments DES+EE2

The authors thank the financial support from the Comunidad Autónoma de Madrid and European funding from the FEDER Programme (project S2009/AGR-1464, ANALISYC-II) and Ministerio de Ciencia e Innovación (project CTQ2008-05821/PPQ). The authors have declared no conflict of interest.

Absorbance (AU)

E2

DES

T

Appendix A. Supplementary data P

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.microc.2013.12.003.

References 0

1

2

3

4

5

6

7

Time (min)

b) DES EE2

Absorbance (AU)

E2

T DES

0

1

2

3

P

4

5

6

7

Time (min) Fig. 3. Chromatograms showing the separation of a mixture of diethylstilbestrol (DES), ethynilestradiol (EE2), 17β-estradiol (E2), testosterone (T) and progesterone (P) hormones using (a) SM-C18-3 and (b) PMO-C18-2 columns. Analysis was carried out using acetonitrile/water (70/30, v/v) as mobile phase at 25 °C. Detection: 249 nm for T and P; 280 nm for DES, EE2 and E2. Flow rate: 0.7 mL/min in PMO-C18-2 column and 1.0 mL/min in SM-C18-3 column.

of C18 groups to obtained mono and bifunctional materials and its great potential of application as stationary phases in RP-HPLC was demonstrated by using a test mixture of 2,4-dimethylphenol, toluene and naphthalene. The SM-C18-3 and PMO-C18−2 columns provided good separation of different steroids, illustrating the applicability of these materials.

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