Preparation and Characterization of Alkylphosphonate-Modified ...

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ANALYTICAL SCIENCES JUNE 2000, VOL. 16 2000 © The Japan Society for Analytical Chemistry

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Preparation and Characterization of Alkylphosphonate-Modified Magnesia-Zirconia Composite for Reversed-Phase Liquid Chromatography Yu-Qi FENG,*† Qing-He ZHANG,* Shi-Lu DA,* and Yan ZHANG** *Department of Chemistry, Wuhan University, Wuhan, 430072, P. R. China **Laboratory of MRAMP of China, Wuhan, 430072, P. R. China

A new material: magnesia-zirconia composite modified by an alkylphosphonate can be used as a reversed-phase stationary phase for high-performance liquid chromatography. The new material was characterized by elemental analysis, FTIR and 13C solid state NMR spectrometry. The pH stability of the new material was investigated using biphenyl and N,N′-dimethylaniline as probes with methanol–water (60:40, v/v) as a mobile phase, after the column was continuously purged with solutions at extreme pH 2 and pH 11. The chromatographic performance of the new material was studied by using polycyclic aromatic hydrocarbons (PAHs) and basic compounds as probes. The results indicate that the new material is of high pH stability and can be used for separation of PAHs and basic compounds. (Received August 27, 1999; Accepted March 1, 2000)

Since Nakamura and co-workers demonstrated zirconia as a packing material suitable for high-performance liquid chromatography (HPLC),1–3 a great deal of attention has been paid to its application to HPLC. However, the applicability of this material is usually limited by the fact that there exist Bronsted acid sites, Bronsted basic sites and Lewis acid sites on the surface of zirconia, which can lead to irreversible adsorption of some compounds which should be separated.4 Therefore, modification of this material is required to block such irreversible adsorption. Many efforts have been made to develop modified-zirconia packing materials for HPLC. Trudinger et al.5 and Yu et al.6 have tried to prepare chemically bonded zirconia with silanes for hydrophobic and hydrophilic stationary phases. However, Zr–O–Si bonds are quite unstable compared with Si–O–Si bonds.5,7 Carr and co-workers have extensively studied carbon8–10 and polymers11–15 coatings on zirconia as surface modifiers of zirconia to create anion-exchange, hydrophobic and hydrophilictype phases with varying degrees of success. Since bare zirconia has a great affinity for Lewis bases, Carr and coworkers have studied modification of zirconia with Lewis bases such as fluoride,16 phosphate17 and ethylenediamine-N,N′tetramethylphosphonic acid (EDTPA),18 and their application in separation of proteins. We have studied preparation of spherical zirconia by sol-gel process19 and modification of zirconia with stearic acid as reversed-phase for HPLC.20 Nevertheless, low column efficiency was obtained due to low surface area and poor pore structure of the zirconia. Particle pore characteristics are crucial factors for chromatographic performance.21,22 Nawrocki and co-workers have described the changes in surface area, pore size, and pore volume of zirconia with thermal treatment.23 Carr and co-workers have compared the pore structures of zirconia synthesized with †To whom correspondence should be addressed. Part of the work was presented at Asianalysis V.

oil emulsion (OEM) and polymerization-induced colloidal aggregation (PICA) methods by means of nitrogen sorptometry, mercury porosimetry, size exclusion chromatography and NMR techniques.24–26 They pointed out that nitrogen sorptometry and mercury porosimetry indicated a significant difference in pore volume and pore size distribution, and the presence of a considerable number of pore constrictions for the zirconia synthesized with the different methods. However, NMR results indicated that they have same average pore size and tortuosity.25 Shalliker and Douglas have reported that the pore diameters, surface area and pore volumes of zirconia could be controlled by sodium chloride impregnation technique, and indicated that poor quality of pore structures was obtained without sodium chloride.27 The crystalline phase of zirconia has also been shown to influence the surface area. Shalliker et al. have recently prepared spherical silica-zirconia composites by coating zirconia microspheres with silica. Their results illustrated that addition of the silica onto the zirconia microspheres prevented the formation of monoclinic zirconia and delayed the crystallization of the tetragonal form to temperatures greater than 700˚C.28,29 Recently, we have prepared spherical silica-zirconia and magnesia-zirconia composites by the coprecipitation of zirconyl chloride with sodium metasilicate and magnesium chloride, respectively.30,31 Those composites crystallized into tetragonal crystal structure after calcination at 600˚C, while the zirconia crystallized into monoclinic phase after calcination at the same temperature. These results suggest that not only silica but also magnesia are able to stabilize the tetragonal phase of zirconia. The silica-zirconia composite has small pore size and low chromatographic performance, which suggests that the composite is an aggregate of very fine colloids of silica and zirconia. Nevertheless, the magnesia-zirconia composite exhibits appropriate surface area, pore size, pore size distribution and a better chromatographic performance.31 In the present study, we report a new material of the porous

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ANALYTICAL SCIENCES JUNE 2000, VOL. 16 Table 1

Physicochemical properties of magnesia-zirconia composite

Mixed oxide Magnesiazirconia

Mg/Zr molar ratio

Average particle size/µm

Average pore size/nm

Specific surface area/ m2 g–1

Acidity at pH 10/ mmol g–1

Basicity at pH 3.5/ mmol g–1

pH of p.z.c.

11.4/88.6

5

17.8

58

Not detected

1.0

11.5

spherical magnesia-zirconia composite modified by an alkylphosphonate, as a reversed-phase stationary phase for HPLC. We have characterized the structure of the modified magnesiazirconia composite by means of elemental analysis, FTIR and NMR. Its chromatographic performance was also investigated with polycyclic aromatic hydrocarbons, positional isomers and some basic compounds as solutes.

Experimental Chemicals All reagents were obtained from commercial sources and were of reagent grade or better unless indicated otherwise. Zirconyl chloride (ZrOCl2·8H2O), polyoxyethylenesorbitan trioleate (Tween 85), sorbitan monooleate (Span 80), methanol, and all the samples were purchased from Shanghai General Chemical Reagent Factory (Shanghai, China). Magnesium chloride (MgCl2·6H2O) was obtained from Guangzhou Chemical Plant (Guangzhou, China). Hexamethylenetetramine (HMTA) and urea were obtained from Chengdu Chemical Plant (Chengdu, China). Cyclohexane was obtained from Tianjing Chemical Plant (Tianjing, China). 1-Dodecanol was purchased from BDH (Poole, England). Zorbax ODS (5 µm) was purchased from Dupont. Fosfomycin, sodium cis-(3-methyloxiranyl)phosphonate, was provided by Northeastern Pharmaceutical Factory (Shengyan, China). Distilled water, before use, was boiled for 15 min to remove dissolved carbon dioxide. Preparation of magnesia-zirconia composite Spherical magnesia-zirconia composite was prepared as Some of the physicochemical described previously.30,31 characteristics of the magnesia-zirconia composite are shown in Table 1. The data have been discussed in detail in another paper.31 Preparation of alkylphosphonate 1-Dodecanol (100 mL) and sodium hydride (2.0 g) was stirred for 30 min at room temperature in an inert atmosphere of nitrogen gas. After 2.0 g of fosfomycin was added, the mixture was allowed to react at 120˚C for 48 h also in an inert atmosphere of nitrogen gas. The reaction was then cooled and adjusted to neutral with hydrochloric acid. Thereafter, the product (alkylphosphonate in 1-dodecanol) was washed five times with distilled water in a separatory funnel to remove unreacted fosfomycin. Preparation of alkylphosphonate-midified magnesia-zirconia The magnesia-zirconia composite was slurry-packed into a 15 cm × 4.6 mm stainless-steel HPLC column. Fifty milliliters of the 1-dodecanol containing alkylphosphonate were dissolved in 300 mL of methanol. This solution was pumped through the column for 4 h at room temperature, followed by washing the column with methanol and water in sequence to remove unadsorbed alkylphosphonate. Elemental analysis of the

alkylphosphonate-midified magnesia-zirconia was performed with a MOD-1106 elemental analyzer (Italy), and Fourier transform infrared (FTIR) spectroanalysis was carried out with an FTIR-8201PC spectrophotometer (Shimadzu, Japan). 13C CP-MAS NMR measurements were performed on a locallyassembled 200 MHz spectrometer (Laboratory of MRAMP, Wuhan, China) on the alkylphosphonate-modified magnesiazirconia composite in a zirconium oxide rotor at a rate of 3500 Hz. The spectrum was recorded with a pulse length of 3 µs and a contact time of 1 ms. Ten thousand scans were accumulated with a repetition rate of 1 s for the spectrum. The NMR spectrum was externally referenced to liquid tetramethylsilane. Chromatography Chromatographic tests were carried out with standard HPLC equipment: a Shimadzu 10A liquid chromatographic pump; SPD-10A UV-Vis photometric detector; Rheodyne 7125 injection system (sample loop, 20 µL); Shimadzu C-R6A integrator (Shimadzu, Kyoto, Japan). The alkylphosphonatemodified magnesia-zirconia column mentioned above was used. Zorbax ODS was slurry-packed into a 15 cm × 4.6 mm stainless-steel HPLC column. The column temperature was controlled at 28±1˚C through a water bath. Methanol and water or buffer solutions were used to prepare mobile phases. Before use, the mobile phases were filtered through a G-4 fritted glass funnel and degassed in an ultrasonic bath for 5 min under reduced pressure. The flow rate of the mobile phase was set at 0.5 mL min–1. The compounds used as samples were dissolved in methanol. The concentration range of samples was approximately 0.5 – 5 mmol L–1. Typically, a 5 µL volume of sample solution was injected. The wavelength used for detection was 254 nm. The retention time of solvent peak was used as dead time for calculation of capacity factors. pH stability of the alkylphosphonate-modified magnesiazirconia composite The pH stability of the alkylphosphonate-modified magnesiazirconia composite column was investigated according the method proposed by Kirkland et al.32 with some modifications. The column was continuously purged at 1.0 mL min–1 with a methanol–HCl solution at pH 2 (50:50, v/v) or methanol–NaOH solution at pH 11 (50:50, v/v) at temperature 28±1˚C. The column was periodically tested with biphenyl and N,N′dimethylaniline as probes with a methanol–water (60:40, v/v) as a mobile phase.

Results and Discussion The Lewis sites on the surface of zirconia can form very strong coordination complexes with hard Lewis bases.33,34 Fosfomycin is a strong Lewis base, which is able to form a very stable complex with zirconium ions.35 Therefore, fosfomycin, after reacting with 1-dodecanol to form an alkylphosphonate, was used to modify the surface of magnesia-zirconia composite in

ANALYTICAL SCIENCES JUNE 2000, VOL. 16 this experiment. The probable structure of the alkylphosphonate used is shown in Fig. 1. Surface coverage of the alkylphosphonate on magnesia-zirconia composite was calculated from elemental analysis data (carbon content). It was

Fig. 1 13C CP-MAS NMR spectrum of the alkylphosphonatemodified magnesia-zirconia composite.

Fig. 2

581 found to be 6.1 µmol m–2, which is similar to phosphate coverage,17 but greater than EDTPA coverage on zirconia.18 The larger coverage of the alkylphosphonate might be ascribed to its linear structure (Fig. 1) and greater concentration of Lewis acid sites of the magnesia-zirconia composite. The FTIR spectrum of the alkylphosphonate-modified magnesia-zirconia composite, after subtraction of the native magnesia-zirconia composite, is shown in Fig. 2. The modified material has peaks at 2800 – 3000 cm–1, which are characteristics of the C–H stretching frequency. The spectrum also shows a broad band at approximately 1100 cm–1 which shows the presence of the P=O group. 13C CP-MAS NMR analysis provides further structure information of the modified material (see Fig. 1). The column efficiency of the alkylphosphonate-modified magnesia-zirconia composite was determined with methanol–water (80:20, v/v) as mobile phase. The number of theoretical plates was found to be 26000 m–1 using biphenyl as solute at a flow rate of 0.5 mL min–1, which is similar to that of Zorbax ODS (5 µm) slurry-packed in our laboratory, but not as good as the commercially available column packed with alkylbonded silica material with same particle size. Higher performance may be achieved through further improvement of its particle size distribution and packing technique. In order to investigate pH stability of the alkylphophonatemodified magnesia-zirconia composite, we have examined the retention of biphenyl and N,N′-dimethylaniline with methanolwater (60:40, v/v) as a mobile phase on the alkylphophonatemodified magnesia-zirconia composite column which was

FTIR spectrum of the alkylphosphonate-modified magnesia-zirconia composite.

Fig. 3 Investigation of pH stability for the alkylphosphonate-modified magnesia-zirconia composite. Purge solution: a, methanol–HCl solution at pH 2 (50:50, v/v); b, methanol–NaOH solution at pH 11 (50:50, v/v). Column temperature: 28 ± 1˚C. Mobile phase for test of solutes: methanol-water (60:40, v/v ).

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Fig. 5 Typical chromatogram of a mixture of polycyclic aromatic hydrocarbons. Mobile phase: methanol–water (70:30; v/v). Flow rate: 0.5 mL min–1; 1, benzene; 2, toluene; 3, naphthalene; 4, biphenyl; 5, fluorene; 6, phenanthrene; 7, anthrancene. Fig. 4 Plots of ln k′ of some basic solutes versus the methanol content of the mobile phase at 0.5 mL min–1 flow rate.

continuously purged with methanol–pH 2 HCl solution (50:50, v/v) and methanol–pH 11 NaOH solution (50:50, v/v), respectively. The results are shown in Figs. 3a and 3b. Little variation of the retention values of the two solutes on the new stationary phase is found after they were purged with the solutions at pH 2 and 11. These results imply that the alkylphosphonate-modified magnesiazirconia stationary phase is very stable against extreme pH solutions. This is different from the organophosphonatemodified zirconia that has limited stability, as reported by Rigney.7 The difference of pH stability might be ascribed to the existence of magnesia on the surface of the magnesia-zirconia composite, which might show higher affinity to the alkylphosphonate. In order to explain why the affinity between the alkylphosphonate and the magnesia-zirconia composite is so strong, further studies are required. The effect of the pH (2 – 10) of mobile phase on the retention of benzene on the new stationary phase was also investigated. We found that an almost constant capacity factor of benzene was obtained over a wide pH range, even though the measurement of retention time of benzene was performed after at least 500 mL of mobile phase at given pH was pumped through the column at flow rate of 0.5 mL. The result indicates that the retention of benzene on the stationary phase is independent of the pH of the mobile phase. The chromatographic performance of the column was evaluated using polycyclic aromatic hydrocarbons, nitroanilines, and some basic compounds as solutes and a mixture of methanol and water as mobile phase. Figure 4, as an example, shows the influences of mobile phase component on the retention of some basic compounds. It can be seen that the retention of the basic solutes decrease drastically with increase of methanol content in the mobile phases, and there was excellent linear dependence of ln k′ on the methanol content. Other solutes tested exhibited similar behavior. These results indicate that the alkylphosphonate-modified magnesia-zirconia is strongly hydrophobic, and behaves as a reversed-phase packing material. A mixture of polycyclic aromatic hydrocarbons (PAHs) can be separated by using methanol–water as the mobile phase on the modified magnesia-zirconia stationary phase. The chromatogram of their separation is shown in Fig. 5. The elution order of the PAHs is same as that on Zorbax ODS column, but their capacity factors are slightly smaller than those on Zorbax ODS under the

Fig. 6 Typical chromatogram of a mixture of basic solutes. Mobile phase: methanol–water (60:40; v/v). Flow rate: 0.5 mL min–1; 1, pyridine; 2, aniline; 3, p-toluidine; 4, o-nitroaniline; 5, βaminonaphthalene; 6, N,N′-dimethylaniline.

same mobile phase condition. This may be ascribed to shorter alkyl chain on the surface of the magnesia-zirconia composite. The typical chromatogram shown in Fig. 6 illustrates the separation of five basic solutes and o-nitroaniline on the modified magnesia-zirconia stationary phase with methanol–water (60:40, v/v) as the mobile phase. They can be separated well and exhibit peaks with slight tailing. For comparison, the same mixture was also supplied to a Zorbax ODS column under the same chromatographic condition to obtain the corresponding chromatogram (Fig. 7). It can be seen from Fig. 7 that pyridine, p-toluidine and o-nitroaniline can not be separated, and N,N′-dimethylaniline has greater retention time than that on the modified magnesia-zirconia column. The reversed elution order of pyridine and aniline was also observed on the Zorbax ODS. The difference in retention behavior of the two solutes on the Zorbax ODS and the modified magnesiazirconia columns may result from the difference in matrices of the two stationary phases.

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Fig. 7 Typical chromatogram of a mixture of basic solutes using a Zorbax ODS column (15 cm × 4.6 mm) with methanol–water (60:40; v/v) as mobile phase at 0.5 mL min–1 flow rate; 1, aniline; 2, pyridine; p-toluidine; o-nitroaniline; 3, β-aminonaphthalene; 4, N,N′dimethylaniline.

In conclusion, we have found that porous particulate of the alkylphosphonate-modified magnesia-zirconia composite behaves as reversed-phase packing, and is of high pH stability. The alkylphosphonate-modified magnesia-zirconia is a viable support for reversed-phase chromatography of neutral and basic solutes. It is expected that organophosphonates can be used as modifiers in preparation of magnesia-zirconia composite-based stationary phase like silanes in silica-based stationary phase.

Acknowledgements The financial supports from the National Nature Science Foundation of China, Hubei Provincial Nature Science Foundation of China, Wuhan University, and the Laboratory of MRAMP of China are gratefully acknowledged.

References 1. M. Kawahara, H. Nakamura, and T. Nakajima, Anal. Sci., 1988, 4, 671. 2. M. Kawahara, H. Nakamura, and T. Nakajima, Anal. Sci., 1989, 5, 485. 3. H. Nakamura, Bunseki, 1991, 1007. 4. B. Lorenz, S. Marmé, W. E. G. Müller, K. Unger, and H. C. Schroder, Anal. Biochem., 1994, 216, 118. 5. U. Trudinger, G. Muller, and K. Unger, J. Chromatogr., 1990, 535, 111. 6. J. Yu and Z. E. Rassi, J. Liq. Chromatogr., 1994, 17, 773. 7. M. P. Rigney, Ph. D. Thesis, University of Minnesota, Minneapolis, MN, 1989. 8. T. P. Weber and P. W. Carr, Anal. Chem., 1990, 62, 2620.

583 9. T. P. Weber, P. T. Jackson, and P. W. Carr, Anal. Chem., 1995, 67, 3042. 10. P. T. Jackson, T.-Y. Kim, and P. W. Carr, Anal. Chem., 1997, 69, 5011. 11. C. McNeff, Q. H. Zhao, and P. W. Carr, J. Chromatogr. A, 1994, 684, 201. 12. L. Sun and P. W. Carr, Anal. Chem., 1995, 67, 3717. 13. C. J. Dunlap and P. W. Carr, J. Chromatogr. A, 1996, 746, 199. 14. J. W. Li and P. W. Carr, Anal. Chem., 1997, 69, 2193. 15. Y. Hu and P. W. Carr, Anal. Chem., 1998, 70, 1934. 16. J. A. Blackwell and P. W. Carr, J. Chromatogr., 1991, 549, 51. 17. W. A. Schafer and P. W. Carr, J. Chromatogr., 1991, 587, 149. 18. A. M. Clausen and P. W. Carr, Anal. Chem., 1998, 70, 378. 19. Q.-H. Zhang, Y.-Q. Feng, and S.-L. Da, Chinese J. Chromatogr., 1999, 17, 284. 20. Q.-H. Zhang, Y.-Q. Feng, and S.-L. Da, Chinese J. Chromatogr., 1999, 17, 229. 21. W. W. Yau, J. J. Kirkland, and D. D. Bly, “Modern Size Exclusion Liquid Chromatography”, 1979, John Wiley & Sons, New York. 22. R. A. Shalliker and G. K. Douglas, J. Liq. Chrom. Rel. Technol., 1998, 21, 2413. 23. J. Nawrocki, M. P. Rigney, A. McCormick, and P. W. Carr, J. Chromatogr., 1993, 657, 229. 24. C. F. Lorenzano-Porras, P. W. Carr, and A. V. McCormick, J. Colloid Interface Sci., 1994, 164, 1. 25. C. F. Lorenzano-Porras, M. J. Annen, M. C. Flicking, P. W. Carr, and A. V. McCormick, J. Colloid Interface Sci., 1995, 170, 299. 26. C. J. Dunlap, P. W. Carr, and A. V. McCormick, Chromatographia, 1996, 42, 273. 27. R. A. Shalliker and G. K. Douglas, J. Liq. Chrom. Rel. Technol., 1997, 20, 1651. 28. R. A. Shalliker, G. K. Douglas, L. Rintoul, P. R. Comino, and P. E. Kavanagh, J. Liq. Chrom. Rel. Technol., 1997, 20, 1471. 29. R. A. Shalliker, L. Rintoul, G. K. Douglas, and S. C. Russell, J. Mater. Sci., 1997, 32, 2949. 30. Q.-H. Zhang, Y.-Q. Feng, and S.-L. Da, Anal. Sci., 1999, 13, 217. 31. Q.-H. Zhang, Y.-Q. Feng, and S.-L. Da, Chromatographia, 1999, 50, 654. 32. J. J. Kirkland, J. B. Adams, Jr., M. A. Van Straten, and H. A. Claessens, Anal. Chem., 1998, 70, 4344. 33. S. Ahrland, D. Karipidis, and B. Noren, Acta Chem. Scand., 1963, 17, 411. 34. W. B. Blumenthal, “The Chemical Behavior of Zirconium,” 1958, Van Nostrand, Princeton. 35. Y.-L. Hu, Y.-Q. Feng, Q.-H. Zhang, and S.-L. Da, Talanta, 1999, 49, 47.