High-performance liquid chromatographic stationary phases based on polysiloxanes with different chain lengths thermally immobilized on silica supports

Journal of Chromatography A, 1119 (2006) 135–139

High-performance liquid chromatographic stationary phases based on polysiloxanes with different chain lengths thermally immobilized on silica supports Edivan Tonhi, Kenneth E. Collins, Carol H. Collins ∗ Instituto de Qu´ımica, Universidade Estadual de Campinas, Caixa Postal 6154, CEP 13084-971, Campinas, SP, Brasil Available online 20 January 2006

Abstract Reversed phases for high-performance liquid chromatography (RP-HPLC) were obtained by thermal immobilization of polysiloxanes having different length chains (C1, C8 and C14) onto HPLC silica particles. The importance both of percent loading of the stationary phase promoted by each immobilization procedure and of the length of the lateral chain of the polymer on the chromatographic performances of the phases obtained is compared and discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Reversed phase; Polysiloxane; Immobilization

1. Introduction Using a pre-formed polymer to cover an oxide support has become an important means for obtaining reversed phases for HPLC. The main advantages of these phases with polymer coatings, compared to chemically bonded phases, are: higher covering of active sites of the support and the possibility of greater selectivity of the stationary phase from an appropriate choice of the polymer. There are several different ways to promote polymer immobilization onto the oxide support. The methods most commonly mentioned in the literature are thermal treatment, with or without the presence of free radical-inducing agents [1–3], use of high energy (gamma) radiation [1,3–5] or use of specific reagents to induce chemical cross linking [1,3]. Besides these, there are other possibilities, such as the use of microwave irradiation [6] and self-immobilization [7,8]. Most early work using thermal treatments to immobilize polymers onto a chromatographic support were carried out in the presence of radical-generating agents, such as azobisisobutyronitrile, dicumylperoxide or allylmethacrylate, to promote cross linking and the consequent immobilization of the polymer onto the support [3,9,10]. ∗

Corresponding author. Tel.: +55 19 3788 3055; fax: +55 19 3788 3023. E-mail address: [email protected] (C.H. Collins).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.12.073

The use of the thermal immobilization of polymers on chromatographic supports, without the presence of radical inducing agents, has also been reported. Schomburg et al. [1] immobilized a polysiloxane on silica by heating to 180 ◦ C. A more recent study [7] used temperatures of 80–280 ◦ C. It was shown that the phases obtained using temperatures below 150 ◦ C promoted the formation of a polymer monolayer on the support and that these phases may be useful in HPLC. However, the phases immobilized using temperatures higher than 180 ◦ C showed the formation of multilayers on the support surface, and the columns obtained with these phases were not adequate for chromatographic use. In this work a comparison between stationary phases based on polysiloxanes with different lateral chains, immobilized by thermal treatment onto porous silica particles, is described. 2. Experimental 2.1. Materials The chromatographic support used to prepare the stationary phases was spherical Kromasil silica (Akzo Nobel) having a mean particle diameter of 5 ␮m, 0.89 mL g−1 specific pore volume and 330 m2 g−1 specific surface area. Poly(dimethylsiloxane), PDMS, (product PS-043), Mr 28,000, poly(methyloctylsiloxane) (PMOS) (product PS-140),

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E. Tonhi et al. / J. Chromatogr. A 1119 (2006) 135–139

Mr 6200 and poly(methyltetradecylsiloxane), PMTDS, (product PS-134), Mr 9400 were obtained from Petrarch/H¨uls America. Methanol (Omnisolv), chloroform (LiChrosolv) and hexane (HPLC-grade) were all from Merck. Distilled, deionized water (Milli-Q Plus, Millipore) was used throughout. The chromatographic test substances uracil (Aldrich), phenol (Labsynth), N,N-dimethylaniline (Fluka), naphthalene (Vetec), ethylbenzene (Merck) and acenaphthene (Aldrich) were analytical-reagent grade and not further purified. 2.2. Preparation of the stationary phases The silica was dried in air at 150 ◦ C for 17 h. Then it was added to a 10% (w/v) solution of the polysiloxane in hexane in the proportion of 1.30 g of PDMS, 1.22 g of PMOS or 1.20 g PMTDS to 1 g silica, respectively. The objective of these polysiloxane/silica proportions was to ensure excess polymer in order to fill the pores of the silica for the preparation of the initial adsorbed stationary phase. The mixtures were stirred for 3 h at 40 ◦ C and the solvent was then allowed to evaporate, without stirring, at 40 ◦ C. The stationary phases obtained by evaporation of the solvent were divided into several portions and each portion was then submitted to a specific thermal immobilization treatment. The stationary phase SiO2 (PDMS) was immobilized at: (1) 80 ◦ C for 30 h; (2) 120 ◦ C for 16 h; and (3) 240 ◦ C for 4 h. The stationary phase SiO2 (PMOS) was immobilized at: (1) 120 ◦ C for 4 h; (2) 120 ◦ C for 16 h; and (3) 220 ◦ C for 4 h. The stationary phase SiO2 (PMTDS) was immobilized at: (1) 80 ◦ C for 30 h and (2) at 120 ◦ C for 16 h. All thermal immobilizations were carried out in the presence of air. After each immobilization procedure, the excess polysiloxane that was not immobilized was extracted from the stationary phase by passing hexane or chloroform (0.5 mL min−1 for 4 h at room temperature), followed by methanol (0.5 mL min−1 for 2 h at room temperature) through the material contained in a column-type washing system. The phases were then dried (40 ◦ C for 12 h) and stored in closed containers until needed. Each immobilized phase was characterized both physically and chromatographically, as previously described [11–13]. 2.3. Preparation of the test columns The columns (50 mm × 4 mm) were made from type 303 stainless-steel tubing with highly polished interior surfaces [14] and downward packed using 10% slurries (w/v) of each stationary phase in chloroform. A packing pressure of 34.5 MPa (Haskel Model 5I769 Packing Pump) was used, with methanol as the propulsion solvent. Columns were conditioned for 4 h with mobile phase (methanol:water 7:3, v/v) at 0.2 mL min−1 at room temperature prior to the chromatographic tests. 2.4. Chromatographic evaluation The columns were evaluated using a modular HPLC system equipped with a Rheodyne model 8125 injector (5 ␮L loop), a Shimadzu model LC-10AD pump and an Alltech model 450 UV

(254 nm) detector with a 0.8 ␮L cell. Data aquisition used Chrom Perfect for Windows, version 3.52 (Justice Innovations), with the Report-Write Plus option for calculation of the chromatographic parameters, installed in a PC compatible computer. The evaluations of the columns packed with immobilizedextracted SiO2 (polysiloxane) stationary phases were based on the separation of a test mixture containing acidic, basic and neutral solutes (uracil, phenol, N,N-dimethylaniline, naphthalene, ethylbenzene and acenaphthene) dissolved in mobile phase (methanol:water 7:3, v/v). Injection of 5 ␮L of this mixture produced satisfactory chromatographic peaks with detection at 254 nm. The separations were carried out at room temperature with a flow rate of 0.5 mL min−1 , optimized by means of a van Deemter curve. The column dead time, tM , was determined using uracil (an unretained compound). The retention factor (k) was determined for each peak and the separation factor (α) was determined for adjacent peaks. 2.5. Stability tests using a neutral mobile phase and an alkaline (pH 8.4) mobile phase at elevated temperature Columns packed with several of the stationary phases were submitted to stability testing by passing 7:3 (v/v) methanol:water at room temperature (∼22 ◦ C) through the column at 1.0 mL min−1 and periodically injecting a test mixture (uracil, phenol, N,N-dimethylaniline and naphthalene) to evaluate column performance as a function of time. For the chromatographic evaluation, the flow rate of the mobile phase was decreased to 0.5 mL min−1 . The chromatographic parameters (retention factor (k), efficiency (N) and asymmetry factor (As) at 10% of peak height) were determined for each peak. The test using alkaline (pH 8.4) mobile phase at 60 ◦ C, developed in our laboratory [15], consists of pumping an alkaline (pH 8.4) mobile phase, 1:1 (v/v) methanol:0.1 mol L−1 sodium bicarbonate, through the columns at 0.6 mL min−1 with the columns inside an oven held at 60 ◦ C. After defined time periods (1 h), the column is removed from the oven and coupled to a HPLC test system, passing a 7:3 (v/v) methanol:water mobile phase at 0.5 mL min−1 for 15 min to remove the alkaline mobile phase from within the column as well as to lower the column temperature. After this time, the detector is connected and the test mixture is injected. After chromatographic evaluation, the column is again placed inside the oven at 60 ◦ C and submitted to the passage of more alkaline mobile phase (0.6 mL min−1 for 1 h). This test continued for approximately 1600 column volumes (Vc ). 2.6. Percent carbon The percent carbon of each SiO2 (polysiloxane) phase was obtained through elemental analysis after polymer immobilization to evaluate the loading of the stationary phases. These determinations were made with a Model 2400 Perkin-Elmer CHN analyzer. From these values, the phase loadings (Table 1) were obtained through the following equation: 
mpolysiloxane × 100 loading = mSP

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Table 1 Physical and chromatographic characterizations of the immobilized-extracted stationary phases SiO2 (PDMS) Thermal treatment

80 ◦ C

%C Loading (%) Specific surface area (m2 g−1 ) Efficiency, acenapthene (N/m) Efficiency, N,N-DMA (N/m)

30 h 9.1 28.2 87 86000 70400

for

SiO2 (PMOS) 120 ◦ C

for

16 h 10.5 32.2 73 94000 72400

240 ◦ C

120 ◦ C

for

4h 11.9 37.2 48 54000 46000

4h 14.5 23.4 157 61400 55000

for

SiO2 (PMTDS) 120 ◦ C 16 h 23.3 37.6 nd 68400 48400

for

220 ◦ C 4h 21.0 34.0 34 nd nd

for

80 ◦ C for 30 h 18.4 26.2 97 89400 75800

120 ◦ C for 16 h 20.6 30.2 78 95800 78200

nd, not determined.

where mSP is the mass of the stationary phase = mSiO2 + mpolysiloxane , mpolysiloxane the mass of the polysiloxane = (%CSP × mSP )/X, where %CSP is the measured percent carbon of the stationary phase and X = 32.5 for PDMS, 62.0 for PMOS and 70.0 for PMTDS, considering that X% represents the fraction of the total mass of each polysiloxane that is carbon. 2.7. Specific surface area The specific surface areas of the unmodified silicas and the stationary phases were measured using the BET method on a Micromeritics model Flowsorb 2300 instrument. 2.8. Thermogravimetric analyses The thermal stabilities of the polysiloxanes were studied using samples of approximately 10 mg, with a heating rate of 10 ◦ C min−1 in an air atmosphere using a TA model TGA 2050 instrument. 3. Results and discussion Table 1 summarizes some of the physical and chromatographic properties of the stationary phases prepared using the three polysiloxanes with different thermal imobilization treatments. The phase loadings shown in Table 1 indicate that polymer immobilization by thermal treatment is a function of both time and temperature. The longer the time and the higher the immobilization temperature, the higher is the amount of polymer immobilized for each stationary phase. For the phases based on PDMS, which possesses the highest thermal stability (Table 2), it was possible to obtain higher loadings using higher temperatures and shorter immobilization times. The three stationary phases prepared with PDMS were all chromatographically useful, with the phase produced using the highest immobilization tempera-

Fig. 1. Chromatograms obtained from columns packed with SiO2 (PDMS), SiO2 (PMOS) and SiO2 (PMTDS) stationary phases immobilized by heat treatment at 120 ◦ C for 16 h. Conditions: columns: 50 mm × 4 mm, mobile phase methanol:water 7:3 (v/v), flow rate 0.5 mL min−1 . Peaks in elution order: (1) uracil, (2) phenol, (3) N,N-dimethylaniline, (4) naphthalene, (5) ethylbenzene and (6) acenaphthene.

ture resulting in a longer retention time for all the compounds of the test mixture, as expected from the % loading obtained. For the stationary phases based on PMOS, the best condition was a longer immobilization time at an intermediate temperature, because above approximately 200 ◦ C the PMOS degrades in an air atmosphere (Table 2). The SiO2 (PMOS) phase immobilized at 220 ◦ C was not suitable for chromatography. For the phases based on PMTDS, temperatures above 120 ◦ C were not tried because this polymer has stability similar to PMOS. In this case, the best condition was also heating at 120 ◦ C for 16 h. Chromatographic evaluations of some of the prepared phases have previously been reported [12,13]. To show the differences between the immobilized-extracted stationary phases prepared with different polymers, Fig. 1 presents the chromatograms of the columns packed with the stationary phases obtained using the same immobilization process (heating at 120 ◦ C for 16 h). It can be seen that the SiO2 (PMTDS)

Table 2 Thermogravimetric determinations of stability of the polysiloxanes

(◦ C)

Temperatura range of initial mass loss Loss (%) Temperature range of second mass loss (◦ C) Loss (%)

PDMS

PMOS

PMTDS

320–350 60 350–800 40

220–250 54 250–800 46

210–230 50 230–800 50

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Table 3 Separation factors between adjacent peaks of the test mixture for the columns packed with SiO2 (polysiloxane) stationary phases immobilized by thermal treatments at 120 ◦ C for 16 h Separation factor

SiO2 (PDMS) (32.2% loading) SiO2 (PMOS) (37.6% loading) SiO2 (PMTDS) (30.2% loading)

Phenol–N,N-DMA

N,N-DMA–naphthalene

Naphthalene–ethylbenzene

Ethylbenzene–acenaphthene

9.0 11.0 9.0

1.8 2.0 2.0

1.7 1.2 1.1

1.0 1.9 2.1

Columns: 50 mm × 4 mm; mobile phase: methanol:water 7:3 (v/v); flow rate: 0.5 mL min−1 .

and SiO2 (PMOS) stationary phases presented quite similar separations, even though the SiO2 (PMOS) stationary phase presents 7.4% more polymeric covering. This effect is due to the length of the carbon chain. In other words, a phase with a higher amount of polymer containing C8 groups is necessary to obtain a separation similar to that seen with a phase with a smaller amount of polymer containing C14 groups. The differences between the loadings of the stationary phases prepared with different polymers are due to the different properties of each polymer, such as viscosity, molar mass, size of the lateral chain, etc. The different separation factors between the peaks from the chromatograms shown in Fig. 1 can be confirmed by the results presented in Table 3. Fig. 2 shows chromatograms of the columns packed with different polysiloxane stationary phases, but having similar percent loadings. The stationary phases were: SiO2 (PDMS) immobilized by heating at 80 ◦ C for 30 h, SiO2 (PMOS) immobilized by heating at 120 ◦ C for 4 h and SiO2 (PMTDS) immobilized by heating at 80 ◦ C for 30 h. The separation factors for adjacent peaks on the chromatograms for the columns packed with each phase are shown in Table 4. As expected, the retention of most compounds increased with the length of the lateral chain of each polymer. If we consider the hydrophobicity as being 0.5(kacenaphthene + knaphthalene ) and the silanol activity as (kN,N-dimethylaniline / kacenaphthene ), it can be noted that, for phases with similar load-

Fig. 2. Chromatograms obtained from columns packed with SiO2 (PDMS), SiO2 (PMOS) and SiO2 (PMTDS) immobilized-extracted stationary phases having similar loadings. Chromatographic conditions and peak identifications as in Fig. 1.

ings, there is an increase of the hydrophobicity and a decrease of the silanol activity as a function of the increase of the carbon number in the lateral chains of the immobilized polysiloxane in the stationary phase (Table 5). Several of the stationary phases (SiO2 (PMOS): 120 ◦ C/4 h; SiO2 (PMOS): 120 ◦ C/16 h and SiO2 (PDMS): 120 ◦ C/16 h) were subjected to stability tests in neutral and basic (pH 8.4) mobile

Table 4 Separation factors between adjacent peaks of the test mixture for the columns packed with SiO2 (polysiloxane) immobilized-extracted stationary phases having similar loadings Separation factor

SiO2 (PDMS) 80 ◦ C for 30 h (28.2% loading) SiO2 (PMOS) 120 ◦ C for 4 h (23.4% loading) SiO2 (PMTDS) 80 ◦ C for 30 h (26.2% loading)

Phenol–N,N-DMA

N,N-DMA–naphthalene

Naphthalene–ethylbenzene

Ethylbenzene–acenaphthene

7.6 6.9 8.3

1.8 1.8 2.0

1.7 1.3 1.1

1.0 1.7 2.1

Chromatographic parameters as in Table 3. Table 5 Hydrophobicity [0.5(kacenaphthene + knaphthalene )] and silanol activity [(kN,N-dimethylaniline /kacenaphthene )] as functions of the carbon number in the lateral chains of the immobilized polysiloxanes in the stationary phases, calculated from the separations shown in Fig. 2 Carbon number in the lateral chain of the polysiloxane

Hydrophobicity Silanol activity

1 (PDMS)

8 (PMOS)

14 (PMTDS)

3.6 0.34

5.1 0.25

9.2 0.21

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phases. No significant changes in retention factor, efficiency (percent change in plate number) or asymmetry factor were seen after the passage of more than 40,000 mL of neutral mobile phase or ∼1600 mL of basic (pH 8.4) mobile phase. These test results are similar to those obtained with a similar column packed with a commercial stationary phase (Rainin C8). 4. Conclusions Comparing the stationary phases having different polymers with similar loadings it was shown that the length of the chain of the polymers promotes different selectivities for the acidic, basic and neutral test compounds and controls properties such as hydrophobicity and the silanol activity of the stationary phases. The degree of separation and the retention times of the compounds of the test mixture can be controlled by varying the loading of the stationary phase. Thermal treatment in the absence of a radical-producing agent is an efficient procedure to immobilize polysiloxanes onto the silica surface and the amount of polymer immobilized is a function of both time and temperature. The longer the time and the higher the temperature, the higher is the amount of polymer immobilized onto the support, although the percent loadings that result from the thermal treatments depend on the specific polysiloxane being immobilized. Acknowledgements The authors thank Dr. Domingo S´anchez of Akzo Nobel for donation of the silica and the Fundac¸a˜ o de Amparo a` Pesquisa

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do Estado de S˜ao Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) for fellowships and financial support. References [1] U. Bien-Vogelsang, A. Deege, H. Figge, J. K¨ohler, G. Schomburg, Chromatographia 19 (1984) 170. [2] L.M. Nyholm, K.E. Markides, J. Chromatogr. A 813 (1998) 11. [3] J. K¨ohler, Chromatographia 21 (1986) 573. [4] M.C.H. Silva, I.C.S.F. Jardim, J. Liq. Chromatogr. Rel. Technol. 21 (1998) 2447. [5] I.C.S.F. Jardim, K.E. Collins, T.A. Anazawa, J. Chromatogr. A 849 (1999) 299. [6] L.S.R. Morais, I.C.S.F. Jardim, J. Chromatogr. A 1073 (2005) 127. [7] K.E. Collins, A.L.A. S´a, C.B.G. Bottoli, C.H. Collins, Chromatographia 53 (2001) 661. [8] K.E. Collins, C.B.G. Bottoli, C.R.M. Vigna, S. Bachmann, K. Albert, C.H. Collins, J. Chromatogr. 43 (2004) 1020. [9] G. Schomburg, J. K¨ohler, H. Figge, A. Deege, U. Bien-Vogelsang, Chromatographia 18 (1984) 265. [10] R. Ohmacht, M. Kele, Z. Matus, Chromatographia 28 (1989) 19. [11] E. Tonhi, S. Bachmann, K. Albert, I.C.S.F. Jardim, K.E. Collins, C.H. Collins, J. Chromatogr. A 948 (2002) 97. [12] E. Tonhi, K.E. Collins, C.H. Collins, J. Chromatogr. A 948 (2002) 109. [13] E. Tonhi, K.E. Collins, C.H. Collins, J. Chromatogr. A 1075 (2005) 87. [14] K.E. Collins, A.C. Franchon, I.C.S.F. Jardim, E. Radovanovic, M.C. Gonc¸alves, LC–GC 18 (2000) 106. [15] D.A. Fonseca, H.R. Guti´errez, K.E. Collins, C.H. Collins, J. Chromatogr. A 1030 (2004) 149.