Chemically Bonded Silica Stationary Phases: Synthesis

Department of Environmental Chemistry, Faculty of Chemistry, Nicolaus Copernicus University,. Gagarina 7, PL-87 100 Torun, Poland, and Department of ...
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Anal. Chem. 1997, 69, 3277-3284

Chemically Bonded Silica Stationary Phases: Synthesis, Physicochemical Characterization, and Molecular Mechanism of Reversed-Phase HPLC Retention Bogusław Buszewski,*,† Renata M. Gadzała-Kopciuch,† Michał Markuszewski,‡ and Roman Kaliszan‡

Department of Environmental Chemistry, Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7, PL-87 100 Torun´ , Poland, and Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdan´ sk, Gen. J. Hallera 107, PL-80 416 Gdan´ sk, Poland

Two types of chemically bonded phases for high-performance liquid chromatography (HPLC) have been prepared: a conventional C18 and AP (N-acylaminopropylsilica), a novel one that contains specific interaction sites localized in the hydrophobic chain. Surface properties of stationary phases, before and after chemical modification, have been characterized by several physicochemical techniques, such as porosimetry, ICP atomic emission spectroscopy, elemental analysis, solid state CP/MAS NMR, and chromatography. For the studies of the reversed-phase HPLC retention mechanism under hydroorganic conditions, a test series of structurally diverse solutes has been selected. Sets of retention parameters and structural descriptors of the test solutes were subjected to multiparameter regression analysis. The quantitative structure-retention relationships derived demonstrated the typical reversed-phase partition mechanism to predominate in the separation on the C18 phases but not on the AP phases. The AP phases were demonstrated to provide significant input to retention due to the structurally specific dipole-dipole and charge transfer interactions with the solutes. The proposed AP phases for HPLC possess distinctive and interesting retentive properties, and chemometric analysis of retention data of appropriately designed series of test solutes appears to be a convenient, objective, and quantitative method to prove a new phase specificity. Most progress in high-performance liquid chromatographic (HPLC) separations has been achieved due to the introduction of numerous silica-based stationary phase materials. Still, however, there is no universal stationary phase material suited to the specific properties of all the possible solutes. It is rather impractical to synthesize an individual phase to solve a given analytical task. To propose a new stationary phase for a common practical use, one should clearly demonstrate its advantage over the existing materials. A single example may not be convincing enough. Having that in mind, Engelhardt et al.1 and Tanaka et †

Nicolaus Copernicus University. Medical University of Gdan´sk. (1) Engelhardt, H.; Low, H.; Gotzinger W. J. Chromatogr. 1991, 544, 371. ‡

S0003-2700(96)01203-6 CCC: $14.00

© 1997 American Chemical Society

al.2 proposed small sets of test solutes of diverse chemical structure to probe the retentive properties of stationary phases. The approach got wide recognition as a simple and convenient method of testing of new stationary phase materials. The classifications based on the Engelhardt-Tanaka procedures are qualitative rather than quantitative. An alternative is a chemometric (statistical) proof of differences in separation provided by the new phases with regard to the standard, well-established materials.3 In modern HPLC, two types of chemically bonded phases are utilized, i.e., the Hala´sz brush phases and the coating-polylayer phases.4 Both types of phases are successfully used in various environmental, medical, pharmaceutical, and other applications. A wide palette of brush stationary phases, such as C18, C8, C2, NH2, CN, DIOL, Ph, etc., are available on the market. Majors5 reported that most recent investigations concentrate on the preparation of the more solvolytically and thermally stable packings prepared using the high-purity (without heteroatoms) silica gel supports. However, many RP HPLC separations of complex organic solutes require selective packings, providing strictly defined interactions between the solutes, the mobile phase components, and the stationary phase surface. A way to improve these separations is to use chemically bonded phases with specific properties. Such appear to be the alkylamide phases which contain terminal alkyl chains attached to the alkylamide groups.4,6-8 Alkylamide phases show interesting chromatographic properties.4,6,9-11 These are ascribed to the participation in the separation process of different interaction sites, e.g., residual, unreacted silanols, and amino groups as well as the hydrophobic chains with amide groups. In addition, the affinity of these phases to solvent molecules differs significantly from that observed for (2) Tanaka, N.; Tanigawa, T.; Kimata, K.; Hosya, K.; Araki, T. J. Chromatogr. 1991, 549, 29. (3) Kaliszan, R. Quantitative Structure-Chromatographic Retention Relationships; Wiley: New York, 1987. (4) Buszewski, B. Chemically bonded phases in chromatographic analysis. Preparation, properties and application; STU: Bratislava, 1992. (5) Majors, R. E. LC-GC 1996, 9 (4), 214. (6) Buszewski, B.; Schmid, J.; Albert, K.; Bayer E. J. Chromatogr. 1991, 552, 415. (7) Feibush, B.; Santasania, C. T. J. Chromatogr. 1991, 544, 41. (8) Unger, K. K. Chromatographia 1991, 31, 507. (9) Buszewski, B.; Gilpin, R. K.; Jaroniec, M. J. Chromatogr. 1994, 673, 11. (10) Kasturi, P.; Buszewski, B.; Jaroniec, M.; Gilpin, R. K. J. Chromatogr. 1994, 659, 261. (11) Buszewski, B.; Lodkowski, R. Analusis 1995, 23, 147.

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Table 1. Characteristics of Bare Nucleosil 100 from Machery-Nagel GmbH, Du 1 ran, Germany (Batch No. 9207) feature

unit

abbreviation

value

particle size specific surface area mean pore diameter pore volume bulk density silanol concentration pH metal impurities

mm m2/g nm cm3/g g/cm3 µmol/m2 pH ppm

dp SBET D Vp σw RSiOH pH Na K Ca Mg Al Fe Mn Ti

4.8 345 9.8 1.01 0.40 4.78 6.15 5.272 1.931 13.373 4.113 3.265 8.477 0.445 2.830

cM

}

measurement technique laser particle counter porosimetry (low temperature adsorption-desorption of nitrogen) picnometry Puls-GC pH-metry ICP

39.700

the conventional alkyl phases. It manifests itself by a good resolution and peak symmetry in the case of individual polar and/ or stereogeometrically differing solutes.4,12-14 These advantages have special importance under hydroorganic conditions with higher concentrations of water in the binary and ternary mobile phases. Therefore, intermolecular interactions determining elution of solutes of different character on alkylamide phases appear interesting from both theoretical and practical points of view. Chemometric techniques have been applied in comparative quantitative structure-retention relationship (QSRR) studies of separation properties of various HPLC stationary phases.3,15-37 Unfortunately, closer examination of some of the published relationships reveals that not every structural descriptor of the solutes considered has been statistically significant. To avoid (12) Czajkowska, T.; Hrabovsky, I.; Buszewski, B.; Gilpin, R. K.; Jaroniec, M. J. Chromatogr. A 1995, 691, 217. (13) O’Gara, J. E.; Alden, B. A.; Walter, T. H.; Petersen, J. S.; Niederla¨nder, C. L.; Neue U. D. Anal. Chem. 1995, 67, 3809. (14) Buszewski, B.; Gadzała, R. M.; Lodkowski, R.; Sander, L. C.; Jaroniec, M. J. Environ. Studies 1994, 3 (3), 9. (15) Buydens, L.; Massart, D. L. Anal. Chem. 1981, 53, 1990. (16) Buydens, L.; Massart, D. L.; Geerlings P. Anal. Chem. 1983, 55, 738. (17) Buydens, L.; Coomans, D.; Vanbelle, M.; Massart, D. L.; Van den Driessche R. J. Pharm. Sci. 1983, 72, 1327. (18) Hasan, M. N.; Jurs, P. C. Anal. Chem. 1983, 55, 263. (19) Chre´tien, J. R.; Szymoniak, K.; Lion, C.; Haken, J. K. J. Chromatogr. 1985, 324, 355. (20) Cserha´ti, T.; Borda´s, B. J. Chromatogr. 1984, 286, 131. (21) Kaliszan, R.; Os´miałowski, K.; Bassler, B. J.; Hartwick, R. A. J. Chromatogr. 1990, 499, 333. (22) Kaliszan, R.; Os´miałowski, K. J. Chromatogr. 1990, 506, 3. (23) Morin-Allory, L.; Herbreteau, B. J. Chromatogr. 1992, 590, 203. (24) Forga´cs, E. J. Liq. Chromatogr. 1993, 16, 3757. (25) Haldna, U.; Pentchuk, J.; Righezza, M.; Chre´tien, J. R. J. Chromatogr. A 1994, 670, 51. (26) Delaney, M. F.; Papas, A. N.; Walters, M. J. J. Chromatogr. 1987, 410, 31. (27) Schmitz, S. J.; Zwanziger, H.; Engelhardt, H. J. Chromatogr. 1991, 544, 381. (28) Righezza, M.; Chre´tien, J. R. J. Chromatogr. 1991, 556, 169. (29) Azzaoui, K.; Morin-Allory, L. Chromatographia 1996, 42, 389. (30) Turowski, M.; Kaliszan, R.; Lu ¨ llman, C.; Genieser, H. G.; Jastorff, B. J. Chromatogr. A 1996, 728, 201. (31) Nasal, A.; Haber, P.; Kaliszan, R.; Forga´cs, E.; Cserha´ti, T.; Abraham, M. H. Chromatographia 1996, 43, 484. (32) Kaliszan, R. J. Chromatogr. A 1993, 656, 417. (33) Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Melander, W.; Horva´th, Cs. Anal. Chem. 1986, 58, 2674. (34) Tan, L. C.; Carr, P. W. J. Chromatogr. A 1993, 656, 521. (35) Abraham, M. H.; McGowan, J. C. Chromatographia 1987, 23, 243. (36) Abraham, M. H.; Roses, M. J. Phys. Org. Chem. 1994, 7, 672. (37) Hsieh, M.-M.; Dorsey, J. G. J. Chromatogr. 1993, 631, 63.

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statistically and physically meaningless correlations, a set of carefully designed test solutes was chosen for this project. Structurally diversified solutes were selected such that, within the series, the intercorrelations were minimized among the individual solute structural descriptors. At the same time, the selection of test solutes was to provide a wide-spanning and even distribution of individual structural descriptor values. EXPERIMENTAL SECTION Chemicals. Specially purified4,38 Nucleosil 100 SG (MacheryNagel, Du¨ron, Germany) was used as a support material for the preparation of the chemically bonded phases. Its physical and chemical characteristics are given in Table 1. The following reagents were used for chemical modification of the support material: octadecyldimethylchlorosilane (MC18 phases) and octadecyltrichlorosilane (PC18 phases), both from Petrarch System (Levittown, PA); γ-aminopropyldimethylmethoxysilane (Wacker GmbH, Mu¨nich, Germany); γ-aminopropyltriethoxysilane (Fluka, Buchs, Switzerland); palmitoyl chloride (E. Merck, Darmstadt, Germany), and a specially prepared39 dry morpholine (Riedel de Hae¨n, Seelze, Germany). Methanol, toluene, and n-hexane of analytical reagent grade purity were purchased from E. Merck. A series of test solutes for QSRR studies formed 21 wellidentified chemicals of various origin. Measurements of Physicochemical Properties of Stationary Phase Materials. The porosity parameters of bare Nucleosil 100 (SBET, specific surface area; Vp, pore volume; D, mean pore diameter) were determined by the low-temperature nitrogen adsorption-desorption method at -197.5 °C using a Model 1800 Sorptomatic instrument (Carlo Erba, Milan, Italy). The concentration of surface silanol groups (RSiOH) was measured by the method proposed by Nondek and Vyskocˇil,40 based on the GC determination of methane formed during the reaction of dimethylzinctetrahydrofuran complex with the accessible silanol groups. Respective numerical data are collected in Table 1. The degree of coverage (RRP) of silica support surface with bonded ligands was calculated by using the equations described in ref 9 from carbon (PC) and/or nitrogen (PN) contents deter(38) Buszewski, B. Chromatographia 1992, 32, 573. (39) Buszewski, B. Solvent purification and recovery, Encyclopedia of Analytical Sciences; Pergamon Press: London, 1995; p 4730. (40) Nondek, L.; Vyskocˇil, V. J. Chromatogr. 1981, 206, 581.

Table 2. Surface Charcteristics of the Chemically Bonded Phases type of phase MAP PAP MC18 PC18

type of structure

nC

PC (%)

PN (%)

RNH2 (µmol/m2)

RRP (µmol/m2)

monomeric polymeric monomeric polymeric

21 19 20 18

10.58 9.08 18.09 15.72

0.99 1.20

2.36 1.90

1.27 1.06 3.075 2.57

Table 3. Detectable 29Si CP/MAS NMR Undersurface Species of Silicaceous Supports and Chemical Shift (δ) Values after Modification with Mono- (M) and Trifuncional (T) Silane

mined by elemental analysis with a CHN analyzer, Model 240 (Perkin Elmer, Norwalk, CT) (Table 2). Solid state NMR measurements, before and after chemical modification, were performed on an MSL 300 spectrometer (Bruker, Rheinstetten, Germany) after placing 200-300 mg samples in the double-bearing rotors of zirconia. Magic-angle spinning (MAS) was carried out at a spinning rate of 4 kHz. 29Si CP/MAS NMR spectra were recorded with a pulse length of 5 ms, with a contact time of 5 ms and a pulse repetition time of 2 s. In the case of the 13C CP/MAS NMR spectra, for control of the efficiency of the stationary phases formation, a contact time of 12 ms was applied. All the NMR spectra were externally referenced to liquid tetramethylsilane, and the chemical shifts (δ) are given in parts per million (ppm) (Table 3). In the inductively coupled plasma (ICP) atomic emission spectroscopy studies, a Model PU-7000 apparatus with an ultrasonic nebulizer, Typ U-5000AT (Unicam, Cambridge, UK), was applied for the determination of metal impurities in the silica

support. The silica sample was prepared using a microwave mineralization system, type MD-2000 (CEM Co., Matthews, NC) (Table 1). Preparation of Chemically Bonded Stationary Phases. The phases with an electric charge localized in the organic ligands of N-acylaminopropylsilica (AP) were prepared in a two-stage reaction. The silica gel support was first heated at 185 °C under vacuum at 10-3 Pa. Next, a portion of the support material was treated with a monofunctional or triethoxyaminopropylsilane modifier. That stage was followed by a reaction of the amino groups with chloride derivative. The procedure, reaction mechanism, and reaction conditions in the case of monomeric (M) and polymeric (P) AP phases have been described in detail previously.4,6,9,41 Similarly, preparation of monomeric and polymeric C18 phases has been described earlier.4,42,43 All the reactions were carried out in a specially constructed glass reactor in the gaseous phase (without solvents) to prevent contact of the reagents with the environment.4 Column Packing. The phases prepared were packed into 125 mm × 4.6 mm i.d. stainless-steel tubes purchased from Bischoff (Leonberg, Germany). The modified materials were shaken in an ultrasonic bath for 5 min with 35 mL of tetrachloroethylene-1-propanol (2:1 v/v). The slurry was then placed in the columns using 150 mL of methanol as a packing solvent. All columns were packed using a DT 122 packing pump (Haskel, Burbank, CA) under a pressure of 50 MPa. Determination of Chromatographic Retention Parameters of Test Solutes. Chromatographic measurements were made using an HP-1050 liquid chromatograph system (Hewlett Packard, Waldbronn, Germany), equipped with a diode array detector and the Vectra QS/HP computer with ChemStation-2 for data collection and control of the process. Solutes were injected using a Rheodyne (Berkeley, CA) Model 7125 sampling valve with a 20 µL sample loop. In all chromatographic investigations, the flow rate was 1 mL/min. Methanol, acetonitrile, and water of HPLC purity (J. T. Baker, Deventer, The Netherlands) were used to prepare the mobile phases. Deuterated water (Fluka, Buchs, Switzerland) was used as the marker in the dead time determinations (Vo). The solutes were chromatographed at four compositions of the methanol-water mobile phase: 60:40, 55:45, 50:50, and 45: 55 (v/v). Based on the linear relationships between the logarithm of capacity factor (log k′) and the methanol concentration in the eluent, the values corresponding to 100% water eluent were obtained by extrapolation (log k′w). Respective data are collected in Table 4. Missing values mean that log k′ was poorly correlated to percent methanol and the extrapolated log k′w was unreliable. Chemometric Comparison of Separation Properties of the Newly Synthesized Phases. Test Solutes and Their Structural Descriptors. A series of 21 test solutes was designed with the welldefined hydrogen bond capacity descriptors derived from the complexation scales of Abraham.44,45 The test solutes were subjected to molecular modeling by the HyperChem package with the extension, ChemPlus (HyperCube, Waterloo, Canada). In effect, a number of quantum chemical and standard additive/ (41) Buszewski, B.; Lodkowski, R. J. Liq. Chromatogr. 1991, 14, 1185. (42) Buszewski, B.; El Mouelhi, M.; Albert, K.; Bayer, E. J. Liq. Chromatogr. 1990, 13, 505. (43) Buszewski, B.; Sien´ko, D.; Suprynowicz, Z. J. Chromatogr. 1989, 464, 73. (44) Abraham, M. H. University College London Data Base, 1996. (45) Abraham, M. H. Chem. Soc. Rev. 1993, 22, 73.

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Table 4. Logarithms of Capacity Factors Corresponding to 100% Water Eluent Determined on the Monomeric (Subscript M) and Polymeric (Subscript P) Phases Studied no.

solute [C.A. code no.]

log k′w(MC18)

log k′w(PC18)

log k′w(MAP)

log k′w(PAP)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1,3,5-triisopropylbenzene [717-74-8] 1,3-diisopropylbenzene [99-62-7] 1,4-dinitrobenzene [100-25-4] 3,5-dichlorophenol [591-35-5] 4-chlorophenol [106-48-9] 4-cyanophenol [767-00-0] 4-iodophenol [540-38-5] 4-nitrobenzoic acid [62-23-7] methyl phenyl ether [100-66-3] benzamide [55-21-0] benzene [71-43-2] chlorobenzene [108-90-7] cyclohexanone [108-94-1] dibenzothiophene [132-65-0] phenol [108-95-2] hexylbenzene [1077-16-3] hexachlorobutadiene [87-68-3] indazole [271-44-3] caffeine [58-08-2] naphthalene [91-20-3] toluene [108-88-3]

3.648 6.582 1.756 5.387 2.334 1.481 2.84

3.626 5.308 1.685 3.326 2.479 1.345 2.99

1.721 0.419 1.75 2.526 0.667 4.256 1.028 1.212 6.598 1.373 0.361 3.108 2.34

2.311 0.921 2.194 3.014 0.791 4.469 1.451 1.49 6.639 1.413 0.548 3.119 2.401

3.122 3.407 0.756 2.404 1.371 1.256 1.804 -0.691 1.059 0.342 1.001 1.175 -0.108 3.633 0.337 3.078 4.085 1.146 0.607 2.583 1.915

3.658 3.958 0.815 1.876 1.599 0.15 2.126 -1.986 1.319 0.165 1.23 1.923 0.131 3.842 -0.304 2.954 3.961 1.209 0.627 2.064 1.311

Table 5. Empirical, Semiempirical, and Calculation Chemistry Structural Descriptors of Test Solutes no.

solute

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1,3,5-triisopropylbenzene 1,3-diisopropylbenzene 1,4-dinitrobenzene 3,5-dichlorophenol 4-chlorophenol 4-cyanophenol 4-iodophenol 4-nitrobenzoic acid methyl phenyl ether benzamide benzene chlorobenzene cyclohexanone dibenzothiophene phenol hexylbenzene hexachlorobutadiene indazole caffeine naphthalene toluene

log Pa

1.46 2.39 1.60 2.91 1.89 2.11 0.64 2.13 2.84 0.81 1.46 1.82 -0.07 3.01 2.69

R2b

π2H c

∑R2H d

∑β2H e

Vxf

EHOMOg

µ(D)h

massi

0.627 0.605 1.13 1.020 0.915 0.940 1.380 0.990 0.708 0.990 0.610 0.718 0.403 1.959 0.805 0.591 1.019 1.180 1.500 1.340 0.601

0.40 0.46 1.63 1.10 1.08 1.63 1.22 1.07 0.75 1.50 0.52 0.65 0.86 1.31 0.89 0.50 0.85 1.25 1.60 0.92 0.52

0 0 0 0.83 0.67 0.79 0.68 0 0 0.49 0 0 0 0 0.60 0 0 0.54 0 0 0

0.22 0.20 0.41 0 0.20 0.29 0.20 0.76 0.29 0.67 0.14 0.07 0.56 0.18 0.30 0.15 0 0.34 1.35 0.20 0.14

1.985 1.562 1.065 1.020 0.898 0.930 1.033 0.671 0.916 0.973 0.716 0.839 0.861 1.379 0.775 1.562 1.321 0.905 1.363 1.085 0.716

-9.214 -9.252 -11.340 -9.537 -9.125 -9.510 -9.244 -10.900 -9.005 -9.941 -9.653 -9.561 -10.310 -8.202 -9.115 -9.299 -9.444 -8.866 -8.945 -8.711 -9.330

0.014 0.232 0 1.408 1.478 3.313 1.586 3.431 1.249 3.583 0 1.307 2.972 0.524 1.233 0.351 0.001 1.546 3.708 0 0.263

204.36 162.27 168.11 163 128.56 119.12 220.01 167.12 108.14 121.14 78.11 112.56 98.14 184.26 94.11 162.27 260.76 118.14 194.19 128.17 92.14

a Logarithm of n-octanol-water partition coefficient.46 b Excess molar refraction.44,45 c Dipolarity/polarizabilyty parameter.44,45 d Effective hydrogen bond acidity.44,45 e Effective hydrogen bond basicity.44,45 f The characteristic volume of McGowan.44,45 g Energy of the highest occupied molecular orbital. h Dipole moment. i Molecular mass.

constitutive structural descriptors have been generated. For 14 solutes of the test series, the reference logarithms of n-octanolwater partition coefficients (log P) were found in the literature.46 The descriptors which were found to be significant in the correlation analysis with the retention parameters are listed in Table 5. Statistical Analysis. Calculations employing the Statgraphics package (Manugistics, Rockville, MD) were run on a personal computer. Initially, a set of four log k′w data and 30 structural descriptors of empirical, semiempirical, and calculation chemistry origin was subjected to a principal component analysis (PCA). The descriptors were identified which were of relevance for the prediction of (46) Craig, P. N. In Comprehensive Medicinal Chemistry, Vol. 6; Hansch, C., Sammes, P. G., Taylor, J. B., Eds.; Pergamon Press: Oxford, 1990.

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retention and which at the same time have not been strongly mutually intercorrelated. Next, the stepwise regression and the multiple regression analysis were carried out keeping the requirements of the meaningful statistics47 in mind. RESULTS AND DISCUSSION In Table 1, the important parameters characterizing surface properties of bare Nucleosil 100 are listed. Analyzing these data, one can state that all the parameters determined, like the specific surface area, mean pore diameter, and pore volume, are within the range of the values corresponding to an ideal adsorbent.4,48,49 (47) Charton, M.; Clementi, S.; Ehrenson, S.; Exner, O.; Shorter, J.; Wolds, S. Quant. Struct.-Act. Relat. 1985, 4, 29. (48) Unger, K. K. Packings and stationary phases in chromatographic techniques; Marcel Dekker: New York/Basel, 1990.

Figure 2. Typical 29Si CP/MAS NMR spectra for bare Nucleosil 100 (a) and for the monomeric (b) and polymeric (c) undersurface structure of the AP phases.

Figure 1. 29Si CP/MAS NMR spectra for bare silica gel (a), silica modified with monofunctional C18 silane (b), and silica modified with trifunctional C18 silane (c).

This is also true for the pH value of 5% aqueous gel suspension, which is considered to be a measure of purity and homogeneity of the surface.4,50,51 This pH differs only slightly from the theoretical value (pH ) 5.6), and the difference is probably due to the presence of eight metal elements listed in Table 1 in trace concentrations. However, the total metal concentration, cM ) 39.706 ppm, was lower than 50 ppm, which is an accepted upper limit for an ideal support.4,48,49 The type and the surface concentration of silanol groups (RSiOH) are important factors determining the homogeneity of adsorbent surface. The well-hydroxylated surface of an ideal adsorbent is characterized by the total concentration of silanols groups near to RSiOH ) 8 µmol/m2.4,48,51,52 The RSiOH value determined for bare Nucleosil 100 by the Nondek method40,53 (Table 1) differs from the theoretical value by about 35-40%. Such a relatively significant difference is probably due to the localization of the active silanols, mainly in small pores (D < 20 Å) of the support.4,48,50,51 These silanols may be unaccessible for the relatively large molecules of organic modifier (molecular length of 20-23 Å). (49) Buszewski, B.; Gadzała, R. M.; Tanaka N. Chemically bonded phase for chromatography and related techniquesstoday and future. In Liquid Chromatography: HPLC and TLC in Pharmacy; E. Merck Verlag: Warsaw, 1996; p 5. (50) Buszewski, B.; Jaroniec, M.; Staszczuk, P.; Gilpin, R. K. Wiad. Chem. 1995, 49, 223. (51) Nawrocki, J.; Buszewki, B. J. Chromatogr. 1988, 449, 1. (52) Nawrocki, J. Chromatographia 1991, 31, 177; 193. (53) Nondek, L.; Buszewski, B.; Berek, D. J. Chromatogr. 1986, 360, 241.

The mechanism of formation of the C18 and AP phases was investigated by 29Si CP/MAS NMR spectroscopy (Figures 1 and 2). Figures 1b and 2b show the spectra obtained for the monomeric C18 and AP phases, and in Figures 1c and 2c, the spectra of the corresponding polymeric phases are presented. The spectra analyzed do not differ significantly one from another. The only evident difference is that, in the spectrum of the trifunctional C18 silane phase, an additional band, T3, is observed (Figures 1c and 2c; Table 3) which is not present in the spectrum of the trifunctional AP phase. That last difference may be treated as evidence for the presence in the materials of specific polar sites localized between two aminopropyl moieties or in the alkyl chains of the ligands. Similar observations were reported earlier.4,41,54,55 Comparing the 13C CP/MAS NMR spectra obtained for the monomeric and polymeric C18 and AP phases (Figure 3), one can see that the materials differ mutually only with respect to the presence of peak A. This peak corresponds to the dimethylsilyl group and is characterized by chemical shift δ ) -2.5 ppm.4,6,56-58 Moreover, in all the spectra within the range of δ values from +13 to +50 ppm, peaks corresponding to the bonded organic moiety can be observed. In the spectra corresponding to the AP phases (Figure 3), an additional peak (δ ) +174 ppm) is observed. This peak (C4) corresponds to an N-acylamino group built into the hydrophobic alkyl chain. In connection to this, one can expect different surface and chromatographic properties of these phases in comparison to the conventional C18, although both types of phases are characterized by similar lengths of grafted chains (54) Carajaval, G. S.; Leyden, D. E.; Quinting, G. R.; Maciel, G. E. Anal. Chem. 1988, 60, 1776. (55) Albert, K.; Brindle, R.; Schmid, J.; Buszewski, B.; Bayer, E. Chromatographia 1994, 38, 283. (56) Maciel, G. E.; Sindorf, D. W. J. Am. Chem. Soc. 1980, 102, 7606. (57) Bayer, E.; Albert, K.; Reiners, J.; Nieder, M.; Mu ¨ller, D. J. Chromatogr. 1983, 268, 197. (58) Buszewski, B. Chromatographia 1990, 29, 233.

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Figure 3. Typical

13C

CP/MAS NMR spectra for monomeric (superscript M) and polymeric (superscript P) AP (a) and C18 (b) phases.

Figure 4. Surface model of the AP stationary phase possessing polar functional groups built in an alkyl chain (a) and of the conventional C18 phase (b).

(LRP ) 20-23 Å).4,59,60 Based on the NMR analysis and on the data collected in Tables 2 and 3, the surface structures of the C18 and AP phases were proposed (Figure 4). Specific retentive properties due to polar functionalities in the ligand of the newly synthesized phases of AP type should manifest themselves in QSRR equations. The QSRR equations describing retention on the AP columns should comprise significant structural descriptors of the solutes, accounting for polar solute-stationary phase interactions. The respective polar terms in the QSRR equations describing retention on the C18 columns should be less significant. To start the QSRR analysis, the intercorrelations were determined among the retention parameters of the test solutes obtained on the four new phases: MAP, PAP, MC18, and PC18. For 20 solutes for which the log k′w values were obtained on all the (59) Berendsen, G. E.; de Galan, L. J. Liq. Chromatogr. 1978, 1, 561. (60) Buszewski, B.; Suprynowicz, Z. Chromatographia 1987, 24, 573.

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Table 6. Intercorrelations between the Logarithms of Capacity Factors Extrapolated to 100% Water Eluent Determined on the New Stationary Phases Studied

(MC18)

log k′w log k′w (PC18) log k′w (MAP)

log k′w(PC18)

log k′w(MAP)

log k′w(PAP)

0.9558

0.8238 0.8420

0.8018 0.8533 0.9387

phases considered, the differences in retention properties are evidenced by the relatively low correlation coefficients (Table 6). As expected, the retention behaviors of the solutes on the two C18 phases are similar (correlation coefficient r ) 0.9558). The same is the case with the two AP phases (r ) 0.9387). Still, however, the separation patterns may change significantly, even within the same kind of stationary phase material. Table 6 clearly

Table 7. Parameters of Linear Relationships, log k′w ) a log P + b, between the Logarithms of Capacity Factors Extrapolated to 100% Water Eluent, log k′w, Determined on the New Stationary Phases Studied and the Logarithms of n-Octanol-Water Partition Coefficients of the Solutes (log P)a

log k′w(MC18) log k′w(PC18) log k′w(MAP) log k′w(PAP) a

a

b

Ra

sb

0.8988 0.8883 0.6044 0.6576

0.0369 0.2676 -0.1455 -0.3887

0.9561 0.9607 0.6592 0.5431

0.2660 0.2473 0.6397 0.9435

Correlation coefficient. b Standard error of estimation.

demonstrates that the AP phases are distinguishable from the C18 phases (r < 0.9). Now, it appeared interesting which retention patterns and to what extent can be predicted by means of structural parameters of the solutes. The solute structural parameters found to be significant in the QSRR analysis are collected in Table 5. As shown in Table 7, the log k′w data determined on individual columns cannot precisely be predicted by the standard hydrophobicity parameter of solutes, the logarithm of the n-octanolwater partition coefficient (log P). Correlation is relatively good (>0.95) in the case of the retention parameters determined on the C18 stationary phases but becomes very weak (