For these reasons mass spectra of individual 5'-n-propyl DTC were run (8) a t different temperatures and various electron energies using direct inlet for liquid samples on the Varian CH07 analytical mass spectrometer. It seems t h a t S-n-propyl esters are stable to electron impact since there is no significant difference between
spectra scanned a t 20 eV and 70 eV. They are also stable up to 250 "C to thermal decomposition.
ACKNOWLEDGMENT The authors thank UniRoyal Ltd. for permission to publish these results and are grateful to Varian Associates of Canada Ltd. for recording mass spectra. Received for review November 29, 1972. Accepted January 22, 1973.
(8) F. I . Onuska, unpublished data, 1972.
Polar Silicone-Based Chemically Bonded Stationary Phases for Liquid Chromatography Milos Novotny,' Susan L. Bektesh, and Kenneth B. Denson Department of Chemistry, Indiana University, Bloomington, Ind. 47407
Karel Grohmann2 and Wolfgang Parr Department of Chemistry, University of Houston, Houston, Texas 77004
It had been felt for a long time that the stationary phases bonded chemically to siliceous surfaces of chromatographic supports may alleviate many practical problems encountered in gas-liquid and liquid-liquid chromatography. The past several years were, in particular, marked by increased activity within this area of research. The reactivity of surface silanol groups offers three main possibilities for chemical modification: the formation of Si-0-C bonds (through esterification); Si-C-C bonds (obtained, for instance, by the reaction of chlorinated surfaces with either organolithium or Grignard reagents; and Si-0-Si-C bonds (through silylation). The use of these general methods for the modification of both adsorbents ( I ) and glass capillary columns in gas chromatography (2, 3) has been reported. Halhsz and Sebestian (4) esterified Porasil C and described the chromatographic properties of this new packing material. The hydrolytic and thermal instability of ester packings are great disadvantages when compared to silicones. Locke et al. (5) applied Grignard reactions to cover the surface of siliceous beads with benzyl and naphthyl functional groups. However, too few suitable reagents and the possible formation of magnesium occlusion salts during the bonding process make this approach much less realistic than esterification or silylation. In situ polymerization of silane compounds on various solid supports was studied by a number of investigators T o whom a l l correspondence should b e directed. Present address, D i v i s i o n o f Biology, California Technology, Pasadena, Calif. 91109. (1) (2) (3) (4) (5)
(6)
(7) (8) (9) (10)
Institute o f
A. V . Kiselev, Advan. Chromatogr., 113 (1967). K. Grob, Helv. Chim. Acta. 51, 718 (1968). M. Novotny and A. Zlatkis, Chromatogr. Rev., 14, 1 (1971). I . Halasz and I. Sebestian, Angew. Chem., lnf. Ed. Engl., 8, 453 (1969). D. C. Locke. J. J. Schermund, and B. Banner. Anal. Chem., 44, 90 (1972). E. W . Abel, 2. H. Pollard, P. C. Uden, and G. Nickless. J. Chrornatogr., 22, 23 (1966). H. N. M. Stewart and S . G . Perry, J. Chromatogr., 37,97 (1968). C. J . Bossart, /SA Trans., 7, 283 (1968). W . A. Aue and C. R. Hastings. J . Chromatogr., 42, 319 (1969). C. R. Hastings. W. A. Aue, and J. M. Augl, J. Chromatogr., 53, 487 (1970),
(6-15). Stewart and Perry (7) prepared a nonpolar chromatographic material by reacting Celite with octadecylchlorosilane and suggested its value for liquid partition chromatography. Following this approach, several packings became commercially available which can be used for reversed-phase separations. Aue et al. published a series of articles (9-12) dealing with the preparation and gaschromatographic properties of nonpolar chemically bonded silicone phases. These articles present the most detailed studies on the mechanism of polymerization on chromatographic supports. Relatively little has been reported on the prepartion of polar silicone chemically bonded phases. Limited availability of commercial silane compounds possessing selective groups in the side chain seems to explain this situation. Bossart (8) was partially successful in the preparation of polar gas-chromatographic packings using certain trimethoxy- and triethoxysilanes. Kirkland described (13) and studied (14) the polar stationary phases with ether and cyanoethyl functions for both gas and liquid chromatography. It is, however, known that the reactivity of silanes is decreasing in the following order: trichlorosilane > dichlorosilane > monochlorosilane > ethoxysilane (16). Consequently, long reaction times and incomplete bonding found by Bossart (8) are not surprising. More recently, Majors and Hopper (15) investigated bonding of a trichlorosilane, possessing a cyano group in the side chain, to highefficiency liquid chromatographic packings. In their recent work, Parr and Grohmann (17-19) synthetized some novel chlorosilanes for the derivatization of a n inorganic silica carrier to form the following structure (11) W. A. Aue, C. R. Hastings, J. M. Augl, M. K. Norr, and J. V. Larsen,J. Chromatogr., 56, 295 (1971). (12) C. R . Hastings, W. A. Aue, and R . N. Larsen. J. Chrornatogr., 60, 329 ( 1 9 7 1 ) (13) J. J. Kirkland and J. J. DeStefano, J. Chromatogr. Sci., 8, 309 (1970), (14) J. J. Kirkland, J. Chromatogr. Sci., 9, 206 (1971). (15) R . E. Majors and M. J. Hopper, 160th National Meeting, American Chemical Society, Chicago, Ill., September 1970, paper No. A42. (16) L. C. F. Blackman and R. Harrop, J. Appl. Chem.. 18, 37 (1968). (17) W. Parr and K. Grohmann, Tetrahedron Lett., 1971, 2633. (18) W. Parr and K. Grohmann, Angew. Chem., Int. Ed. Engl. 11, 314 (1972). (19) K. Grohmann, Ph.D. Thesis, University of Houston, 1972. A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 6, M A Y 1973
971
-Si-
I I
Table I. k Values for Columns Prepared by Different Procedures
0
k
alcohol
Phenanthrenequinone
3.06 2.90
6.04 14.00
9.85 12.70
11.70 10.40
4.15
9.20
12.00
12.60
4.73
13.70
10.70
11.60
5.20
14.00
14.30
15.10
4.40
10.20
12.20
13.30
Column
I -Si I for further use in the solid-phase peptide synthesis. The structure suggests a n obvious universal utility in the preparation of polar chemically bonded stationary phases as the chloromethyl terminal groups can be modified in a number of ways. Consequently, it should be possible to prepare a variety of chromatographic columns with different selectivities based on this approach. In this article, we wish to report the first state of investigations carried out with trichloro[3-(4-chloromethylpheny1)propyllsilane and dichloro[3-(4-chloromethylphenyl)butyl]silane as monomers. Bonding and subsequent polymerization were carried out with porous and superficially porous spherical chromatographic supports. This procedure was followed by the hydrolysis of the terminal chlorine to provide -CeHd-CHz-OH selective groups. In order t o achieve a n optimum consistency of the polymeric coating, different packings were prepared with various ratios of monomers and evaluated chromatographically in terms of both selectivity and efficiency. T h e basic column characteristics (the capacity ratio k and the number of theoretical plates per second) were further studied as functions of operating conditions (the mobile phase composition and temperature) t o provide some insight into the separation mechanism and show the utility of these chemically bonded phases for analytical purposes.
EXPERIMENTAL Preparation of Columns. Porasil C (37-75 p ) and Corasil I p ) were used for the experiments described. Both materials were obtained from Waters Associates, Framingham, Mass. Porasil C was dried at 200 "C overnight before use. The monomers were reacted with solid supports in the tetrahydrofurane solution (5% of the weight of the support) for 8 hr followed by further overnight heat treatment (100 "C) in order to complete polymerization. The experiments were carried out primarily for comparative purposes and no attempts were made as yet to optimize the reaction conditions. Chlorine analyses obtained by the Volhart titration method indicate that only about 50% of the monomers had reacted. This is not surprising in view of Bossart's experience that a relatively long period is needed for a complete reaction (8) The materials were further refluxed with solvents of different polarity to remove any unreacted material. The hydrolysis of terminal chlorines was carried out in a slightly alkaline solution of sodium carbonate in the mixture of tetrahydrofurane and water (1:l) at 40 "C. Although the reaction seems to proceed quite fast (as evidenced by the evolution of gas and a change in affinity of the beads to the hydrolytical solution), the chlorine content was only about 50% of the value expected from the results of the analysis of bonded. material. No substantial change was noticed at higher pH values. This might be explained at least partly by a previous hydrolysis during the process due to traces of water, loss of chlorine during the heat treatment, or a limited penetration of the aqueous solution into the hydrophobic polymer structures. After excessive washing with a series of solvents and drying, the materials were packed into the stainless steel tubes (2-mm i.d. X 80 cm) by a conventional dry filling procedure and conditioned with a mobile phase prior to chromatography. Liquid Chromatographic Measurements. A Varian 4100 highpressure liquid chromatograph with a UV monitor was used throughout the experiments. Different proportions of n-heptane and isopropanol were used as mobile phases; both were spectroquality solvents. Samples of o-cresol, phenol, benzylalcohol, and phenanthrenequinone were used to measure the separation characteristics of Porasil columns with and without bonded phases. (37-50
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 6, M A Y 1973
No
1 2
3
4
5 6
Packing Porasil C 50% dichloroand 50% trichloromonomer; not polymerized at high temperature 2 0 % trichloromonomer and 80% dimethyldichlorosilane 1 0 0 % dichloromonomer 1 0 0 % trichloromonomer As 2, but polymerized at high temperature
values
o-Cresol Phenol
Benzyl
Naphthoquinone and o-, m-, and p-nitroaniline were chosen for experiments with the treated Corasil column. The measurements shown in Table I and Figure 3 were carried out using 0.25% isopropanol in n-heptane.
RESULTS AND DISCUSSION For good column performance in liquid-liquid chromatography, the chemically bonded stationary phases should resemble the liquids with minimum viscosity as much as possible. This study, as well as the previous work of Kirkland (13, 14), indicates t h a t we are dealing with resinous films rather than true liquids. It is known from the technology of silicones t h a t the properties of polymers can be to a great degree controlled by the type of monomer or the ratio of different monomers (e.g., proportions of methyland phenylsilanes or dichloro- and trichlorosilanes) as well as the conditions of polymerization. A number of column materials were prepared in this study in order to determine differences among chromatographic properties of the systems with different ratios of monomers. Six representative examples are shown in Table I where the retention characteristics of individial columns are compared with each other and Porasil C (50 m2/gram). Repeated syntheses of the packing materials resulted in products with similar properties. While the k values were not identical (most likely reflecting different thicknesses of the polymer layer), their relative proportions and the order of elution of tested compounds were maintained. All prepared columns showed high selectivity for polar solutes, but poor efficiency with nonpolar mobile phases ( e . g . , hexane or heptane) quite consistently. Even a small addition of isopropanol to the carrier (0.25% in heptane) considerably shortened the retention times of studied solutes and improved somewhat the column efficiency. Selective properties of these chemically bonded phases are demonstrated by increased retention for the hydroxylated compounds as compared to Porasil. Reversed order of elution of phenol, benzylalcohol, and phenanthrenequinone with columns 2 and 4 as compared to 1 (Table I) can be seen. Another comparison is interesting: although the k values are quite
-
1.5
1.0
-
0.5
-
I 0
T
!
0.5
1.0
-
I 2.0
1.5
t of isopropanol
Figure 1. Dependence of the number of theoretical plates per second on concentration of isopropanol in the mobile phase Conditions: Columns (2 mm. i.d. X 80 cm) with different polymers bonded on Porasil C (37-75 p ) ; flow rate 1.0 ml/min; temperature 23 "C. (-) column with the ratio of dichloro- and trichloromonomer 1 : 1 , not polymerized at high temperature. ( ) column with the ratio of dichloro- and trichloromonomer 1 : 1, polymerized at high temperature. (0) phenol. ( 0 )cresol.
-- -~
#
I
SO
k
1
1
55
40
I 45 t
12 -
I
I
so
5s
4C
Figure 3. Dependence of k values on temperature 10
Column: 50% trichioro- and 50% dichloromonomer; flow rate: 1.0 ml/ min, ( 0 )phenol, ( m )cresol
-
? -
4 -
1 -
L 0
I
0.5
I
1.0
1.1
T
2.0
t of isopropanol
Figure 2. Dependence of k values on concentration of isopropa-
nol Conditions and symbols as in Figure 1
'
different, the order of elution on columns 5 and 6 resembles the one obtained with Porasil C. As a higher degree of cross-linking is known to take place with a trichlorosilane than dichlorosilane alone or the mixture of both, a different degree of cross-linking may explain these phenomena. However, more comparative experiments will be necessary to reveal the exact nature of these layers. Presently, it seems highly probable t h a t columns prepared with dichlorosilanes or their proper mixtures with trichlorosilanes represent systems more penetrable for a polar component of the mobile phase-a property which is essential for proper chromatographic function of these substrates. The columns with suspected cross-linking also exhibited somewhat lower efficiencies-presumably the effect caused by reduced mass transfer. A 20% addition of trichloro[3-(4chloromethylphenyl)propyl]silane to dimethyldichlorosilane in column 3 does not seem to provide sufficient selectivity. Kirkland's findings (14) t h a t the limited penetration of solvent into the polymer layer is responsible for poor in-
6
i
Figure 4. Chromatogram of the model mixture on Corasil I column ( 2 m m , i.d. X 80 cm) bonded with 2% of the mixture of trichloro- and dichloromonomer (1 : 1 ) Mobile phase 0.5% isopropanol in heptane: flow fate 1.0 mlimin.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 6 , M A Y 1973
973
teraction of solute molecules with the stationary phase and increased resistance to mass transfer have been confirmed with our systems. Generally, a rapid increase of column efficiency with the polarity of mobile phase can be observed. It must be pointed out, however, that such effect was grossly different for the columns with different types of polymers (see Figure 1).The number of theoreti.cal plates per second as a function of isopropanol concentration is different for two different stationary phases, while the dependence of k values on the concentration of isopropanol is essentially the same in both cases (Figure 2). Comparison of the two graphs also suggests that a compromise between column efficiency and selectivity must be carefully chosen. The resinous state of these polymeric layers is also suggested by a relatively long time of equilibration of newly prepared packings with a mobile phase. This may limit the usefulness of these columns in gradient elution experiments. k values of o-cresol and phenol on the column prepared with a 1:l mixture of dichloro- and trichloromonomer were investigated in the temperature range of 30-55 "C (Figure 3). No sudden change of efficiency or selectivity occurred within this temperature range which would indicate a phase transition. A gradual decrease in k values with temperature is in agreement with the results of Schmit et al. (20) obtained with reversed-phase systems. The separation of model compounds was carried out on a Corasil I column reacted with 2% of the mixture of dichloro- and trichloromonomer (Figure 4).
(20) A. Schmit, R. A . Henry, R. C. Williams, and Diekman, J . Chromatogr. Sci. 9 , 6 4 5 (1971).
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A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 6, M A Y 1973
CONCLUSIONS The advantages of chemically bonded stationary phases have been widely discussed elsewhere (9, 13, 21). It is our opinion t h a t even with column efficiencies lower than those obtained with conventional liquid-liquid systems, the chemically bonded nonhydrolyzable phases have a great potential for analytical work. Their remarkable selectivity combined with new high-efficiency columns (22, 23) and powerful gradient techniques will make them particularly suitable for the analysis of complex mixtures such as lipids or polycyclic compounds. The approach undertaken by ourselves has great promise of universality as further modifications (other than hydrolysis described in this article) can be quite easily made. It is necessary to stress that no optimum conditions were developed as yet, and additional work will be needed to improve on separation efficiency in particular. The results also suggest t h a t different procedures may be employed to control the selectivity of packings (types of monomers and their ratio, polymerization conditions, etc.). It is evident that a better understanding of the polymerization mechanism will be essential for further progress in this area. Specialized surface spectroscopic techniques and thermal analytical methods are a natural choice. Received for review November 30, 1972. Accepted January 29, 1973. This work was supported by the National Science Foundation Grants No. GP-33751 (to M. Novotny) and GP-26019 (to W. Parr). (21) "Modern Practice of Liquid Chromatography," J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971. (22) R. E. Majors, Anal. Chem., 44, 1722 (1972). (23) J. J. Kirkland, presented at the 9th International Symposium on Chromatography, 1972, Montreux, Switzerland, 1972.
