1239
Anal. Chem. 1882, 64, 1239-1245
Superficially Porous Silica Microspheres for the Fast High-Performance Liquid Chromatography of Macromolecules J. J. Kirkland’J E. I. DuPont de Nemours and Co., Central Research and Development, Experimental Station, P.O.Box 80228, Wilmington, Delaware 29880-0228
New wperfkiaily porous “Pororheii” particles have been synthdzed and tested for HPLC separations of macromob eculer. Spraydrying techniqueswere used to prepare silica mkrosphores that typically have a 5-pm solid core and a -1-pm-thlck outer shell wlth 300-A pores. Compared to conventlonai totally porous partickr of the same &e, Pororh.li partkks show improved mass-transfer kinetic prop ertiea that permtlmore rapid separations of macromolecules. Tho UHllty of tho new PorosheW cokwnn packlng k demonstrated with uparatlons of proteins and synthetic polymers.
INTRODUCTION Columns containing 30-50-pm pellicular and superficially porous silica particles greatly stimulated early interest in HPLC separations. Horvath and co-workers developed pellicular (thin-skin) ion exchangers for nucleosides.1p2 The pellicular particle conformation also was proposed as an optimum support for affinity chromatography and as a vehicle for heterogeneous catalysis.3~4Superficially porous particles with thicker outer shells were used extensively for liquid-liquid chromatography,“ and as the support for early bonded-phase packings in reversed-phase HpLc.8~9 These pellicular and superficiallyporous materials showed significant advantages in separation speed and reproducibility over the larger irregular, totally porous column supports used in the initial stages of HPLC. In addition, improved column stability and reproducibility resulted because of the spherical particles and greater mechanical strength. In the mid-19705, however, columns of spherical, totally porous silica particles of 20-pm pellicular and superficially porous particles, because of significantly improved separation speed.11.12 This improvement largely resulted from the much shorter diffusion paths for molecules within the porous structure of the smaller particles.12 Compared to pellicular particles, the higher surface + Current address: Rockland Technologies, Inc., 538 First State Blvd., Newport, DE 19804. (1)Horvath, C. G.; Preiss, B. A.; Lipsky, S. R. Anal. Chem. 1967,39, 1422. (2)Horvath, C. G.; Lipsky, S. R. Anal. Chem. 1969,41,1227. (3)Horvath, C. G.; Engasser, J. M. Znd. Eng. Chem. Fundam. 1973, 12,229. (4)Horvath, C. G . Biochim. Biophys. Acta 1974,358, 164. (5)Kirkland, J. J. Anal. Chem. 1969,41,218. (6)Kirkland, J. J. J. Chromatogr. Sci. 1969,7 , 7. (7)Kirkland, J. J. In Modern Practice of Liquid Chromatography; Kirkland, J. J., Ed.; John Wiley: New York, 1971;Chapter 5. (8)Kirkland, J. J.; DeStafano, J. J. J. Chromatogr. Sci. 1970,8,309. (9)Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLCMethod Deuelopment; John Wiley: New York, 1988; Chapter 3. (10)Kirkland, J. J. J. Chromatogr. Sci. 1971, 9,206. (11) Kirkland, J. J. J. Chromatogr. Sci. 1972,10, 129. (12)Snyder, L. R.;Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; John Wiley: New York, 1979,Chapter 5.
0003-2700/92/0364-1239$03.00/0
area of totally porous microspheres also permitted higher sample loads without compromising separation efficiency. Studies have shown that extremely small (lo0 OOO MW probably could have been eluted without difficulty. It is speculated that polymers of much higher MW might be characterized by this approach using Poroshell particles with thinner porous shells and weaker stationary phases.
CONCLUSIONS Superficially porous Poroshell silica particles with wide pores were successfullysynthesized by a spray-dryingmethod. Typical particles had a total diameter of about 7 pm and a 1-rm porous shell of -300-A pores. Particles of this configuration exhibit the desirable separation characteristics of 1-2-rm totally porous particles for large macromolecules, without the associated experimental disadvantages. These (28) Kirkland, J. J.; Rementer, S.W. Anal. Chem. 1992,64, 904. (29) Glkkner, G.; Stickler, M.; Wunderlich, W. Fresenius' 2. Anal. Chem. 1988,330,46. (30)Gl&kner, G.;MUer, A. H. E. J.Appl. Polym. Sci. 1989,38,1761. (31) Glkkner, G.; Barth, H. G . J . Chromatogr. 1990,499, 645.
1245
new highly-purified silica particles and resulting columns were not optimized during this study. Still, results clearly show the advantages of this particle configuration for HPLC separations of both synthetic and natural macromolecules. Compared to similar columns of totally porous microspheres, the separating efficiency of Poroshell columns was clearly superior for higher molecular weight macromolecules. Columns of the new Poroshell particles can be used generally in HPLC, including size-exclusion and reversedphase chromatography. Similar sample-loading capacity in size-exclusion chromatography was exhibited for totally porous and Poroshell particles with the same pore size. The applicability of the new Poroshell particles in reversed-phase separations is demonstrated by rapid separations of protein mixtures and broad molecular weight distribution synthetic polymers. The practical aspect of these new highly-efficientparticles is an important feature. Columns of Poroshell particles are packed efficiently by typical slurry-packing methods. Columns of conventional dimensions can be optimally used with ordinary HPLC equipment. Compared to 5-rm totally porous particles, it is expected that larger Poroshell particles (e.g., L7-pm diameter) should result in less shear for large macromolecules separated at equivalent mobile-phase velocities.
ACKNOWLEDGMENT I thank J. J. DeStefano of Rockland Technologies, Inc. for particle-size distribution measurements and for the sample of experimental Zorbax Rx-SIL. I also thank M. L. and M. J. van Kavelaar of DuPont for the electron microscopy studies. Finally, the assistance of G. R. Wooler with the experiments is greatly appreciated. RECEIVED for review December 17, 1991. Accepted March 16, 1992. Presented in part at the American Chemical Society Chromatography Award Symposium, 203rd National Meeting of the American Chemical Society, San Francisco, CA, April 6, 1992. Registry No. SiOz, 7631-86-9; poly(vinylcarbazo1e) (homopolymer), 25067-59-8.
