Anal. Chem. 1987, 5 9 , 2501-2506
2501
amine and 1,5-diaminonaphthalene, suggesting that two adjacent amino groups were simultaneody anchored to one large cavity, while dibenzo-30-crown-10fit snugly to l,&diaminonaphthalene rather than o-diamine compounds (6). The present method can afford structure-recognizing resolution of amino compounds, and the combined use of crown ether with an ion-pair agent (5) provides wider selectivity in controlling retention and separation in reversed-phase HPLC.
(A) (V
LITERATURE CITED
I-
-
P 0
4
Time
8
(min)
0
4
Time
8
(min)
Figure 11. Separation profiles of amlnocresol isomers in IPFPLC (A) and CERPLC (6). For HPLC conditions, see Table I.
8/2 (v/v), pH 2.5) contained neither sulfonate nor 18-crown-6, the mutual separations among methoxyaniline isomers, between AC I and V, and among AC 11,111, and I V were incomplete. By the addition of 10 mM of pentanesulfonate into the mobile phase, m-methoxyaniline was resolved from the other isomers, and AC I and V were separated, whereas the peaks of 0-and p-methoxyanilines and of AC 11,111, and IV were not resolved (Figures 10A and 11A). However, addition of 1mM 18-crown-6 into the mobile phase instead of pentanwulfonate (Figure 10B) resulted in the complete separation of methoxyaniline isomers in a shorter period of time than the result shown in Figure 10A, and addition of 2 mM 18crown-6 (Figure 11B) resolved five aminocresol isomers in almost the same period of time as shown in Figure 11A. As for diamino compounds, we reported previously that 24and 27-membered crown ethers fit o-phenylenediamine and 2,3-diaminonaphthalene rather than m- and p-phenylenedi-
(1) Karger, B. L.; LePage, J. N.; Tanaka, N. In High-Performance LlquM Chromatogfaphy Advances and Perspectives; Horvath, C., Ed.; Academic: New York, 1980; Vol. 1, Chapter 3. (2) Nakagawa, T.; Mizunuma, H.; Shlbukawa, A,; Uno, T. J . Chromatogr. 1081, 277, 1. (3) Nakagawa, T.: Shibukawa, A.; Uno, T. J . Chromatogr. 1082, 239, 695. (4) Nakagawa, T.; Shlbukawa, A.; Uno, T. J . C h m t o g r . 1085,254, 27. (5) Nakagawa, 1.;Shlbukawa. A.; Murata, H. J . Chromatogr. 1083, 280, 31. (6) Nakagawa, T.; Murata, H.; Shlbukawa, A.; Murakemi, K.; Tanaka, H. J . Chromatogr. 1085, 330, 43. (7) Nakagawa, T.; Shibukawa, A.: Kalhara, A.; Itamochl, 1.;Tanaka, H. J . Chromatogr. 1088, 353,399. (8) Nakagawa, T.; Shlbukawa, A.; Kalhara, A.; Tanaka, H. Nippon Kagaku Kakhl1088, 1002. (9) Iwagami, S.; Nakagawa, T. J . Chromatogr. 1086, 369, 49. (10) Wiechmann, M. J . Chromatogr. 1082, 235, 129. (11) Debowskl, J.; Sybilska, D.; Jurczak, J. Chromatographla 1082, 16, 198. (12) Debowskl, J.; Jurczak, J.; Sybilska, D. J . Chmmatogr. 1083, 282. 83. (13) Sybilska, D.; Debowskl, J.: Jurczak, J.; Zukowski, J. J . Chromatogr. 1084, 286, 163. (14) Zukowskl, J.; Sybllska, D.; Jurczak, J. Anal. Chem. 1085, 57, 2215. (15) Debowski, J.; Sybllska, D. J . Chromatogr. 1086, 353, 409. (16) Qazdag, M.;Szepesl, G.; Huszar, L. J . Chromatogr. 1088 377, 227. (17) Tanaka, M.;Mlkl, T.; Shono, T. J . Chromatogr. 1085, 330, 253. (18) Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. J . fharm. Dyn. 1081, 4 , 87. (19) Cram, D. J.; Cram, J. M. Sclence 1074, 783,803. (20) Horvath, C.; Melander, W.: Molnar, I.; Molnar, P. Anal. Chem. 1077, 49, 2295. (21) Izatt, R. M.; Lamb, J. D.; Izatt, N. E.; Rosslter, B. E.. Jr.; Chistensen, J. J.; Haymore, B. L. J . Am. Chem. SOC. 1070, 701, 6273. (22) HONath, C.; Melander, W.; Molnar, I . Anal. Chem. 1077, 49, 142.
RECEIVED for review March 24,1987. Accepted July 1,1987.
Synthesis and Characterization of 2-pm Wide-Pore Silica Microspheres as Column Packings for the Reversed-Phase Liquid Chromatography of Peptides and Proteins Neil D. Dadelson*' and J. J. Kirkland E. I. du Pont de Nemours and Company, Central Research and Development Department, Experimental Station, Wilmington, Delaware 19898
Slllca microspheres have been prepared wlth wlde (200 A) pores for the hlgh-speed reparatlon of blomacromolecules. These 1-2-pm partkles are synthesized In a narrow partlcle-slze dlstrlbutlon at relatlvely high yleld by significantly modtfylng the coacervation reactlon used to produce Zorbax (Du Pont) chromatography packlngs. Reversedphase columns prepared from 2 - r n partkles exhlblted > I 3 0 000 plrtw/m with good permeablltty and peak symmetry. *par a t b of multkompnent peptide and protein mixtures were petfofmed in less than a minute wtth good recoveries of such
solutes. Permanent address: Department of Chemistry, Miami University, Oxford, OH 45056. 0003-2700/87/0359-2501$01.50/0
The theoretical desirability of using very small particles (e.g.,
