557th MEETING, LIVERPOOL
775
Haslam, J. M., Cobon, G. S. & Linnane, A. W. (1973~)Biochem. SOC.Trans. 2,207-209 Haslam, J. M., Perkins, M. & Linnane, A. W. (19736) Biochem. J. 134,935-947 Haslam, J. M., Spithill, T. W., Linnane, A. W. & Chappell, J. B. (1973~)Biochem.J. 134,949-957 Marzuki, S., Hall, R. M. & Linnane, A. W. (1974) Biochem.Biophys. Res. Commun.57,372-378 Marzuki, S., Cobon, G. S.,Crowfoot, P. D., & Linnane, A. W. (1975) Arch. Biochem. Biophys. in the press Proudlock, J. W., Haslam, J. M. & Linnane, A. W. (1971) J. Bioenerg. 2,327-349 Subik, J., Kolarov, J. & Kovac, L. (1972) Biochem. Biophys. Res. Commun.49,192-198 Watson, K., Haslam, J. M., Veitch, B. & Linnane, A. W. (1971) in Autonomy and Biogenesis OfMitochondria and Chloroplasts (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds.), pp. 162-174, North Holland, Amsterdam
High-Performance Liquid Chromatography of Ubiquinones, Ficaprenols and Dolichols PETER L. DONNAHEY and FRANK W. HEMMING Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. High-performance liquid chromatography offers several advantages over other conventional chromatographic systems. These include speed, direct quantitative assay of solutes, ease of recovery of solutes and the absence of a requirement of volatility of the solute. Separation of the isoprenologues of families of polyisoprenoid compounds requires reversed-phase partition chromatography, and in this area the availability of stable chemically bonded stationery phases for use in high-performance liquid chromatography is an added advantage. We are reporting here some preliminary observations relating to the application of this technique to the analytical and preparative separation of the components of families of the biologically important polyisoprenoid compounds the ubiquinones, the ficaprenols (Stone et al., 1967)and the pig liver dolichols (Hemming, 1970). Conventional reversed-phase partition t.1.c. was used to check the purity of the recovered solutes (Dunphy et al., 1966). The chromatographic system used was a Waters Associates model ALC 100 liquid chromatograph fitted with a refractive-index detector, a U.V. monitor (254nm) and an M6000 pumping system. Of several bonded reversed-phase stationery phases tried, p-Bondapak C,,/Porasil ( 4 0 p m ; Waters Associates) was the most successful, and results mentioned here were obtained with a pre-packed column (30cm x 5mm internal diam.) at room temperature. Ubiquinones were detected and assayed with the U.V. monitor and the polyisoprenoid alcohols with the refractive-index detector. The separation of a synthetic mixture of ubiquinones-7, -8, -9 and -10 was very successful. However, with a single solvent adequate separation of ubiquinone-7 and ubiquinone-8 resulted in long retention times for ubiquinone-10. For this reason a linear gradient of solvent composition from methanol-water (9:1, v/v) to dry methanol at 4ml/min (to = 0.75min) over 1h was found convenient. With approx. 25pg of each component, ubiquinones-7, -8, -9 and -10 had retention times (tR)of 8, 12.5, 19.5 and 24min with band-widths (tw)of 1.5, 2.0, 2.5 and 2.5min respectively. Reversed-phase t.1.c. confumed the complete separation of each component. This procedure appeared capable of satisfactory operation in the long-1 mg range. The upper limit and speed of the separation could probably be increased by further modification of the parameters. The method revealed the presence of several unsuspected minor contaminants. The separation of the components of a natural mixture of ficaprenols-10, -1 1 and -12 was less good than that of the ubiquinones, but was still complete when assessed by reversed-phase t.1.c. The most satisfactory result was achieved with methanol-water (1 :24, v/v) as solvent at 2ml/min (to 1.5min). A load of 3mg of ficaprenol mixture gave for ficaprenols-10, -11, and -12 tRvalues of 14,23 and 37.8min with t , values of 1.5,2.4 Vol. 3
776
BIOCHEMICAL SOClETY TRANSACTlONS
and 3.6min respectively. The sample appeared to be more heterogeneous than expected and some tailing of bands was apparent. Analytically the procedure was still satisfactory with dry methanol as solvent (2ml/min) with a decreased analysis time of approx. 15min. Sample load limits were probably between 200pg and 5mg. A natural mixture of pig liver dolichols (dolichols-17, -18, -19, -20 and -21) presented the major problem for the procedure, and in fact complete separations, as judged by reversed-phase t.l.c., were not achieved. The most satisfactory arrangement was to use as solvent chloroform-methanol (9:41, v/v) at 1ml/min (to = 3.lOmin) with a 2mg sample of the mixture. Under these conditions dolichols-17, -18, -19, -20 and -21 had t, values of 24.2, 30.5, 39.7, 49.5 and 61.5min and apparent t , values of 3, 4, 5, 5.75 and 6.5min respectively. However, bands appeared to be slightly distorted and tailing was apparent. Also, there was a surprisingly high degree of contamination of the bands eluted later by those eluted early, made apparent by reversed-phase t.1.c. of the recovered fractions. Attempts to improve the method by variation of column temperature, solvent composition and flow rate were not successful. Calculation of the chromatographic parameters indicated that possibly this difficult separation requires longer columns or recycling (Snyder & Kirkland, 1974). The problem is still under investigation. It was concluded that high-performance liquid chromatography can be used to advantage, with saving of time and labour, for the analytical and preparative chromatography of families of ubiquinones and ficaprenols. Partial separation of the components of families of pig liver dolichols can also be achieved. This work received financial support from the Science Research Council. Dunphy, P. J., Kerr, J. D., Pennock, J. F. &Whittle, K. J. (1966) Chem. Ind. (London) 15491552
Hemming, F. W. (1970) Biochem. Soc. Symp. 29,105-115 Snyder, L. R. & Kirkland, J. J. (1974) Introduction to ModernLiquidChromatography,pp. 17-90, John Wiley and Sons, New York Stone, K. J., Wellburn, A. R., Hemming, F.W. & Pennock, J. F. (1967) Biochem. J. 102,325330
1975
775
Haslam, J. M., Cobon, G. S. & Linnane, A. W. (1973~)Biochem. SOC.Trans. 2,207-209 Haslam, J. M., Perkins, M. & Linnane, A. W. (19736) Biochem. J. 134,935-947 Haslam, J. M., Spithill, T. W., Linnane, A. W. & Chappell, J. B. (1973~)Biochem.J. 134,949-957 Marzuki, S., Hall, R. M. & Linnane, A. W. (1974) Biochem.Biophys. Res. Commun.57,372-378 Marzuki, S., Cobon, G. S.,Crowfoot, P. D., & Linnane, A. W. (1975) Arch. Biochem. Biophys. in the press Proudlock, J. W., Haslam, J. M. & Linnane, A. W. (1971) J. Bioenerg. 2,327-349 Subik, J., Kolarov, J. & Kovac, L. (1972) Biochem. Biophys. Res. Commun.49,192-198 Watson, K., Haslam, J. M., Veitch, B. & Linnane, A. W. (1971) in Autonomy and Biogenesis OfMitochondria and Chloroplasts (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds.), pp. 162-174, North Holland, Amsterdam
High-Performance Liquid Chromatography of Ubiquinones, Ficaprenols and Dolichols PETER L. DONNAHEY and FRANK W. HEMMING Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. High-performance liquid chromatography offers several advantages over other conventional chromatographic systems. These include speed, direct quantitative assay of solutes, ease of recovery of solutes and the absence of a requirement of volatility of the solute. Separation of the isoprenologues of families of polyisoprenoid compounds requires reversed-phase partition chromatography, and in this area the availability of stable chemically bonded stationery phases for use in high-performance liquid chromatography is an added advantage. We are reporting here some preliminary observations relating to the application of this technique to the analytical and preparative separation of the components of families of the biologically important polyisoprenoid compounds the ubiquinones, the ficaprenols (Stone et al., 1967)and the pig liver dolichols (Hemming, 1970). Conventional reversed-phase partition t.1.c. was used to check the purity of the recovered solutes (Dunphy et al., 1966). The chromatographic system used was a Waters Associates model ALC 100 liquid chromatograph fitted with a refractive-index detector, a U.V. monitor (254nm) and an M6000 pumping system. Of several bonded reversed-phase stationery phases tried, p-Bondapak C,,/Porasil ( 4 0 p m ; Waters Associates) was the most successful, and results mentioned here were obtained with a pre-packed column (30cm x 5mm internal diam.) at room temperature. Ubiquinones were detected and assayed with the U.V. monitor and the polyisoprenoid alcohols with the refractive-index detector. The separation of a synthetic mixture of ubiquinones-7, -8, -9 and -10 was very successful. However, with a single solvent adequate separation of ubiquinone-7 and ubiquinone-8 resulted in long retention times for ubiquinone-10. For this reason a linear gradient of solvent composition from methanol-water (9:1, v/v) to dry methanol at 4ml/min (to = 0.75min) over 1h was found convenient. With approx. 25pg of each component, ubiquinones-7, -8, -9 and -10 had retention times (tR)of 8, 12.5, 19.5 and 24min with band-widths (tw)of 1.5, 2.0, 2.5 and 2.5min respectively. Reversed-phase t.1.c. confumed the complete separation of each component. This procedure appeared capable of satisfactory operation in the long-1 mg range. The upper limit and speed of the separation could probably be increased by further modification of the parameters. The method revealed the presence of several unsuspected minor contaminants. The separation of the components of a natural mixture of ficaprenols-10, -1 1 and -12 was less good than that of the ubiquinones, but was still complete when assessed by reversed-phase t.1.c. The most satisfactory result was achieved with methanol-water (1 :24, v/v) as solvent at 2ml/min (to 1.5min). A load of 3mg of ficaprenol mixture gave for ficaprenols-10, -11, and -12 tRvalues of 14,23 and 37.8min with t , values of 1.5,2.4 Vol. 3
776
BIOCHEMICAL SOClETY TRANSACTlONS
and 3.6min respectively. The sample appeared to be more heterogeneous than expected and some tailing of bands was apparent. Analytically the procedure was still satisfactory with dry methanol as solvent (2ml/min) with a decreased analysis time of approx. 15min. Sample load limits were probably between 200pg and 5mg. A natural mixture of pig liver dolichols (dolichols-17, -18, -19, -20 and -21) presented the major problem for the procedure, and in fact complete separations, as judged by reversed-phase t.l.c., were not achieved. The most satisfactory arrangement was to use as solvent chloroform-methanol (9:41, v/v) at 1ml/min (to = 3.lOmin) with a 2mg sample of the mixture. Under these conditions dolichols-17, -18, -19, -20 and -21 had t, values of 24.2, 30.5, 39.7, 49.5 and 61.5min and apparent t , values of 3, 4, 5, 5.75 and 6.5min respectively. However, bands appeared to be slightly distorted and tailing was apparent. Also, there was a surprisingly high degree of contamination of the bands eluted later by those eluted early, made apparent by reversed-phase t.1.c. of the recovered fractions. Attempts to improve the method by variation of column temperature, solvent composition and flow rate were not successful. Calculation of the chromatographic parameters indicated that possibly this difficult separation requires longer columns or recycling (Snyder & Kirkland, 1974). The problem is still under investigation. It was concluded that high-performance liquid chromatography can be used to advantage, with saving of time and labour, for the analytical and preparative chromatography of families of ubiquinones and ficaprenols. Partial separation of the components of families of pig liver dolichols can also be achieved. This work received financial support from the Science Research Council. Dunphy, P. J., Kerr, J. D., Pennock, J. F. &Whittle, K. J. (1966) Chem. Ind. (London) 15491552
Hemming, F. W. (1970) Biochem. Soc. Symp. 29,105-115 Snyder, L. R. & Kirkland, J. J. (1974) Introduction to ModernLiquidChromatography,pp. 17-90, John Wiley and Sons, New York Stone, K. J., Wellburn, A. R., Hemming, F.W. & Pennock, J. F. (1967) Biochem. J. 102,325330
1975
![A sensitive quantitative assay method for dolichols, cholesterol and ubiquinone using high-pressure liquid chromatography [proceedings].](/assets/img/hold.png)
