often prohibitively expensive, do not need a piston or cushion fluid of greater density to unload the rotor, since either air pressure or water supplied t o the core is satisfactory. Zonal systems offer biomedical research a new outlook for the isolation and study of cellular and subcellular systems. The ultracentrifuge is a sophisticated research instrument whose capabilities are ever increasing t o meet the demands of research. If a complete un-
by Ralph
R.
derstanding of human disease mechanisms and their treatment is to be achieved and the level of disease mechanisms is ultimately at the macromolecular level, then separation of cellular and subcellular species is of the utmost importance. References
tional Cancer Institute Monograph 21, p 41, 1966. (3) S. P. Spragg, R. S. Harrod, and C. T. Rankin, Jr., “The Optimization of Density Gradients for Zonal Centrifugation (19691,”in press. Suggested Reading
(1) Hans Xoll, Nature, 215, 360, 1967. (2) A. S. Berman, “Theory of Centrifugation : Miscellaneous Studies,” Na-
National Cancer Inst,itute Monograph No. 21, 1966. The Development of the Zonal Centrifuge.
ings proved t o be unsatisfactory (loss of sphericity at high speed?). Rotors with definite ellipticity rather than exact cylindrical shape were capable of higher speed before fpacture occurred. More recent observations have shown a decrease in temperature of the rotor upon acceleration from rest to 60,000 rpm. This was attributed t o stretching of the rotor and a consequent adiabatic change, causing a temperature drop of about - 1 . O O C . This was contested by Hiatt ( 2 ) but completely confirmed by Biancheria and Kegeles ( 1 ) by melting point techniques and by Pickles ( 3 ) by direct measurement with a thermistor mounted in the rotor. I t has been shown that the temperature decrease is linear with the square of the rotor speed, and the cooling for acceleration t o 60,000 rpm is very close t o 0.8”C. -4ccording t o Schachman (4)much of the data in the literature must be modified because of neglect of this factor. Svedberg made extensive studies of frictional losses of all kinds. Windage losses were examined in detail. By operation in hydrogen a t reduced pressure, the loss was strictly proportional to the 3/2 power of hydrogen pressure. Today, operation in a good vacuum is preferred. Speed control and speed measurement have been a continuing problem. The earliest control method involved a differential gear arrangement in which one input shaft was driven by the main motor or turbine, the other input shaft by a synchronous motor of known speed. The output shaft of the differential gear drove a rheostat controlling the main motor. Rotational speed was measured stroboscopically. Today, the measurement and control of speed can be as precise and elegant as one can afford. For example, a single reflecting spot on the rotor can be scanned photoelectrically, yielding an output pulse for each revolution. By
substituting an encoding disk for the single spot, several hundred pulses per revolution can be obtained. The pulses can be counted directly or after preliminary electronic pulse shaping. A scaler can indicate the total number of counts for a precisely defined time interval. I n the limit, an 8-decade scaler with crystal controlled counting of time (operation a t 100 MHz) can measure speed to 1 part in 108. This is quite feasible but not necessarily sensible economically. Speed control can be realized indirectly by other means. Thus, in the magnetically levitated rotor, designed by J. W. Beams, the rotor is brought u p to speed by an air turbine. After drive cutoff, the rotor is operating in a high vacuum and its decrease in speed can be as low as 0.3 rps per day! The researches of Jesse Beams a t the University of Virginia over a period of more than 30 years provide another classic in the field. Numerous key references are to be found in Ref. (4). The resources of physical optics have been adequate since the very beginning of ultracentrifuging techniques. Some of them, notably light absorption, have been vastly improved by modern methods of photoelectric photometry. The Jamin, Mach-Zender, and Rayleigh interferometers provide elegant means of following boundaries during ultracentrifugation. Since one can measure to within 004 of an interference fringe, with the sodium line equal t o 58936, this amounts to a distance of about 4 x 10-7 in. The original Lamm-scale method yielded excellent results but was exceedingly tedious and time consuming. The schlieren technique of T6pler and Thovert as modified by Philpot, Svensson, and Longsworth is widely used for viewing and analyzing refractive index gradients. Excellent examples of the patterns given by the various methods
H. Muller
CASCIATO’S discussion emphasizes
D the importance of large-scale zonal
ultracentrifugation permitting, as it does, the separation of cellular and subcellular species. I n his opinion, this is the ultimate role of the ultracentrifuge in biomedicine. In a preparative sense, this might well be true, but so much information is obtained during the course of ultracentrifugation that we wonder i f biochemists would agree with the finality of this significant achievement. I n the 46 years of development since The Svedberg’s pioneer n-ork, enormous advances have been made. Fern methods or techniques in science borrow so heavily on related disciplines. The ultracentrifuge has never been a simple device. Even the mere task of spinning a relatively heavy rotor smoothly a t speeds approaching 100,000 rpm is an engineering feat of the first order. Even the earliest investigations of Svedberg could have been published as outstanding examples of engineering, applied optics, or physical chemistry, entirely aside from the main problem of ultracentrifugation. Most experts seem t o agree that the 1940 summary of the field by Svedberg and Pedersen (S)is much more than a historical review; it is a classic which embodies practices and concepts still accepted and only greatly improved upon with the passage of time. The earliest studies coincided with our graduate studies a t Columbia University and they were required rezding particularly since our research was concerned with the optical and electrical properties of colloidal dispersions of gold. -411 students were urged t o study these papers as models of skilled research, interpretation, and documentation. Detailed mechanical studies with the original oil turbine-driven centrifuge revealed interesting effects. Ball bear-
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
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Commentary
are shown in the frontispiece of Ref. ( 4 ) . Absorption methods are being revived for two reasons. The first is the great advance in photoelectric photometry and the second arises from the use of much shorter wave lengths in the ultraviolet for which the absorbance by proteins and related substances is very high. This has permitted operation in the pg/ml range. We refrain from a discussion of the biochemical applications of the ultracentrifuge because our ignorance of microbiology and biochemistry is so great. These things have been going on for 45 years and many of the details are fully comprehended only by experts. We are enthusiastic about the field because it is so refreshing to note how good engineering and inspired instrumentation have contributed to the solution of such vital problems. Many years ago, Wolfgang 0stvc.ald wrote a n intriguing little book entitled, “The World of Neglected Dimensions,” referring, of course, to colloidal dispersions. Modern developments and refined concepts have differentiated between ill-defined lumpy aggregates and true, but gigantic molecules. That these things are at once of interest to the polymer chemist, the biochemist, and the clinician is obvious. Even the casual reader can profit from a reading of Refs. (4-6). D r . Schachman’s book ( 4 ) gives an excellent balance between technique and theoretical interpretation and, as the table implies, deals with biochemistry. Beyond this, he has contributed heavily t o the field. Ref. (6) covers a symposium, somewhat more recent, by experts lending their varied experiences in the field. The editor, John Warren Williams, is one of the pioneers in ultracentrifugation. All reviews, monographs, and summaries are out of date by the time they are published, particularly if the field is an active one. This does not diminish their value for those of us who hope to get caught up in a strange field. In this one, the periodic bulletins, entitled “Fractions” published by the Spinco Division of Bechman Instruments, Inc., of Palo Alto, Calif., is a useful source of reports on current developments. Literature Cited
(1) Biancheria, A,, and Kegeles, G., J. Am. Chem. Soc., 76,3737 (1954). (2) Hiatt, C. W., Rev. Sei. Insts., 24, 182 (1953). (3) Pickels, E.G.(1953). See Ref. (4). (4) Schachman, Howard K.,“Ultracentrifugation in Biochemistry,” Academic Press, Yew York and London, 1959. ( 5 ) Svedberg, The, and,, Pedersen, Kai, “The Ultracentrifuge, 1940, Oxford, Clarendon Press. (6) “Ultracentrifugal Analysis,” 1963, J. W. Williams, Ed., Academic Press, New York and London.
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