OPAQUE ULTRACENTRIFUGES FOR DIRECT ANALYSIS’,2 JAMES W. McBAIK
Department of Chemistry, Stanford University, California Received July I, 1998
Upon a recent occasion Kraemer (6) stated that nowadays the first instrument which any colloid laboratory should acquire is an ultracentrifuge. This must have seemed to many listeners and readers a counsel of perfection unattainable without special financial resources. It is the purpose of this presentation before the Colloid Symposium t o exhibit some of the inexpensive alternatives which are now available to any scientific laboratory that has only modest means, but yet is not satisfied with anything less than the highest degree of accuracy so far attained in this field. The opaque ultracentrifuges3 designed and developed by the author and his collaborators at Stanford University are all based on the direct airdriven spinning top of Henriot and Huguenard (5). They possess no optical system, and they are run at any desired constant temperature, in the open air (not i n vacuo), the driving air being passed through a coil of copper pipe immersed in a thermostat. They run at 125,000 to 186,000 R.P.M. with an air pressure of 100 lb. per square inch, but lower pressures of air are usually employed. The steel must be protected with many t,hin coats of Bakelite lacquer, each baked on at 135°C. With these simplest of means, both sedimentation velocity and sedimentation equilibrium are readily measurable with great accuracy. The sedimentation equilibrium of sucrose (9) yielded a molecular weight of 341 instead of the theoretical value 342. The sedimentation velocity of egg albumin ( 7 ) measured in rotor V (described below) gave szoo = 3.56 x 10-13 Presented a t the Fifteenth Colloid Symposium held at Cambridge, Massachusetts, June 9-11, 1938. Transparent ultracentrifuges are not discussed here, but it may be mentioned that the original air-driven ultracentrifuge of McBain and O’Sullivan (J. Am. Chem. Sac. 67,780, 2631 (1938)) has been so improved that i t is available for every purpose; for example, the sedimentation velocity of hemoglobin was found by A. H. Lewis to be s z o o = 4.65 X in agreement with the value 4.63 X published by Steinhart ( 3 . Biol. Chem. 123,513 (1938)), rather than the previous value of 4.5 x lo-” obtained a t the University of Upsala. a Eight different designs of these ultracentrifuges were exhibited at the Colloid Symposium, in addition to the transparent rotor of McRain and O’Sullivan. The external diameter of the rotors was usually 37 mm. 1063
1064
JAMES W. MCBAIN
in good agreement with the definitive value 3.55 X published by workers a t the University of Upsala. An ultracentrifuge avoids convection in the sedimenting liquid. This is achieved either by careful avoidance of temperature fluctuations and of vibration, or by allowing the sedimentation to take place within narrow radial spaces mechanically shielded from convection. With the direct air-drive w e employ, the temperature is as constant and fixed a5 the thermostat, I. PRIMITIVE ONE-PIECE ROTOR
We may begin with a reference to the very simplest hollow one-piece rotor with which many problems can be solved. The system may be immobilized by the use of a jelly or curd, the method introduced by McBain and Btuewer (8). This enables the sedimentation equilibrium of any substance present to be measured, but it is unsuitable for the measurement of wdimeritation velocity except that of the jelly structure itself. With 0.3 pi’r cent agar jelly it gave the same sedimentation rate (65 X as was gi7F.n (63 x 10-13) by the transparent ultracentrifuge of McBain and O’Sullivan. Swelling pressures of the jellies were also m e a ~ u r e d . ~Soap curd was used in the sedimentation equilibrium of sucrose (9). Details and drawings of the one-piece top may be found in earlier articles (8, 9). 11. ROTOR WITH ANNULAR WASHERS FOR
SEDIMENTATION EQUILIBRIUM
In this rotor, fully described by McBain and Tostado (9), the immobilized sedimenting liquid lies between horizontal annular washers spaced a t uniform known distances apart by using alternately narrow and wide washers. Convection is permitted in the liquid in contact and in equilibrium with the innermost part of the sedimentation column. Analysis of this liquid before and after gives the molecular weight of any monodisperse substance, such as sucrose, or larger particles. Dilute agar jelly has been used in the National Institute for Medical Research, London (Schlesinger: Naeure 138,519 (1936); Schlrsinger and Galloway: J. Hyg. 37, 445,463(1937)) to convert the Sharples Super-centrifuge into a convectionless ultracentrifuge. Five cubic centimeters of virus solution gelatmized with dilute agar lines the rlosed bowl t o a depth of 0.18 mm. Anolher 5 cc is then added and the film is so thin that convection does not occur, thus allowing both rate and equilibrium t o be measured. Virus of foot-and-mouth disease is measured after 3 min. .4n antibody required only 30 min. for sedimentation equilibrium. It is necessarily assumed that the agar jelly is of such concentration that i t neither swells nor sediments. This, however, ran be verified by direct experiment and adjusting the concentration of agar to the requisite value. Any influence of the agar on the absolute rate has to be tested by comparison in some other ultracentrifuge. Sedimentation equilibrium is of course unaffected (McRain: Science 87, 2250 (1938)).
OPAQUE U I . 1 ' I l A C E N T I l l ~ U ~ E S
1W5
Ill. ROTOR WITH ANNULAR WASHERS FUR SEDIMENTATION EQUILIBRIUM
This insertion in a rotor, designed by Tostado, is a modification of rotor 11, permitting analysis of thr liquid'ahove and below the sedimenting column, especially for use io polydisperse uyntems. Here the annular wavhers arr all alike and are inerrly piled loosely upon each other. They are kept rentered by perforatrd buttresses or ba.ses on the container, a5
FIG.1. Rotor with m n u l s i w.nehrrs ior w t l i m m t i i t i m evqiiilil~iiiini I>,li,l cirw,l,,r tliw.
is SIIOWII in figiirr I . 'l'la- liquid frmi tlic! iuiddlc. nlid Ila. iixtrrior hiw I t ) hr ritlalra\\wi a t approximntrly thr snnw rntc to avoid mixing. 1 1 1 using rotors 11, 111, IY, V, and VI1 thc rotor is stopprd I d o r e analysis, atid
experience shows that this is ea-ily accomplished without mixing. With rotor V I the samples must br takrn while running; in the case of rotor I this is merely a matter of optional ronvrnirncr, using the twhniqne described elsewhere (8, 9).
IV. CONCENTRIC BAFFLh RIXGS FOR ANAL1 SCS O F THE DIFFEREKT FRACTIONS
O F THE SEDIMENTATION COLUMK
I n certaiii cases such as the soaps it is advisable to analyze all portions of the sedimenting column. The method suggested by my former collaborator, Dr. Tirey Foster Ford,5is to use concentric metal rings closely fitting and bored radially with numerous holes Our own method, also exhibited, i i to use instead concentric piles or nests of loosely piled washers, each pile being held as a unit by three vertical pins connecting the uppermost and lowermost washer of that set. This has the advantage of providing radial spaces for sedimentation. RCTOR FOR SEDIMEKTATION VELOCITE OR EQUILIBRIUM USING SOLID
T.
CIRCULAR DISCS
?‘hc simpleit a i d for most purposts the best design (7) is that of NllcBain and Lcyda (figure 2). The cells are ideally radial, consisting of a central pilr of discs about 0.08 mm thirk, alternately wide and narrow, with a large disc. j u 5 t fitting the container a t top and bottom to keep them central 1 vcitienl axial pin runs through the central solid pile of metal, holding it together. Tlie sedimenting liquid is immobilized between the sucressive . Both the liquid outside the discs and that within them is anaiyzed. About 2 2 cc. of liquid has been employed Yolydisper>e systems require that runs be made a t several speeds There IS also the adTantage that every constituent may be analyzed for independently Surh opaque ultracentrifuges possess the further advantage over any transparent ultracentrifuge that the whole of the column of liquid from its upper surface outwards and from the very beginning of thc sedimentation ii available, without distortion, for exart measurement Incidentally, the agreement of the measurements of sedimentation velocity made with this cell with those obtained by the JIrBain and O’Sullivan transparent ultracentrifuge and those of Svedberg shows that there is no wall effect upon velocity of sedimentation or diffiirion where the spaces are as srnall as 0 08 nini , as would indeed be expected from hydrodynamic theory. Tlir protciii partic1lt.i or inolec~ule~ arc 10,000 times smaller than the rapillary s p w w ~ R t w i i l l y Swdkwrg and hi%cdahoratori (14) haw I)een nixkiiig rffrc*ti\( x I I W of :t iiniilar nnalyticd method by putting N partitioii oI filtvr p:il)(v iii the, ~inddlcuf their trxn\parrnt dtracentriluge cell. Attention is also directed to the design of the rotor by which a perfectly tight seal has invariably been obtained. The lower part of the rotor, ruggedly made of 4 UMA steel, has placed upon it in succession a narrow loose washer to cause slipping when assembling, a thin metal disc, a disc 6
Present addless. Shdl I)e\elopment Compaiiy, Emei y~ ille, Callforma
OPAQUE ULTRACENTRWQES
1067
of thin rubber or Pliofih, or rubberized Cellophane, etc., and upon this the upper part of the rotor is directly screwed down. Its thinner longer annular wall bends outwards in the centrifugal field, engaging still more tightly in the lower piece. The rotor is assembled and opened upside down so as not to disturb the cell and its contents. Two small nicks are made in the periphery of the cell to enable the liquid to be withdrawn before taking out the cell if desired. VI. ONE-PIECE STEEL R O M R WITH WINDOW AND WITH SECTORIAL RAQFLES
When it is inconvenient to make 8 two-piece rotor, or where with B non-aqueous solvent a suitable seal cannot readily he found, the simplest one-piece rotor (as in I) may be used for sedimentation equilibrium (as in 11) by simply inserting sectorial baffles (cut radially from annular
.......
SECTORIAL BAFFLES.
FROM ABOM Fra. 3. One-pieoe steel rotor with window and with sectorial bslles
washers) piled like open brickwork. For sedimentation velocity it is necessary first to place in the rotor a circular distributing disc. When platinum is used for this purpose the rotor must not he more than 20 nun. in diameter, since larger pieces of such pure soft metals as silver and platinum flow freely in the centrifugal field. In 8 two-piece rotor any strong metal or plastic may he used. A window must be left in the upper part of the two-piece rotor. The platinum disc is inserted in the one-piece 20-mm. rotor folded like an umbrella and then opened out. Through its center there is a smooth hole with upturned edges. Upon the distributing disc is piled the annular circle of sectorial b&es (6gure 3). To make a measurement of sedimentation velocity; only sufficientliquid is put in to fill the spaces between the baffles. After the top has been spinning for a suitable time, the Cellophane window is opened with a flame or
1068
JAMES W. MCBAIN
a razor (8, 9) without stopping the rotor. Then a heavy liquid such as carbon tetrachloride is inserted through the hole in the platinum, which instantly distributes it outside all the baffles, displacing an equal volume of liquid inwards. This is collected with a glass capillary scraping pipet. When a colored material is sedimenting, such as the respiratory protein of earthworm blood, no precautions whatever are taken except to stop when the pipet begins to collect some colored liquid. The sedimentation velocity obtained is naturally too large. In the case of earthworm blood such crude measurements (even with thick baffles) yield values of 80 to 90 X instead of Svedberg’s value of 61 X 10-13. However, even an approximate analysis as by color, or color reaction etc., enables correction to be made to the actual distance the meniscus has sedimented, the total volume displaced being known from the carbon tetrarhloride added. Method VI is far inferior to V when thick baffles with essentially liquid only between their edges are employed, but with thin baffles, as in I1 to V, placed just so as just t o overlap, it is much improved and contains much more liquid. 111. A LARGER ROTOR WITHIN INVERTED IMJIERGED .METAL
T C B E S FOR
VELOCITY OR EQUILIBRIUM
This is a modification by a number of workers a t Stanford University of the idea first employed by Elford and his collaborators (2, 3, 4, 13) and of the rotor of McIntosh and Selby (10, 11). VIII.
FORD’S MODIFICATION FOR GLASS CAPILLARY TUBES
The lower part of the rotor has a flat cylindrical depression upon its upper surface. In this rests a thin tray carrying two circular flanges through which holes are bored radially. Through these are placed glass capillary tubes closed a t the outer end. The tray is held down by a broad-headed screw. This is suitable for velocity using Elford’s method of observing the position of the boundary after the rotor is stopped, either by eye, using the color or scattered light or fluorescent light or cutting the tube in two for analysis, or by photography along with a series of tubes of different concentrations. This latter method has also been used by Dr. Ford for observing sedimentation equilibrium of hemolyzed beef blood. IX. THE BECHHOLD-SCHLESIhrGER
CONVECTIVE PROCEDURE
This method was originated (1, 12) in 1931. It encourages slow convection. Originally a commercial centrifuge was used. During the following years various quantitative and semiquantitative observations of its occurrence were made in the author’s laboratory at Stanford, in that of Beams a t Virginia, and also by Gratia in Belgium, using the simplest form of the one-piece hollow rotor I. Although admitting of quantitative results this
OPAQUE ULTRACENTRIFUGES
1069
is not an ultracentrifuge, and the periphery must be of, or lined with, some material which like filter paper holds all particles sedimented into it. McIntosh and Selby (10, 11) obtained quantitative results for the sedimentation velocity of bacteria, viruses, phages, and oxyhemoglobin, and also measured the actual specific gravity of the sedimentingo particles. For example, McIntosh and Selby obtained a diameter of 56 A. for oxyhemoglobin, identical with that quoted by Svedberg. Where in the absence of convection the concentration of the homogeneous part of the liquid after time T would become proportional to R ( l - e-"'), where R is the radius, Bechhold and Schlesinger have shown that with conR
vection it will be proportional to h ( 1 - e - b ' KT), where h is the inner radius of the sedimentiiig liquid. But after a given time in an ultracentrifuge, all the particles will have been centrifuged out, whereas in the centrifuge the concentration is asymptotically impoverished. Bechhold and Schlesinger show that therefore, if the centrifuging is greatly prolonged, the residual concentration of the convecting liquid becomes exceedingly sensitive to the size of the sedimenting particles, a change of twice the diameter soon making a difference of 105-foldin the concentration or as much more as is desired. This is valuable with virus or phage where the analysis may be good only to a single power of 10. I n conclusion, it may be noted that even the Bechhold-Schlesinger method may be used for distinguishing between monodisperse and multidisperse systems. It is easy to ascertain whether or not two substances are combined or associated with each other or sediment separately (as was done, for example, by Gratia in 1934). It is evident that the problem of obtaining exact quantitative data on sedimentation velocity, sedimentation equilibrium, the actual density or the true partial specific volume has been completely solved by simple means within the reach of every scientific laboratory, and that the time is approaching for this to be a routine experiment in courses of physical or colloid chemistry. SUMMARY
A number of simple and very inexpensive opaque rotors are now available with which measurements both of sedimentation equilibrium and of sedimentation velocity have been made with an accuracy at least as great as that obtainable with the best transparent ultracentrifuges. REFERENCES (1) BECRHOLD AND SCHLESINGIER: Biochem. Z. 236, 392 (1931);Z.Hyg. Infektions-
krankh. 112,668(1931);116,342,354(1933);Phytopathology 6,627 (1933). (2) ELFORD: Brit. J. Exptl. Path. 17,399(1936). (3) ELFORD AND ANDREWES: Brit. J. Exptl. Path. 17, 422 (1936). (4) ELFORD AND GALLOWAY: Brit. J. Exptl. Path. 18, 155 (1937).
1070
JAMES W. MCBAIN
(5) HENRIOTAND HUQUENARD: Compt. rend. 180, 1389 (1925); J. phys. radium 8, 433 (1927). (6) KRAENER:Ind. Eng. Chem., Anal. Ed. 10, 128 (1938). (7) MCBAINAND LEYDA:Nature 141,913 (1938);J. Am. Chem. SOC.80 (1938),communicated. (8) MCBAINAND STUEWER: Kolloid-Z. 74, 11 (1936). (9) MCBAINA m TOSTADO: Nature 139, 1066 (1937); J. Am. Chem. Soc. 69, 2489 (1937). 110) MCINTOSH: J. Path. Bact. 41. 215 (1935). (llj MCINTOSH AND SELBY:Brit. J . Exptl. Path. 18, 162-74 (1937). (12) SCHLESINGER: Z. Hyg. Infektionskrankh. 114, 161 (1932); Biochem. Z. 264, 6-12 (1933); Kolloid-Z. 67, 135 (1934);Biodynamica 1936, 1. (13) TANG,ELFORD, AND GALLOWAY: Brit. J. Exptl. Path. 18, 269 (1937). (14) TISELIUS, PEDERSEN, AND SVEDBERG: Nature 140,848 (1937);Ind. Eng. Chem., Anal. Ed. 10, 116 (1938).
