ANALYTICAL EDITION
12
OTHERSUQGESTED APPLICATIONS The method, it would seem, is admirably adapted to the study of slow chemical changes with time and to the detection of intermediate compounds in certain chemical reactions. The author has not had occasion, however, to do work of this. character, although one such experiment is under way a t present. Other possible uses of the method than those mentioned no doubt suggest themselves. There is certainly a need for more trained workers in order that the usefulness of this method as a research tool may be still more definitely determined and in order that there may be developed further
Vol. 4, No. 1
improvements that will render the procedure less difficult for the observer both visually and photoelectrically. LITERATURE CITED (1) Allison, Phys. Reu., 30,66 (1927). (2) Allison, Christensen, and Waldo, Ibid., 37, 1003 (1931). (3) Allison and Murphy, Ibid., 35,285 (1930). (4) Allison and Murphy, Ibid., 36, 1097 (1930). (5) Allison and Murphy, J. Am. Chem. Soc., 52,3796 (1930). (6) Allison, Murphy, Bishop, and Sommer, Phw. Rev., 37, 1178 (1931). RECEIVED August 20, 1931.
The Ultracentrifuge and Its Field of Research J. B. NICHOLS, E . I, du Pont de Nemours 4 Company, Wilmington, Del. T H E U L T R A C E N T R I F U G E was developed to provide a means for accurately characterizing the distribution of particle size in many natural and synthetic substances. Two types of centrifuge are described, one for yielding moderate forces u p to 10,000 times that of gravity and the other capable of producing an action 100,000 times that of gravity. The most important features of ultracentrifugal analysis developed up to the present are: First, the possibility of deciding whether a substance is monoor polydisperse and, if polydisperse, the range of particle sizes or number of molecular species pres-
e
ent; second, the uncovering of structural relationships as evidenced by the classification of many proteins into groups which are integral multiples of a molecular weight of 34,500; third, the separation and optical analysis of mixtures of micelles; fourth, the study of the efect of p H on the molecular condition or aggregation of a substance; j f t h , the determination of the molecular weights of complex dyes, which opens the way to a study of indicator action and the effect of salts on association; and sixth, Ihe study of the formation of primary colloidal particles and the factors influencing the subsequent growth.
THE ULTRACENTRIFUGE last five, there has come a realization of the imporThe development and refinement of the ultracentrifuge by tance of an accurate characterization of the distribu- Svedberg and his associates ( l a ) have provided a group of tion of particle size in many naturally occurring materials methods for answering such questions. Furthermore, these and synthetic substances designated as macromolecular com- methods promise to avoid many of the weaknesses of the pounds by the organic chemist, or as lyophilic colloids by the earlier methods and to constitute the most reliable and adaptcolloid chemist. Many methods have been used to gain a able technic for investigating dispersity in colloidal solutions. knowledge of particle size, but few have given more than an estimate of the mean size present, and most of them have PRINCIPLES INVOLVED failed to yield satisfactory answers to certain basic problems The principle of the ultracentrifuge is illustrated in Figure 1. under investigation in different fields of chemistry. The biochemist and the medical man would like to ascertain the A transparent cell containing the solution or suspension to be size of the unit present in filterable viruses and in the bacterio- studied is rotated at a speed sufficient to produce a centrifuphages. They would also like to know whether a rigorously gal force 1000 to 100,000 times that of gravity. A beam purified protein has changed its aggregation as a result of the of light passes up through the cell and permits the colorimetric process. The cellulose chemist wonders whether the cellu- determination, either by visual examination or by photolose molecule changes in size when treated with chemical graphic recording, of the changes undergone by the solution reagents, or whether the reactions are mainly topochemical. while the centrifuge is in action. If the solution contains The fubber chemist would like to obtain information on the equal-sized particles and the centrifuging is rapid enough, a differences in polymerization of rubber, balata, and gutta- sharp boundary moves outward. The particle size can then percha, and to decide whether viscosity changes produced by be determined by measuring the displacement of the boundaging or oxidation reflect changes in micellar size, The proc- ary xz-zl in the time interval tz-tl, and applying a modified ess of formation of primary particles and subsequent growth form of Stokes’ law. With nonuniform material, however, a and aggregation forms an important phase of colloid chem- partial separation is effected, giving one an opportunity to istry which has hitherto been difficult of attack. The indi- determine the distribution of sizes present from an analysis of cator reaction of dyes may be either a structural change or a the radial variation in concentration (29). From the molecular-kinetic standpoint there is no dischaege in molecular size. The setting of a paint film is s u p posed to be a colloidal process with the production of high tinction between a colloidal particle and a molecule in solution (g). The kinetic energy of any suspended unit which polymers.
URING the last fifteen years, and especially the
D
January 15, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
retains its identity is the same as that of a molecule; therefore, the term “micellar weight” will be used as a generic name for the weight of either a suspended particle or a molecule in solution. “Particle size” will be used to designate the equivalent radius of the molecule or particle. Depending upon the experimental conditions, micellar weight or particle size
FIGURE 1. PRINCIPLE OF ULTRACENTRIFUGE
may be determined either by the establishment of a sedimentation equilibrium between centrifuging and diffusion or by the measurement of the sedimentation velocity and of the diffusion (13).
13
LOW-SPEED TYPEOF ULTRACENTRIFWGE
An improved form of ultracentrifuge used a t the du Pont Experimental Station will serve as an example of the machines developed for investigations requiring moderate centrifugal forces (23). This machine was constructed to permit the use of either a colorimetric or a refractive-index method for measuring concentration changes. I n some systems, for example, rubber in benzene, cellulose nitrate in acetone, or cellulose in cuprammonium solution, the light-absorption method is not applicable because the absorption coefficient of tlhe dissolved substance is not sufficiently greater than that of the solvent in any usable region of the spectrum. If the solution is not turbid, the index-of-refraction method is more generally applicable because there is more frequently a sufficient difference in the refractive indices of solvent and solute. The method involves the measurement of the displacement of the lines of a micrometer scale photographed through the solution in the centrifuge cell (5, 6). I n order to increase the sensitivity of the method and to insure freedom from parallax, a photographic lens of rather long focal length is used-i. e., about a meter and a half.
REQUIREMENTS FOR SATISFACTORY OPERATION
A centrifuge developing a centrifugal force of 1000 to 10,000 times that of gravity renders possible the study of the sedimentation velocities of the lyophobic inorganic colloids and the sedimentation equilibria of the proteins and other organic lyophilic substances of micellar weight greater than 10,000. For the determination of the sedimentation velocities of these lyophilic substances and for the sedimentation equilibria of most substances in the range 10,000 to 1000, centrifugal forces of about 100,000 times that of gravity are required. The most important conditions for satisfactory ultracentrifugal analysis are : First, no vibrational or thermal effects should disturb the sedimenting system; second, the
FIGURE 3. ROTOR OF OIL-TURBINE ULTR A-CENTRIFUGE
A photograph of the arrangement of the centrifuge is shown in Figure 2. The machine is built on the base of B rayonspinning centrifuge designed by Siemens and Schuckert (3). Any tendency to injurious vibration is immediately counteracted by a rubber ring on which the synchronous motor is supported by means of a wide flange. The speed is regulated by means of a frequency transformer which permits a variation in speed from about 1500 to 12,000 r. p. m. Exposures are made by means of electromagnetically operated shutters. Speed is indicated by means of a magneto connected to the frequency transformer. As in the earlier types of centrifuges, thermostatic control is provided from 0” to 50” or 60” C., and hydrogen or helium is blown through the rotor chamber to cut down frictional heat. HIGH-SPEED TYPEOF MACHINE
For some purposes, however, it is desirable to have available extremely high centrifugal forces of the order of 100,000 times that df gravity. Then it is possible to determine the sedimentation velocity of lyophilic substances in two or three hours which might require from two to three days to attain sedimentation equilibrium with a low speed. If we are working with unstable materials that change continuously, this is a very decided advantage. It is also possible to obFIGURE2. PHOTOGRAPHIC REPRESENTATION OF LOW-SPEED ULTRACENTRIFUGE. A, front end of camera; B, centrifuge tain more exact information about mixtures of micelles because of the greater separation in the higher centrifugal field. proper; C , illumination unit The oil-turbine type of ultracentrifuge was developed by amplitude of vibrations of the rotor must be small enough and Svedberg and Lysholm (18,21) to produce fields of force up to the optical system well enough defined that sharp pictures 100,000 times that of gravity and yet not heat injuriously. can be obtained; third, cells should be constructed to with- Figure 3 shows the rotor. Figure 4 gives a diagrammatic stand the pressures of 100 to 200 atmospheres produced when representation of the centrifuge. The rotor is made of the centrifugal force reaches the magnitude of 100,000 times chrome-nickel steel and has openings for four cells. It is that of gravity; and fourth, the substance must undergo no driven by oil under a pressure of twelve atmospheres impinging against two eight-bladed turbines, one a t each end deterioration during the centrifuging.
14
ANALYTICAL EDITION
Vol. 4, No. P
the solution, pp the density of the particle] pm the density of the solvent, w the angular velocity, and q and c1 the concentrations a t the points zzand z1 distant from the axis of rotation of the centrifuge. The concentration is determined by the light-absorption method or in terms of the refractive index by L a m ' s method (6). No assumptions have been made in Equation 1 as to form and structure of the molecule or particle. I n addition, there are no membrane complications as in the osmotic-pressure method of determining micellar weight, and the presence of electrolytes is beneficial rather than harmful in that electrical potentials due to the separation of electrical charges h a v i n g d i f f e r e n t inobilities are repressed. FIGURE4. CROSS-SECTIONAL VIEW OF OIL-TURBINE ULTRA- On the assumption that the fricCENTRIFUGE. A, rotor; B, cell tional force per mole is the same for sedimentation and for diffusion, the of the shaft. The rotor revolves in hydrogen or helium following simple s e d i m e n t a t i o n 6 . SEDIMENgas a t a reduced pressure. Under optimum conditions the v e l o c i t y e q u a t i o n s for s m a l l TATIONEQUILIBRIW temperature difference between the rotor and the casing is $-intervals are obtained ($0): OF HEMLOCKExonly 1.5' a t 40,000 r. p. m. The oil from t h e bearings is preTRACT vented from entering the rotor chamber by an elaborate system of deflectors. The temperature is adjusted by cooling the oil before it is admitted to the turbine chambers. The observation windows are protected from oil mist by or r=-\i 97s (24 electromagnetically controlled shutters. The speed of the 2 ( P P - Pm) rotor is determined by means of a stroboscopic tachometer. The complete optical system and stroboscopic device for Both D, the diffusion constant, and s, the sedimentation measuring the speed of the oil-turbine centrifuge is shown in constant (l/w4dz/dt),characteristic of each molecular species or particle size, are calculated directly from the data obtained Figure 5. during the centrifuging ($1). FIELD OF RESEARCH I n order to give a concrete idea of the range of problems to which the ultracentrifuge technic has been applied, a, number of examples are given to illustrate some of the salienf features. Many of the illustrations are drawn from the protein investigations carried out by Svedberg and co-workers (Id, Id), and the others from work carried out at the du Pont Experimental Station. The technic of study brought out in the FIGURE 5. OPTICALSYSTEM FOR OIL-TURBINE ULTRACENTRIFUGE examples drawn from the protein work should be considered A Water-oooled lamp house H Objective as of equal applicability to the whole field of natural and J ' Motor B' Lamp or mercury arc synthetic polymers and colloidal material. Other phenomena. Ir? Strohosaope disk C: Water filter D Llght filter L' Resistance which can be studied by means of the ultracentrifugal methE' Shutter d Magneto generator N,' Hot wire ammeter, readings in r. p. m. F' Centrifuge ods, such as thermodynamic and molecular-kinetic properties G: Camera of large molecules, and certain chemical and physical problems have been collected in an earlier paper (9). THEORY At sedimentation equilibrium the change in free energy, dF, or the change in concentration with time, dcldt, becomes equal to zero at every point in the cell; integration of the basic differential equations give the following relations for micellar weight ($0):
For particle size the relation is
where M is the micellar weight, r the effective radius of the particle considered as a sphere, R the gas constant, 83.2 X 108, T the absolute temperature, N the Avogadro number, V the partial specific volume of the sutstance, p the density of
FIGURE 7. SEDIMENTATION OF CARBON MONOXIDE-HEMOGLOBIN
INDUSTRIAL AND ENGINEERING CHEMISTRY
January 15, 1932
To illustrate the appearance of a Sedimentation equilibrium, Figure 6 is reproduced. It refers to a centrifuging carried out on a 2.74 per cent solution of hemlock extract a t a centrifugal force approximately 7500 times that of gravity, with the use of the strong absorption of the material in the blue region of the spectrum for the photographic determination of concentration. Exposure 1 represents the start of the
0
ea5 &3t’wcI
om
azT
15
POLYDISPERSE SUBSTANCES-GELATIN
Although many proteins have been found to be monodisperse, such substances as gelatin, gliadin, and muscle globulin are polydisperse and usually are unstable. Krishnamurti (4) found gelatin to contain a range of micellar weights from 10,000 to a t least 70,000 a t a temperature of 20’ C. by the sedimentation-equilibrium method and to show large variations in sedimentation velocity with hydrogen-ion concentration. Figure 9 presents his data on the change of the sedimentation constant of gelatin with pH at 20’ C. It is evident that in strongly acid solution the gelatin decomposes to relatively simple molecules in contrast to the action of egg albumin in strongly acid solution (Figure 10). At the
i o em
from M**,3SU*
FIGURE 8. SEDIMENTATION CURVESOF L CARBONMONOXIDE-HEMOGLOBIN
centrifuging, and exposure 2 the final equilibrium state 44.5 hours after the start. A value of approximately 10,000 was obtained for the micellar weight of this tannin body. The red coloring matter of the extract was found to be either an integral part of the micelle or so strongly adsorbed as to appear to be. As an example of the sedimentation-velocity method, Figure 7 gives a reproduction of the photographic record of the centrifuging of a 1 per cent solution of carbon monoxidehemoglobin subjected to a field of force 87,000 times that of gravity. It shows the sedimentation of the solution 0.5, 1.0, . . 3 hours after the start of the run. The top row is the scale of concentrations in fractions of 1 per cent.
.
MONODISPERSE SUBSTANCES-HEMOGLOBIN By means of the ultracentrifuge it is possible to ascertain whether a substance exists as ansinglemolecular species or is made up of several. Figure 8 gives the variation of concentration with distance after 0.5,1.0, . 3 hours of centrifuging
..
FIGURE10. DISTRIBUTION CURVEFOR EGGALBUMIN IN 0.1 N HYDROCHLORIC ACID
isoelectric point the gelatin tends to precipitate out, but in the moderately alkaline region there is practically no aggregation. I n contrast to gelatin, egg albumin has the same sedimentation constant over the rather wide pH range of 2.0 to 9.0 (7,261. EFFECTOF ACIDSAND ALKALIES ON MICELLES
OF SEDIMENTATION CONSTANT, FIGURE9. VARIATION s, OF GELATINWITH PH (4)
of carbon monoxide-hemoglobin, a photographic record of which was given in the previous figure (21). The dotted curves for two and three hours represent the theoretical diffusion curves for a substance with a uniform molecular weight of 68,000 subjected to the same experimental conditions; therefore, hemoglobin consists essentially of a single molecular species.
Many materials undergo large changes when subjected to strong alkalies or acids. For instance, after native cellulose has been steeped in strong alkali or treated with strong acids, the x-ray diagrams are different from that of the native cellulose. Strong acid produces an aggregating effect on egg albumin, in contrast to the decomposition that usually occurs in strongly alkaline solutions of proteins. Figure 10 shows the denaturing action of 0.1 N hydrochloric acid on an originally monodisperse solution of recrystallized egg albumin with a molecular weight of 34,500. The strong acid caused the egg albumin to aggregate to gel clumps containing an average of about seven molecules per particle, which increased in size during the determination (7). SEPARATION OF MIXTURES OF MICELLES relative proportions of mixtures of substances, either si ilar or divergent in character, may readily be determined. With the ultracentrifuge, efficient separation can be made of
Vol. 4, No. 1
ANALYTICAL EDITION
16
TABLE I. MICELLAR WEIGHTSOF NATURALLY OCCURRINGSUBSTANCES (SVEDBERG AND CO-WORKERS) AND MOLECULAR WEIGHTSOF COMPLEXDYES APPROXIMATE MULTIPLE
MICELLAR WEIQHT
SUBSTANCE
MONODISPERSE PROTEINS
Class I Class I1 Class I11 Class I V Class V
Egg albumin, insulin, and Bence-Jones' protein Hemoglobin and serum albumin Serum globulin Edestin and seven plant globulins Hemooyanins
1 2 3 6
34,600 68,000 104,000 208 000 2 - 5 ' x 106
X X X X
34,600 34 600 34:500 34,600
POLYDIBPERBBI PROTEINS
Casein, laotalbumin, gelatin, musole globulin, and ten others
Mean micellar weight falls in approx. same range as monodisperse proteins
CELLULOSE
40 000 40'000 and smaller 33'000 approx.)
Cotton linters Wood Viscose (oellulose xanthate) Cellulose nitrate (largely degraded)
1o:ooo [approx.)
COMPLEX DYES
substances which would be difficult to separate by ordinary recrystallization or fractionation procedures. Figure 11 gives an example of such a separation. It shows the concentration gradient plotted against distance along the cell for a mixture of equal parts of serum albumin and edestin investigated by Time of Ccnlrifuq~n~
I hr.
2.25 hr.
ni"
2.5 ht:
1
,
3hr'
2 X formula wt. oa. 2 X formula wt.
1 389 1:682 and larger
Sodium eosinate (retrabromofluoresoein) Sodium eryrhrosinate (terraiodofluoresoein)
an assumption verified by the experimental findings of Svedberg and his associates (24). Astbury and Woods attempt to explain the fundamental unit of weight by suggesting that the primary-valence chains are limited to a length corresponding to 34,500 owing to vibrational instability. Svedberg's study of the molecular complexity of insulin (16) would seem to prove that the possibility of synthesizing this hormone is extremely remote CELLULOSE STUDIES I n his ultracentrifugal investigation of the micellar weight of cellulose in cuprammonium solution, Stamm (11) found that concentrations of cellulose above a few hundredths of a per cent had a marked depressing effect on the values obtained for the diffusion constant and the sedimentation constant, a phenomenon which occurs when some interaction between the micelles occurs or incipient gelling effects arise. I n this case it was likely due to the elongated shape of the micelles rather than to their size. The variation of the specific sedimentation velocity and of the diffusion constant with cellu1
CE FROM MENISCUS, Xm
-
FIGURE 11. SEPARATION OF MIXTURE OF SERUM ALBUMINAND EDESTIN PRODUCED IN ULTRACENTRIFUGE (5). A , serum albumin, molecular weight, 67,500; B, edestin, molecular weight, 208,000 Lamm (6). The two maxima become more and more separated as the centrifuging progresses. After three hours the edestin has been practically completely removed. Lamm's calculations showed that the movement of the two proteins was nearly but not completely independent of each other. REGULARITIES IN PROTEIN STRUCTURE Table I summarizes the regularities observed by Svedberg and his co-workers (12,14, 11, 8), in the molecular weights of proteins and other complex substances. The micellar weights of the monodisperse native proteins, with the exception of the hemocyanins, fall into classes of one, two, three, and six times 34,500. This rather surprising regularity and others found by Svedberg (16) remained without explanation until some recent considerations put forth by Astbury and Woods (1). They suggest that the sequence of numbers, 1, 2, 3, and 6, may arise from the crystallographic configurations that neighboring peptid chains can assume when linked together and -NHgroups. by the secondary valences of -GOSuch molecular associations might be expected to be reversible,
CELLULOSL COHCLNrRATfON e $OLW~ON 15 m o L n
IN PLRCLNT
O S O L U r l o n 16 WR5OLD
FIGURE 12. VARIATIONOF SEDIMENTATION CONSTANT, s, AND OF DIFFUSION CONBTANT, D, WITH CELLULOSE CONCENTRA TION (11)
-
lose concentration is shown in Figure 12, both for solutions centrifuged immediately after preparatioh and for solutions sixteen hours old. The degradation produced by the exposure to the air during the sixteen-hour period produced very little effect on the sedimentation velocity. However, an appreciable increase in the diffusion constant occurred, caused probably by the formation of a small amount of decomposition products of low molecular weight. Stamm found the micellar weight of cotton linters alpha cellulose (Table I) to be 40,000 on a copper-free basis, whereas wood cellulose contained in addition some micelles 20,000 in weight and a small amount of degraded material. Viscose was found to have a slightly smaller micellar weight than cotton linters alpha cellulose.
January 15, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
1
71 1
I
17
When ferric chloride is hydrolyzed at 100” C. the solution first assumes a clear red color but becomes turbid on continued boiling and finally forms a sediment, the rate depending on the original concentration of ferric chloride used. Figure 14 shows the large effect that continued digestion at 100’ C. had on an 0.037 M ferric chloride solution. The distribution
I
I
1
I
,
I
1
FIGURE 13. WEIGHT-OPTICAL DISTRIBUTION CURVES OF GRADED SERIESOF GOLDSOLSPREPARED BY NUCLEAR METHOD SOL
MEANRADIUS mN
c1
cs C8
C4
cs
s
2.9 4.1 6.9 9.4 15.6
AREA
% 108 114 106 96 102
MOLECULAR WEIGHTSOF COMPLEX DYES
It is possible to determine centrifugally the molecular weights of substances of small enough weight to give fairly reliable results by the ordinary methods of physical chemistry. The results obtained from the boiling-point elevation and from the sedimentation equilibrium in a field of force 9000 times that of gravity for the dyes sodium eosinate and erythrosinate agreed rather well (8). The ionization of the dye of course affected both methods. However, the opposing electrical potential arising in the centrifuge from the partial separation of the large dye ions from the small sodium ions may be repressed by the addition of 1 per cent sodium chloride. Under these conditions the dyes were found to consist in solution for the most part of molecules twice the formula weight with a small amount of more highly associated material for the iodocompound (Table I). The successful application of the ultracentrifuge method to the study of these dyes enables us to attack the special problems-degree of uniformity, purity, ionization, and the effect of substituent groups-of substances of molecular weight of the order of 1000 from two converging angles. FORMATION AND GROWTH OF PARTICLES The mechanism entering into the formation and growth of colloidal particles forms a rather important phase of colloid chemistry. A graded series of gold sols prepared by successive depositions of gold on the particles of a nuclear gold sol, which contained practically the smallest particles that can exist without re-solution in a medium, is shown in Figure 13. This series was used by the x-ray laboratory of the station in the analysis of the methods for determining particle size by the broadening of the x-ray diffraction lines. Rinde (IO) has studied the various theories of crystal growth in the light of his comprehensive investigations on the variation in the distribution curves of gold sols with conditions of preparation.
OF TIME OF HYDROLYSIS ON FIGURE 14. EFFECT WEIGHT-OPTICAL DISTRIBUTION CURVESOF FERRICOXIDE I FROM 0.037 M FERRIC CHLORIDE
SAMPLB We-6) We-9)
TIMEOF HYDROLYSIS
MEAN RADIUS
AREA
Hours
mp
%
1 8
4.4 132
90 89
curve of the hydrous ferric oxide formed from one hour’s hydrolysis still represents primary particles of 4.4 millimicrons radius. Continued digestion a t 100” C. for eight hours gave a coarse, brick-red sediment. The large growth from 4.4 to 132 millimicrons was largely caused by the secondary processes of dehydration and development of crystallinity.
LITERATURE CITED (1) Astbury and Woods, Nature, 127,663 (1931). (2) Einstein, Ann. physik., [4], 17, 549 (1905). (3) Elsasser, Siemens-Z., 12,580 (1925). (4) Krishnamurti, J . Am. Chem. SOC.,52, 2897 (1930). (5) Lamm, 2. physik. Chem., 138A, 313 (1928). Lamm, Ibid., 143A, 177 (1929). Nichols, J . Am. Chem. Soc., 52,5176 (1930). Nichols, Nature, 125,841 (1930). Nichols, “Sixth Colloid Symposium,” pp. 287-308, Chemical Cataloe. - 1928. Rinde, “Distribution of Sizes of Particles in Gold Sols,” pp. 146-65, Dissertation, Upsala, 1928. Stamm, J. A m . Chem. Soc., 52, 3047, 3062 (1930). Svedberg, “Colloid Chemistry,” 2nd ed., pp. 146-167, Chemical Catalog, 1928. Svedberg, Kolloid-Z. (Erganzungsband), 36, 57 (1925). Svedberg, Ibid., 51, 10 (1930). Svedberg, Nature, 123,871 (1929). Svedberg, Ibid., 127,438 (1931). Svedberg and Heyroth, J. Am. Chem. SOC.,51, 550 (1929). Svedberg and Lysholm, Nova Acta Regiae SOC.Sci. Upsaliensis (Volumen Extra Ordinem Editum), 1927. Svedberg and Nichols, J. A m . Chem. SOC.,45, 2910 (1923). Svedberg and Nichols, Ibid., 43, 3081 (1926). Svedberg and Nichols, Ibid., 49, 2920 (1927). Svedberg and Rinde, Ibid., 46, 2677 (1924). Svedberg and Sjogren, Ibid., 51,3594 (1929). Svedberg and Sjogren, Ibid., 52,279 (1930). Svedberg and Sjogren, Ibid., 52, 5187 (1930). RECEIVED August 20, 1931. Communication No. 7 1 from the Experimental Station of E. I. du Pont de Nemours & Company.
Other papers of this Symposium on New Research Tools will be found in INDUSTRIAL AND ENGINEERING CHEMISTRY, 23, 1223 and 1366 (1931); 24, 89 (1932); and in the February, 1932, issue.
