ma
ANALYTICAL CHEMISTRY
acid, silver yielding a black zone, chromate a violet-red zone. For spraying, the reagent solution was placed in an atomizer (De Vilbiss) operated with compressed gas a t 5 to 10 pounds’ pressure. A typical separation is shown in Figure 1. In this separation, the silver chromate solution was 0.03 111. I t flowed into the cell a t the rate of about 5 ml. per hour. The electrolyte flowed a t about 270 ml. per hour, and about 1.7 hours were required for the flow of wash liquid from the top to the bottom of the cell. The electrical potential was 260 volts and the current was 55 ma. Safety Precautions. The high potential and large power output of the electronic rectifier (Figure 3) are a great hazard in the operation of the electrochromatographic cells. Care must be taken to avoid simultaneous contact with opposite sides of the moist cells or with the solutions flowing from the cell. Wires to the electrodes should be heavily insulated, especially where there is danger of contact with the cell support. DISCUSSIOK AND STRUCTURAL DETAIL
The separations illustrated by Figures 1 and 2 depend upon the delicate adjustment of many variable conditions such as electrical potential, the electrolyte and its concentration, the flow of t h e electrolyte, and the concentration and flow of the solution of the mixture. When all these conditions have been adjusted, continuous and complete separations of many ions may be effected. In conventional chromatography each zone of solute is contaminated by traces of less-absorbed solutes that form more rapidly migrating zones. In the continuous electrochromatographic method, by contrast, the ions follow separate paths through the cell; hence, the separation of ions by the continuous procedure should be absolute, as is indicated by the radioautograph in Figure 2. The separability of ions in the continuous electrochromatographic procedure is a function of their separability and sequence in one-way electromigration and in conventional chromatog-
raphy. As shown already, ions that separate to the same degree and in the same sequence by electromigration and by chromatography do not separate in the continuous method ( 5 ) . Ions that separate to the same degree but in reverse sequence should be readily separable in the continuous method. Scale drawings of the steel cell support and a wiring diagram and specifications of the electronic rectifier are presented in an unclassified document issued by the Atomic Energy Commission (Z), which contains a discussion of the effects of concentration of mixtures upon their separability, and includes a section on the selection of electrolytes. It also contains a discussion of the separability of mixtures by chemical precipitation, by conventional chromatography, and by continuous electrochromatography. independent methods that supplement one another. A part of this report concerns the nomenclature of separations based upon differential migration of solutes caused by flow of solvent and by flow of electrical current (4). ACKNOWLEDGMENT
The authors wish to thank Jane K. Glaser for preparing the photographs for this paper. LITERATURE CITED
(1) Sato, R.. and Norris, W. P., Quarterly Report, Division of
Biological and Medical Research, Argonne National Laboratory, ed. by A. AI. Brues, ANL-4531, 153-5 (August-October 1950)
(2) Sato, T. R., Norris, W. P., and Strain, H. H., Office of Technical Services, Department of Commerce, Washington, D. C., C. S. Atomic Energy Commission, Document ANL-4724 ( 1 9 5 1 ) . (3) Snyder, R. H., Lawrence, B., and Finkle, R. D., U. S. Atomic Energy Commission, MDDC-270, 1-7 (1946). (4) Strain, H. H., ASAL.CHEM.,2 3 , 2 5 (1981); 2 4 , 5 0 , 3 5 6 (1952). (5)
Strain, H. H., and Sullivan, J. C., Zbid., 2 3 , 8 1 6 (1951).
RECEIVED for review
September 4 , 1951. Accepted January 17, 1952.
Automatic Determination of Radioactivity on Filter Paper Chromatograms LOUIS B. ROCICLAND’, JOSE LIEBERJIAN, AND bI.-iX S. DUNS Chemical Laboratory, Linicersity of California, Los Angeles, Calif.
D
IRECT counting of radioactivity from isotopically labeled compounds separated on filter paper chromatograms is increasing in use for metabolism and other studies (1,3-5,9-11). Recently, Muller and Wise (6) have developed an automatic recording bet,a-ray densitometer nith which the radioactivity of labeled organic compounds may be determined with increased precision and economy of time. The automatic recording Geiger-Muller counter and automatic sample changer described in this paper were designed to determine the radioactivity of isotopically labeled compounds separated by the authors’ (6-8) small scale filter paper chromatographic techniques. The background counting rate is approximately one third that attained by Muller and Wise and the precision appears to be higher, especially for low-activity samples. DESCRIPTION AND OPERATION OF APPARATUS
K i t h minor alterations in the sample changer circuit, Scaling Cnit Model 163 (Nuclear Instrument and Chemical C o p ) has been used satisfactorily. The automatic sample changer was constructed for use with 1 Present address, Fruit a n d Vegetable Chemistry Laboratory, U. 9. Department of Agriculture, Pasadena, Calif.
Tracerlab equlpment including an SC-1-4 Autoscaler, an SC-4 Eagle preset counter, an SC-9 manual sample changer containing a TGC-2 Geiger-Muller (1.86 mg. per sq. em.) tube, an SC-5A printing interval timer, and an SU-38 laboratory monitor as well as with a General Electric photoelectric continuous pen recorder (Model 8CE 1 DPIB-2, D’Arsonval 2.5-0-2.5 pa.). The sliding platform of the manual sample changer was replaced by the parts shoivn in Figure 1. The new platform, e, was designed to permit movement of a brass bar, c, in small incrementa on a predetermined schedule governed by the radioactivity of the sample and the preset count. The scheduled movement of the bar (with attached filter paper chromatogram) under the slit, g, and the Geiger tube, b, is regulated by the dimensions of the ratchet (paired n-ith the slit) and by electronic actuation of the device shown in Figure 2. Paired slits and ratchets are employed interchangeably to vary the increment counted during a cycle from to 1 inch. As indicated in Figure 3, the apparatus is operated continuously and automatically by means of a series of switches, relays, and minor modifications of the stated commercial units.
The movement of the sliding bar, c, is synchronized with the sample number indicated on the printing interval timer and printed on the paper tape after a count is completed. The power supply is shut off automatically by means of the microswitch, k,
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2
779
An automatic sample changer was developed to permit the rapid survey and accurate counting of filter paper chromatograms with the aid of two types of recording counters and other standard apparatus for the determination of radioactivity. Examples are given of radioactive counts made of glycine-2-C14 spotted at five concentrations on Whatman No. 1 filter paper and of a typical phenol-developed test tube chromatogram of glycine-2-C14.The average recovery of radioactivity in glycine-2-CI4spotted at the origin was 85 * 6%. The R, value (0.48) of a chromatogram stained with ninhydrin coincided with that estimated from the peak of the radioactive curve. This apparatus may be emplo) ed to measure radioactivity of compounds containing higher energy, as well as carbon, radioisotopes on one- or (sectioned) two-dimensional chromatograms.
which is actuated by lever arm h after minimum extension of the ratchet, j or, in case of power failure, by the release of a locked-in relay, K-13. In constructing the apparatus the wires to pins 1 and 2 on the Jones plug of the Autoscaler were reversed without disturbing the relay points connecting pin 3 to pin 2. The Eagle preset counter was modified by soldering the incoming wire directly to the “clear” switch to eliminate the use of terminal 2 as a tie point, adding a jumper wire between terminals B and L2, installing outgoing wires from terminal 4,terminal L1, and the ground, and adjusting the contacts to permit operation according to the following schedule: B a n d L2
Operation Normal Counting Counting completed
Open Closed Open
Contact Points 1a n d 3 Open Open Closed
sigma relay, K-1, the time clutch solenoid, K-6, and the print solenoid, K-3, are de-energized, causing the timing t o stop and the print hammer to strike the paper. Because, a few milliseconds later, the Simplex print relay, K-2, disconnects the print solenoid, the latter is energized only momentarily. Simultaneously, relay K-1 applies a pulse to the count solenoid, K-14, causing the count to be stored in the preset counter, SC-4. If the count is n
!I”
2 and 4 Closed Closed Open
Sequence of Electrical Operations. When the predetermined number of counts set on the Autoscaler dial has been made. the
,
Q
I
b
Figure 2. \
Left, side view. Right, top view a t o 0 , see Figure 1
\
Figure 1.
Diagram of Automatic Sample Changer for Filter Paper Chromatograms
Lead shield Geiger-Mtiller tube, thin mica end window Sliding platform d . Removable brass mounting strip Stationary platform Slit holder 0. h. Interchangeable slit Microswitch actuator for breaking current a t end of run a.
b. C.
:;
Diagram of Actuating Mechanism for Automatic Sample Changer
i.
Spring Interchangeable ratchet Microswitch, current breaker 1. Ratchet actuating lever m. Ratchet release n. Relay mounting bracket o. Ratchet-actuating relay
j.
k.
p. y.
Ratchet-holding bar Restoring spring8
less than the preset number, counting continues, but if the preset count is attained an applied voltage shuts off the Autoscaler and resets the scale to zero. At the same time the time delay relay, K-11, closes, the reset clutch solenoid, K-4, is energized, and the time scale of the printing mechanism is reset to zero. A delay period of half a second allows sufficient time for the print hammer to strike the paper before the reset mechanism is actuated. When the reset mechanism starts to rotate, the microswitch, 8-5, is actuated and, after one cycle of operation has been completed, the reset clutch solenoid, K-4, is disconnected. Restarting of the Autoscaler is delayed until the Tracergraph hrts completed its reset cycle by application of voltage to the sequence relay, K-7. The latter remains closed by means of a pair of its own contacts until the reset mechanism completes its cycle and the microswitch, 8-5, returns to its original position. When the circuit is completed to the scaler start relay, K-8, the latter operates and counting by the Autoscaler begins. While the reset mechanism is being actuated voltage is applied to the solenoid, K-12, if the scale switch, 8-6, is in the “Off” position, causing a new sample to be moved into counting position. If the scale switch, 8-6, is in the “Scale” position, only alternate pulses cause the sample carrier to move, thus permitting the sample to be counted twice. The scale of two, comprised of relays K-9 and K-10 and resistors R-2 and R-3, is actuated
780
ANALYTICAL CHEMISTRY Operation of Automatic Sample Changer and Automatic Recording Counters. 4 filter-paper chromatogram is placed on a 3 / 4 X 6 inch strip of black glossy paper, which is mounted on a 3 / 1 8 X 15 inch brass strip, d. The brass strip with attached papers is placed in the recess of the sliding platform, c, and is secured a t each end with pressure adhesive tape. The sample changer is put in operating position by pulling c to its distal point against the tension of the springs, z. The printing interval timer and the index suitch, S-9, are set for manual operation, the Autoscaler and the Traceigraph are tuIned on, the momentary switch 8-10, IS depiessed, the Autoscaler is set at calibrate, the sample number IS set a t 1, and the Geigei-llullei tube is brought to its operating
by pulses from the scale of two-pulsing relay K-16. Since this relay is connected in parallel M ith the print relay, K-2, it operates a t the time the count is printed. Khen the sample changer solenoid, K-12, operates, the index advance solenoid, K-5, is energized and the index-print R heel is advanced to the next number. Power is applied to the entire system through the “Stop” switch, 8-11, and the contacts of the holding relay, K-13, which is operated by momentary closing of the “Start” switch, 8-10. The apparatus remains in operation until the microswitch, S-11, is opened by contact with the sample carrier entering the last position or until there is an interruption of power. If interrupted, the latter remains off until the “Start” switch, S-10, is closed.
G O E 0 A
0
-
K O
EXISTING TERM. STRIP IN TRACERGRAPH I
1 I
I
1 I
’ 0 -2 3 4-
33
0 0 T \ A
A
A
8 7
10
9
;=11
I 2 3 4
AUTOSCALER
TRACERLAB, S C - 4
MICROSWITCH
___ _ _ 5 6
4
I 2
--
--~
.__ _-.
3 pL-3
T O JONES PLUG ON AUTOSCALER
Figure 3.
Diagram of Circuit for Control of Automatic Sample Changer, Autoscaler, and Tracergraph Printing Interval Timer
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2 voltitge. After a warm-up period of 30 iiiinutes the scaler is adjusted to its predetermined count (4096). The indes reset, S-9, and the .4utoscaler cycle sivitcheP, S-8, are changed froni the iiianual to the automatic positions and a s e r k of four or five automatic calibration counts is made as a check on the operation of the scaler and the printinginterval tinier. The cycle switch, S-8, is placed in the manual position, the Autoscder is adjusted to Geiger-NCiIler counting. and, if desired, the scaling tact,or is altered to a new predetermined count. The cycle switch, S-8, and the index switch, S-9, are changed to automatic operation. Following these manipulations the apparatus operates automatically, recording the time interval to reach the predetermined count (singly or in duplicate as desired) of each increment of the filter-paper chromatogram until the entire strip has been counted and the apparatus is inactivated by pressure of the lever arin, h, against the microswitch, k.
781 Table I. Glycine Solution" per spot, 311. X 10-4 20 40 60 80
100 hrerage
Glycine-2-C14Counting Data
Single Aliquotsb, Counts/Min. Strip A Strip B 1 2 1 2 3 . 6 l d 3 . 2 3 3 . 8 1 3.73 3.97 3.58 4 . 0 0 3 8 0 3.77 3 . 8 2 4 03 4 . 0 8 3.68 4 . 1 5 3 . 8 0 R.95 3.94 4.14 3 . 8 2 4 2 6 3.70 3 . 7 8 3.8'1 3 . 9 7
Multiple Ali uotsc, counts/>?in. Strip A Strip B 1 2 1 2 3.70e 4 . 2 0 4 . 0 9 3 . 8 2 3.90 4 . 1 8 4 . 7 3 4.12 4.70 4.50 4.25 8.62 4 . 2 4 4.03 4 . 1 5 4.11 4.07 4 . 5 0 3 . 9 4 3 . 7 2 4,14 1 . 2 8 4.10 4 . 0 1
~~
Average, Counts/Jlin.
_ _Single __~____ A li i It i piealiquot 3 . 6 0 h 0 23/ 3.84jz0.17 3.93 rtO.13 3.89=0.18 4,0420.17 3 . 8 8 =t 0 2 0 1
aliquots 3 95 + O 20 I.llr0.31 4 20&Oo.53
414=tO.O8
406+0.28 4 13 2 0 3 0 g
i d . of ylycine-Z-CI', activity approximately 14,000 C / M 'ing. .Lpplied with Giliiiont 0.Ol-ml. ultramicroburet. .Lodied in aliouots of 20 X ml. with Gilrnont 0.01-nil. ultrainicrobiiret alloivine thoroiiuli drv" ing of's'pots between additions. Spot sizes were approximately e ual in area. d Total counts over background per ml. of glycine solution. 8ackground counts, average of 25 indii.idual determinations of 4096 counts determined in areas between radioactireupots, n-ere 24.15 124.12 and 24.17) for strip 4 and 23.32 (23.24 and 23. 41) for strip B. e Counts and backgroiind made as in d . Background counts, a v e r a g e !\-err 26.02 (26.03 and 26.02) for strip A and 24.20 (24.13 and 24.33) for ;lrir, B.
3.98 mg. per
h
C
/ Standard deviation where u = B
I.":
,
'
IL
= 4.
Standnrd deriation where n = 20.
The localization of radioactivity of a paper strip chromatogram niay be determined rapidly on the basis of a relatively small n u n her of predetermined counts recorded automatically as numbers on paper as described, or as curves on paper with the aid of a General Electric photoelectric pen recorder and a Tracerlab lalioratory monitor.
The pen recorder is connected by iiieans of a 100-K variable rrsistor to the milliameter of the monitor. The Geiger-Muller tube of the monitor is removed and the cable is connected by nienns of a four-pronged plug directly to the tube, b, in the shielded-sample changer, a. The tube is brought to its operating voltage hy adjusting the set-screw at the rear of the monitor wing a vacuum-tube voltnwter as aid in t'he adjustment. The .iutosralrr is set at calibrate sii(-iithat a normal counts per second
~
~
~~
~
~
45 40
c/M 35 30
25 0
5
IO
15 2 0 2 5 SEGMENT
30
35
4. Total Radioactivity of Glycine-2-C14 Spotted on Filter Paper with Multiple ,iliqiiots a t Five Concentrations Figure
P-3. Littlefuse 342001,3 A fiise post K-7. A d r a n c e K-1504 ( l l % v o l t ) , D . P . D . T . relay K-8. Advance K-1502 (115-voIt), S.P.S.T. relar K-9. Leach 1120 (50-volt), make before break relay K-10. Leach 1127 (50-volt). D.P.D.T. relay K-11. Adrance 304 B (115-volt a.c.), D.P.D.T. time delay relay K-12. Allen-Bradley Bulletin 702, Series 460, OBT-3 contactor K-13. Advance 953 B (115-1wlt a.c.1, S.P.D.T. relay K-16. Advance K-1501 S (lI.j-voltJ, S.P.S.T., 3.0.relay P L - 1 (male). Jones P-310-RP. 10 contact plug P L - 1 (fenialel. Jones S-310-CCT, 10 contact plug PL-2 (male,. Jones P-304-CCT, 4 contact plug PL-2 (female,. Jones S-304-.4B. 4 contact nlua Pi.-3 (male). ' Existing Jones p l u g o n SC14 'Eaile 'eset coilnter P L - 4 (fenialel . Amphenol 6 1-3IIP-6 1F pliig R-2. I . R . C . DI-I.1. 420-ohul resistor I . R . C . DG, 750-ohm resistor R-3. Microswitch BZ-2R1. S.P.D.T. sivitch S-5. S-6. I.C..4. No. 1299 switch s-7. I.C.A. S o . 1299, D.P.D.T. switch S-8. 1.C.A No. 1296, S.P.S.T. switch s-9. I . C . A . No. 1296, S.P.S.T. switch s-io. I.C.A. No. 1282, S.P.S.T., N.O. switch s-11. Microswitch BZ-2RI.. S.P.D.T. switch TB-1. Existing Jones terminal strip in Tracergrai printing inter, .a1 ti mer T B - 2 . .Jones 4-141. 4 terminal R t r i n 1'3-3. 5.i41,' j t & , l ~ ~ ;st;(; ~~f Pilot light. Johnson 147-1032 L n m p . lZO-volt, G w a t t . clear, candelabra base, S-6 bulb I.'-1. Existing fuse F-1 in Tracergraph printing interval timer F-2. Exjsting fuse F-2 in Tracergraph prinring interval timer K-1. Existing relay K-1 in Tracergraph printing interval timer K - 2 . Existing relay K-2 in Tracergraph printing interval timer K - 3 . Existing relay K-3 in Tracergraph printing interval timer K - 4 . Existing relay K-4 in Tracergraph print,ing interval timer K-6. Exjsting relay K-5 in Tracergraph printing interval timer K-6. Existing relay K-6 in Trarergrsph printing interval timer K-14. Existing count solenoid in S C - I Eagle preset counter K-15. Existing clutch solenoid in SC-4 Eagle preset counter P L - 3 (female). Existing Jones plug on Autoscaler R-1. Existing resistor E-1 in Tracergraph printing interval timer S-1. Existing switch S-1 in Tracergraph printing jnterval timer 3'-2. Existing switch S-2 in Tracergraph printing interval timer S-3. Existing switch S-3 in Tracergraph printing interval timer S-4. Existing switch S-4in Tracergraph printing interval timer 5-12. Existing "set" switch in SC-4 E a le reset counter S-13. Existing "olear" s n i t c h in 8C-4 8agfe preset counter
.J&
C M (total c o u n t s per m i n u t e c a l c u l a t e d from t o t a l c o u n t of 4096 per segment u n i t ) , v e r t i c a l axi9; segment ( u n i t &of 1 ' 8 inch), hori,ontal axib; d o t t e d l i n e , averagc- of b a c k g r o u n d c o u n t i n g
r a t e lietween radiodcti7e peal\.
pulse is fed into its counting circuit. The ilutosraler functions only as a preset recycle timer, the time cycle being adjusted by the selected predetermined count. IYhen connected to a Tracerlab SC-4 Eagle preset counter, the Autoscaler has a t'iniing range of 0.07 second t o 7.5 hours per sample. Since the Autoscaler is synchronized with the sample changer and the printing interval timer, the apparatus operates continuously and autoniatically as previously described, except that the printing interval tinier records the actual time required for the predetermined number of count,s per second pulses of each sainple-counting interval to accrue. While t'he actual time each sample is monitored is not critical as long as sufficient time is allowed for the integrating ty1)e meter to reach equilibrium, the printed record may he used as a check on the operation of the Autoscaler. Determination of Radioactivity on Filter Paper Chromatograms. Strips (each 10 X 18 X 130 mm.) of Whntman S o . I filter paper were spotted each a t 20-min. intervals with 20 x 40 X 60 X 10-5,80 X and 100 X 10-5 ml. of a solution containing 5.98 nig. per nil. of glycine-24". Other identical strips were spotted similarly with the same quantities of the radioactive glycine but using from one to five 20 X 10-6 ml. sliyuots (allowed tso dry thoroughly bet\yeen additions) of the glycine solution. The radioactivity of forty '/*-inch increments of each strip was counted automat.ically over a period of about 96 hours to a predetermined count of 4096. An example of the counting rates is shown in Figure 4. The average number of total counts above background observed a t
ANALYTICAL CHEMISTRY
782 each level of glycine is shown in Figure 5 . The total counts above background were related linearly to the quantities of radioactive glycine present in each spot. Examples of the counting data, calculated to a unit volume of radioactive glycine solution, are shown in Table I.
50
ADDENDUM
-in automatic device for scanning and determining radioactivity in filter paper chromatograms has been described recently ( 2 ) .
I
t-
the electrical circuits and to Theodore Winnick for the glycineof activity approximately 14,000 counts per minute per mg. under his counting conditions.
-1
LITERATURE CITED
(1) Block, R. J., and Stekol, J. d.,Proc. SOC.Esptl. Bid. M e d . , 73, 391 (1950). (2) Friersen and Jones, AXAL.CHEM.,23, 1748 (1951). (3) Keston, A. S.,Udenfriend, Y.,and Le\y, RI.,Federation Proc., 7, 164 (1948).
(4) Keston, A. S., Udenfriend, S.,and Leby, M., J . Am. Chem Soc., 69, 3151 (1947).
30 background 1
*
I
45
20
-40 -
solvent boundary
~
-35
-30 25 F jure 5 , Radioactivity of Glycine-2-CI4 Spotted on Filter Paper at Five Concentrations with Single and Multiple Aliquots
The radioactivity found on a typical phenol-developed test nil. tube chromatogram (8) spotted with three aliquots of each of the glycine solution described above is shown in Figure 6. The same chromatogramstained withninhydrin solutiongave apurple spot whose R, value (0.48) coincided with that estimated from the peak of the radioactive curve. The average recovery (three separate counts of radioactivity) of radioactivity initially spotted a t the origin was 85 =k 6%. DISCUSSION
The described automatic apparatus for measuring and recording radioactivity increases the speed and efficiency of small scale filter paper chromatography and extends the usefulness of such techniques for metabolic and other types of investigations. The authors' apparatus has been operated almost continuously for nearly two years in counting samples, some of which could not be counted readily, if a t all, by manual operation owing to the very low radioactivity. The electrical circuits and sample changing devices are relatively simple and inexpensive to construct. This apparatus has been employed only for determination of C i 4 on one-dimensional chromatograms, but i t should be applicable to other higher energy radioisotopes and to sectioned two-diniensional chromatograms. ACKiYOWLEDGLMENT
The authors are indebted to Karl K. Jensen and Ross W. Farmer for technical assistance in the design and construction of
20
RF*+
0.48
C / M (counts per m i n u t e corrected for background), u n i t s o n vertical axis; m l . X 10-4, units of volume glycine-Z-ClP solut i o n on horizontal axis; o p e n circles, single aliquots, a n d closed circles, m u l t i p l e aliquots. Each point represents average o f duplicate counts of 4096 each on each of t w o strips
15 I
-
origin
-10
-5 +
35 30 29 counts/min. Figure 6. Rj Value qnd Radio, activity of Glycine-2-C'd Determined on Filter Paper Chmmatogrqm For notations see Figure 4
( 5 ) Mdler, R. H., and Wise, E. N., ANAL.CHBM., 23,207 (1951). (6) Rockland, L. B., Blatt, J. L., and Dunn, h'f. S., Ibid., 23, 1142 (1951). (7) Rockland, L. B., and Dunn, M.S., J. Am. Chem. SOC.,71, 4121 (1949). (8) Rockland, L. B., and D u n , hf. S.,Science, 109, 539 (1949). (9) Taurog, A., Tong, A,, and Chaikoff, I. L.,!Nature, 164, 181 (1949). (10) Tomarelli, R. M., and Florey, K., Science, 107, 630 (1948). (11) Weland, T., Schmeiser, K., Fischer, E., and Maier-LeibnitqlH., Naturwissenschaften, 36, 280 (1949).
RECEIVED for review June 10, 1951. Accepted Novernber29,195l.l fPapeP 84 in a series. For the preceding related paper (Paper 78) see (6). Work aided by grants from the National Institutes of Health, Public flealth Sew' ice, and the University of Califqrnia.
