(11) Reed, G. W., Kigoshi, K., Turkevich,
A., U. S. Atomic Energy Comm. Rept. A/CONF-l5/P/Q53 (1958). (12) Seyfang, A. P.. Smales, A. A., Atomic Energy Research Establishment, Harwell, Berks. (England), Rept. AERE-C/R-980 (October 3, 1952). ( 13) Singer, S. F., OEce of Naval Research, London (England), Rept. ONRL76-52 (August 1, 19ii2).
(14) Smales, A. A., Atomic Energy Research Establishment, Harwell, Berks. (England)] Rept. AERE-c/R-930 (May 13,1952); The Analyst 77,778 (1952). (15) Verstegen, J. P. M. J., Ph.D. Thesis, University of Amsterdam The Netherlands, 1960. (16) Welford, G., Alercie, J., U. S. Atomic Energy Comm. Rept. NYO-4000 (June 1952).
(17) Whitson, D., Kwasnoski, T., I bid., K-1101 (June 16, 1954). (18) Zanten, B. van, Decat, D., Leliaert, G., Talanta 9,213 (1962). RECEIVEDfor review May 24, 1962. Accepted January 17, 1963. Presented at the Symposium on Activation Analysis, Grenoble, France, May 4-5, 1961.
Apparatuls for Extraction of Gases for lniection into the Gas Chromatograph Application to Oxygen and Nitrogen in Jet Fuel and Blood SAMUEL NATELSON and RODNEY L. STELLATE Department o f Biochemistry, Roosevelt Hospital, New York, N. Y.
b A
practical instrument has been designed for the purpose of extracting gases from relcttively large volumes of solvents for injection into the gas chromatograph. This instrument permits the anaerobic sampling of the specimen, evacuation with stirring to release the gases, removal of the solvent below the lower two-way stopcock, and injection of solvent free gases into the heliuin stream flowing into the gas chrcmatograph. The dissolved oxygen arid nitrogen in jet fuel is readily deiermined b y this technique. For blood an acid ferricyanide reagent is first added to liberate the oxygen and carbon dioxide, before injection.
Ideally a system should have a carrier gas flow t h a t is uninterrupted. Further, the volatile gas being analyzed should be injected into the system only after it has been completely separated from the solvent. This report concerns an attempt to approach such a system. The principle used is t o introduce the specimen into a microgasometer previously described (4). After release of the gas under vacuum, the solvent is sequestered in a storage chamber, and the gas is injected into a flowing stream of carrier gas. While the major purpose of this paper is t o describe the mechanism used, application to jet fuel for oxygen and nitrogen and to blood for oxygen estimations is also described below.
S
EVERAL SYSTEMS hzve
been suggested for the extraction of small volumes of volatile constituents from a relatively large volume of solvent for injection into the gas chromatogrE,ph. One system comprises passing the carrier gas over the agitated specimen (9). This has the disadvantage that often the volatile material is not released instantaneously, resulting in trailing in the chromatograph patterns. Another system uses a bypass to equilibrate the sample with the carrier gas and then passes the carrier gas over the spetinien ( I ) . It has been our experience that this has the disadvantage that the pressure changes in the system, nhen E,oing from regular flow to bypass and t m k again, result in patterns with a changing base line. This is so because i t takes some time for the column to eq iilibrate with the gas at different pressures. Others have used the Van Slyke manometric appar:ztus to evtract the gas before injection into the ga> chromntoSraph ( 2 , 7‘).
EXPERIMENTAL
Saponin-Ferricyanide Solution. One gram of saponin is dissolved a n d made t o 100 ml. with 0.9% sodium chloride solution. Potassium ferricyanide (1.2 grams) is dissolved and made t o 100 ml. with water. On t h e d a y of t h e test 1.5 ml. of t h e ferricyanide solution is mixed with 10 ml. of t h e saponin solution in a small glass-stoppered bottle and covered with a 1-em. laver of caprylic or octyl alcohol. Potassium Hvdroxide 3 N . KOH (16.8 grams) dissolved and made to 100 ml. I’ ith water. This solution is diluted threefold to make the 1N KOH. Some of the 3N KOH is transferred to a test tube containing 5 ml. of mercury and covered with light - mineral oil t o a depth of 1cm. Sodium Hvdrosulfite Solution. In a test tube is ;laced 2 ml. of mercury, I gram of sodium hydrosulfite, and mineral oil t o a depth of 1 em. Five milliliters of deaerated 1N KOH is now
Reagents.
added under the mineral oil. This reagent is prepared daily. Lactic Acid, 1N. Nine milliliters of 85% lactic acid is made up to 100 ml. with water. Deaerating the Reagents. Into a vacuum desiccator are placed the hydrosulfite reagent, the 3N KOH, the saponin-ferricyanide reagent, a small bottle containing 20 ml. of water covered with a 1-cm. layer of mineral oil, a bottle containing 10 nil. of IN KOH covered with a 1-em. layer of mineral oil, and another bottle containing 20 ml. of 1N lactic acid covered with a 1-em. layer of mineral oil. The vacuum desiccator is fitted with a pressure gauge, a pressure regulator, and a water aspirator or pump. The pressure is reduced to just below the vapor pressure of water, for the temperature of that particular day. Lower pressures will cause boiling. The desiccator is rotated occasionally or may be mounted on a mechanically rotated support which moves slowly to gently swirl the reagents without breaking the oil surface of the solutions. The gases form bubbles tvhich move u p and through the mineral oil or caprylic alcohol. After 20 minutes no more bubbles will be observed and the reagents are deaerated. During the deaeration process the sodium hydrosulfite mixes and dissolves in the I N KOH. Apparatus. The Gas Chromatograph. T h e gas chromatograph used was t h e Fisher Clinical Gas Partitioner #6-390, with constant temperature stabilizer. This instrument uses a silica gel column followed by a niolecular sieve column. A thermistor bridge is used as the detector and results are read on a Texas recorder. The reaction chamber mas removed from the instrument and the helium flow arranged to flow through the flow chamber attached to the microgasorneter at the rate of 100 ml. of helium per minute. This required a gas pressure of from VOL 35, NO. 7, JUNE 1963
847
r
1
I
Figure 2.
W Figure 1.
Number of divisions read for oxygen
Gas extractor and injector
1. Container with sample 2. Graduations 3. Bent pipet 4. Ball joint 5. 0.1 2-mi. mark 6. Reaction chamber 7. lower 2-way stapcock 8. Bypass tube 9. Ball joint 10. Connecting tube 1 1 . Rotating magnet 12. Reservoir 13. Glass barrel 14. Piston 15. Movable guide
16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26. 27.
Pulley wheel Hand pulley Motor Belt Motor Manometer Stopcock for drain Connecting tube Platinum contact Ball joint Injection capillary Flow chamber (8mm. bare) 28. Connecting tube to chromatograph 29. Gas chromatograph
18 to 22 lb. per sq. inch. The microgasometer was obtained from Scientific Industries, Springfield, Mass. The reaction chamber i* the Special Reaction Chamber 31-373-3 of their catalog. The blood rotator used for saturating blood with oxygen n a s also obtained from this same company. Procedure. Sampling. I n sampling with the instrument the upper two-way qtopcock (Figure 1) is turned so t h a t thP pipet communicates with t h e reaction chamber. The lower stopcock is turned so t h a t t h e storage rescrroir communicates with t h e reaction chamber. The mercury is advanccd with t h e picton by turning t h e whcel until a bubble of mercury protrude.; from t h e tip. T h e t i p is dipped into the specimen and the specimen is sampled to the required mark on the pipet. The pipet is then lon-ered into the mercury contained in the bottom of the test tube, nhen a fluid, such as jet fuel, is being sampled. Ten microliters of mercury is now sampled so as to trap the sample free of air. If blood is heing sampled, the blood is brought just above the desired mark. The tip of the pipet is then dipped in mercury contained in a separate tert tuhe, and the piston is advanced to the mark. The piston is then withtirairn
848
Reproducibility obtained for oxygen with gas chromatograph
Peak on extreme left i s combined oxygen and nitrogen peak. peak is indicated in figure
ANALYTICAL CHEMISTRY
so as to sample 10 pl. of mercury as a seal. Subsequent reagents are then sampled in a similar fashion. When a reagent, such as the saponin-ferricyanide reagent is sampled for blood analysis, caprylic alcohol is sampled first, then the reagent, and finally caprylic alcohol as a seal, before withdrawing the pipet from the test tube. Caprylic alcohol separates the mercury from the blood to maintain t'he interfacial tension and thus the meniscus. Reagents and sample are now drawn into the reaction chamber by dipping the pipet into a pool of mercury, drawing the mercury beyond the first stopcock to the mark at 0.12 nil. The upper stopcock is now closed by turning 90" and the sample is brought into t'he react>ionchamber by means of the piston, evacuating simultaneously until the mercury level is at the 3-ml. mark. The sample and reagents are now in the lower part of the reaction chamber. The mercury above the 0.12ml. mark drops down to leave the upper part of t,he reaction chamber and the tube clear. Release of Gases and Sequestering of Reagents. l'he stirrer is started and allover1 to operate for 3 minutes. The upper level of the reagents is brought to the 0.12-ml. mark and the pressure on the manometer is noted. The sample and reagents are again brought don-n to the reaction chamber and again mixed for 1 minute. The pressure is measured again. Khen two consecutive readings are the same, the gazes are considered completely released. The reagents and sample are now brought below the lover stopcock. The reagents are now in the storage chamber. The lower stopcock is now turned 180' to isolate the reagents from the instrument, bypassing the storage chamber. Injection of the Gas into the Chromatograph Stream. The piston is acivanced so t h a t the mercury in the manometer reads 300 mm. 'I'hc gas a t this point is still under reduced pressure. 'l'he upprr stolwork is now turned 90"
to communicate with the capillary protruding into the gas stream. Rlercury which extends above this stopcock to the capillary tip, from a previous run, is pushed back into the instrument by the gas pressure. One now retreats nith the piston to cause all the mercury to coalesce at a point halfway into the reaction chamber. At this point the piston is ad1 anced to inject the gases into the gas stream of the chromatograph, until the mercury reaches the tip of the capillary. K i t h the motorized unit, the motor is started. When the mercury reaches the tip of the capillary the motor is stopped by contact with a platinum wire which closes the circuit. ?'he upper stopcock is now turned 90" to isolate the instrument from the gas fl0T.
Preparing the Instrument for the Succeeding Sample. Khile the pattern 15 developing, a second sample is prepared for injection. This is done by retreating with the piston until the mercury is a t the loner part of the reaction chamber. The Ion-er stopcock is turned 180" so that the storage chamber now communicates uith the reaction chamher. The piston is advanced so that the reagents reach almost to the upper stopcock. The upper stopcock is nom turned 90" to arrange communication between the pipet and the reaction chamber. The reagents are ejected and discarded, along nith excess mercury added during the process. The instrument is non- rimed with deaerated water and if blood is being analyzed, nith 1.L' lactic acid. 1'0ensure that no air has hem entrapped in the n-a>hing process it i> adLisable to close the upper itopcock, evacuate, and then eject the last traces of air and reagent, after oliening the upper stoproc k to the pipet side. The Standard. Thirty microlitrrs of air is sampled in the wme way as the unknown saml)k and injected into the ga' (*hiomat ogrq )h. (T'p of uatei a t room temp.) X (relativc hiirnitiity) = parti:d p r v w i w of \\:itri \:qm (1'70).
Ihromctric. prcssuIc - Pw = corr. ~)ressure( P ) , Obtain factor (f) from “Handbook of Chemistry and Physics” to correct to 0” and 760 mm. from P and room temp. S701umc of air injectccl X f = corr. voluiiie of air injcctec! (V corr.). V corr. X 0.21 = volume of oxygen inject’ed. V corr. X 0.751 = volume of nitrogen injected. Prepare a standard curve for oxygen and nitrogen injecting 10, 20, 30, 50, :ind 60 pl. of air at room temperature. The standard curve is a straight line which does not go through the origin. Oxygen and Sitrogen in J e t Fuel. Using the techniques described above, the jet fuel is added to a test tube containing approximately 1 ml. of mercury. The fuel (0.2 ml.) is :ampled and sIt-ept into the dry instrument with mercury. The instrument is (evacuated and the gas extracted as described above, sequestering the fuel in the storage chamber (#12, Figure 1) as described above. ‘l’he volume of gas is estimated from the peak heights obtained for oxygen and nitrogen, comparing 11-ith the stantlard curve. When the gas chromatograph was not used the oxygen was determined in the jet fuel by absorbing i.n sodium hydrosulfate and measuring the pressure changes as described for blood (4). Residual gases were estimated by ejecting the residual gas, measuring the pressure after ejection of the gas and calculating as for oxygen. Oxygen Capacity of Whole Blood. After rinsing the instrument with deaerat’ed 1N lactic acid, 10 pl. of caprylic acid is sampled followed by 30 p l . of blood saturated with oxygen, 10 p1. of caprylic acid, 0.2 ml. of saponinferricyanide mixture, and 10 p1. of caprylic alcohol. The blood and reagents are swept into-the reaction chamber with enough mercury to bring below the upper stopcock and to the 0.12-ml. mark. The gases are liberated and injected into the gas (chromatograph as described. Correcti0.n is made for a n y trapped air from the matio of the height
Table 1.
Comparison of Values Obtained for Oxygen in Whole Blood by Microgasometer and Gas Chromatograph.
Gasometer Chromatograph X-rayb Ilemoglot)in, Hemoglobin, Hemoglobin, Hemagrams/100 grams/100 tocrit, Blood grams/100 KO. Vol. yo nil. Vol. yo ml. ml. 5% 1 10.45 7.78 10.22 7.62 7.85 22 2 10.88 8.12 10.02 7.60 8 01 23 __ 3 26. io 19.47 27.40 20 40 19.13 56 4 18.00 13.43 18.86 14.07 13.80 40 5 18.78 14.00 18.83 14.04 14.20 42 6 18.06 13.47 17.73 13.00 13.10 40 7 18.48 13.78 I 7 64 13.01 13.02 40 8 20.93 15.61 20.29 15.14 15 01 43 Values are mean of triplicates in each case. Factor for conversion of vol. % t o hemoglobin value was 0.746. Calculated from iron content, 50 mp./100 nil. Fe = 14.7 grams hemoglobin. Std. dev. (precision) for the values in vol. yG,for gasometer f0.21, for chromatograph f0.25. For hemoglobin by x-ray &0.12.
:
of the oxygen peak to the nitrogen peak observed with the air standard. This is subtracted from the observed ouygen peak. This factor with the Fisher instrument n as consistently 0.295. I3lood oxygen peak - (nitrogen peak) 0.295 = corr. oxygen peak. The corrected oxygen peak so obtained is compared to the standard curve to calculate the blood oxygen content. Oxygen Content of Blood. The hlood is drawn from a vein in the conventional manner into a syringe. The needle is removed and the syringe is covered n ith a metal cap full of mercury. This forces some mercury into the syringe. The syringe is now placed on the rotator (Scientific Industries, Springfield, Ill.) and rotated for 10 minutes at 60 r.p.m. Sampling is done by removing the metal cap while the syringe is held in a vertical position. This leaves a drop of mercury at the tip. A Tygon funnel is fitted to the tip and additional mercury is added to the funnel to create a mercury seal. The Tygon funnel is made from tubing which is forced on to the tip of a pipet and heated in boiling water to retain its shape.
-El-
-81s 7
46. 5
JET (J
FUEC
-8-
P 6) -8-
-8-
- N _
IL’
28
s
I
inject
L1
MI NUT E S
Figure 3. Chromatograph tracing for jet fuel, demonstrating that after evacuation, substantially all oxygen and nitrogen have been removed
The barrel of the plunger is nom advanced to move the blood into the funnel. Thirty microliters of blood is promptly sampled from below the surface of the blood, followed by 10 PI. of mercury a5 a seal. The blood is now assayed for oxygen as described above. The Tygon funnel is now removed, leaving a drop of mercury a t the tip or the syringe as a seal. The blood in the funnel is discarded. The syringe is sealed again with the metal cap full of mercury, and the syringe is replaced on the rotator during the analysis to mix the blood prior to taking a second sample. Iron with the X-Ray Spectrometer. Blood 10.1 ml.) is mixed with 0.4 ml. of water to hemolyze the cells. Tm-entyfive microliters of the mixture is measured into a confined spot as described hefore (5) on Whatman #40 paper. l’he iron is then estimated in the Philips x-ray spectrometer aq described previouqly (3, 6). using a 5-mil titanium filter over the x-ray tube wind ow. DISCUSSION
The instrument of Figure 1 is a schematic representation of the setup used. One can see that the injection chamber comprises a glass tube with a capillary sealed into it. -4ball joint permits it to be attached to the microgasometer previously described (4). Ball joints at the entrance and exit of the flow chamber (not shown in Figure 1) permit easy connection and removal for cleaning from the source of gas and the chromatograph. The shape of the peak is influenced by the shape and capacity of this chamber. Sharper peaks are ohtained as the capacity of the chamber is decreased. Sampling is rapid and accurately controlled with the piston. The total time required for gas release and injection is usually less than that required for t h e development of the chromatogram. Further, one can prepare the second sample for injection during the developVOL. 35, NO. 7, JUNE 1963
* 849
-2 JET A5hland -m
FUELO i l (JP6)
-
, \ a [id Drop
Hg. )lets
B I ood Barrel
luneer
Figure 5. Sampling of blood for oxygen content before
hydrosulfite O M I N U T E S
J o-
Syringe full of blood is held in vertical position Blood sampled is trapped between two columns of mercury
after
'
"
'
Figure 4. Chromatograph tracing for dissolved oxygen and nitrogen in jet fuel Curve on right is obtained after oxygen has been removed with sodium hydrosulfite
ment of the first chromatogram. These advantages added to that gained by a continuous flow of gas through a single system has resulted in reproducible results for oxygen in blood as seen in Figure 2. It is of interest to note that just prior to injection, the gas sample is pushed down into the reaction chamber by helium. Thus the gas is separated from the flowing stream by a layer of helium extending through the capillary. The sample need not be injected immediately. If allowed t o remain with the upper stopcock open for a few minutes and then injected, the same pattern is obtained. This is so because diffusion through the fine capillary is slow. The reproducible shapes of the curves obtained permit the use of peak heights for assay. These heights were compared to that obtained for an air standard described above. Table I indicates that the results obtained are satisfactory for practical use in the clinical laboratory. Just before sampling, the microgasometer needs to be cleaned of air by evacuation and injection. If some air is left entrapped in the mercury or introduced during sampling this will demonstrate itself by an elevated nitrogen peak as shown in Figure 2. To correct for this variable the peak height ratio of oxygen to nitrogen for air was measured. This factor (usually 0.295 for the instrument used) was multiplied
850 *
ANALYTICAL CHEMISTRY
by the height of the nitrogen peak and this amount subtracted from the height of the oxygen peak. Thus, in Figure 3 where the nitrogen peak is six divisions, 1.8 divisions would be subtracted from the oxygen peak. The validity of this procedure was checked by adding varying amounts of air up to 6 11. t o a single sample being assayed. When corrected in this way reproducible results were obtained for oxygen in the original sample. Thus if the reagents become contaminated with a small amount of air the analysis is still valid For larger amounts of air its source should be eliminated. If the tip of the pipet is touched when wiping, after sampling, some of the sample will be removed and replaced by a micro bubble of air which may escape notice. This was also observed in our earlier experiments, when using a syringe with metal tip for injecting a liquid sample. In this case one could easily overlook the minute bubble of air a t the tip because of the opaque needle. Again this is visualized on the recorder as an elevated nitrogen peak. To avoid introducing air into the system a mercury bubble should project a t the tip when dipping into the next reagent for sampling. n'ormally arterial blood contains 0.25 ~01.7~ of dissolved oxygen while venous blood contains approximately 0.1 vel.%. For dissolved nitrogen low values are also observed (8, IO). For this reason
corrections for dissolved oxygen and nitrogen were ignored. Another source of error is the fact that the dry air contains 0.93% by volume of argon. Dissolved argon in blood may be ignored. I n the air standard argon would represent 4.4% of the oxygen peak, since argon runs with oxygen in the system used. Heat conductivity of argon is somewhat less than that of oxygen. For example, the heat conductivity ratio of argon to helium is 39 to 340, while the oxygen to helium ratio is 57 to 340. Thus an error of approximately 5% in the direction of obtaining lower results should be obtained when an air standard is used for the calculation of results. Comparative results are listed in Table I for 8 consecutive blood analyses for oxygen by gasometer and chromatograph, compared to the iron content determined by x-ray spectrometry (3, 6). Results are converted to grams of hemoglobin for comparison. Calculation was based on the air standard described above. One would expect higher values for the chromatograph for oxygen. Actually no significant difference is observed among the three methods when the t test is applied for significance of mean differences. This may be partly explained by the fact that the correction we apply ignores dissolved nitrogen (which would depress the values, while the argon effect operates in the opposite direction) and partly by inherent errors in the precision of the three techniques. Figure 3 illustrates the results obt,ained with Ashland oil jet fuel (JP6). A 0.2-ml. sample of jet fuel was used for the analysis. Note that the disturbance of the pattern produced by the injection is not excessive. After in-
Table II. Oxygen and Nitrogen Content in Jet Fuel"
8
m
B CONTENT
8
52.7
I
CAPACITY
Microgasometer R e s i d u a l Gas chromatograph _____ Oxygen, gas,* Oxygen, Kitrogen, vo1./100 vo1./100 vo1./100 vol./lOO nil. rnl. nil. ml. 4.51 10.23 4.35 9.12 . 4.51 10.78 4.39 8.99 4.51 10.78 4.37 5.98 a Each value, mean values of triplicate determinations. All gases other than oxygen.
rapidly. For volatiles other than oxygen the gas chromatograph is a n obvious advantage. The reaction chamber may be electrically heated to temperatures of the order of 60" C. when less volatile materials such as ethyl or methyl alcohol are being assayed from blood. The instrument described is thus flexible and convenient for the purposes described above. ACKNOWLEDGMENT
Figure 6. Tracing of same blood for oxygen before and after blood has been saturated with oxygen
The authors are indebted to B. W. Taylor of the Fisher Scientific Co. for assistance and technical advice in setting up and usinglthe gas chromatograph. LITERATURE CITED
jection, the residual gas, after evacuation, was injected $1 second time to demonstrate t h a t substantially all the gas had been extracted. Note also t h a t results are reproducible. Figure 4 indicates that oxygen is being assayed, since after treatment of the gases with 30 pl. of sodium hydrosulfite the oxygen peak is practically eliminated, the nitrogen peak remaining constmt. The small residual peak contains some argon since argon is not separated from oxygen with the columns used. The values w e compared to those obtained with the microgasometer in Table 11. In sampling of blood for oxygen content it is essential that the blood be kept away from air. When mixing the blood on a rotator, inconsistent results were first obtained. This was soon shown, when red-cell counts were performed on the samplt, specimens, t o be caused by erratic sampling. By increasing the speed of the rotator from 18 r.p.m. to GO r p.m. and intro-
ducing globules of mercury into the specimen, adequate mixing was obtained. The method of sampling is illustrated in Figure 5. At all times the blood in the syringe is kept away from the air by a mercury seal. Sampling is taken from below the level of the blood and once the blood is pushed into the funnel and sampled i t is discarded. Figure 6 shows a comparative chromatogram of blood after oxygenation and before oxygenation. Note the increase in oxygen content and loss of C02 after oxygenation. A measured amount of air (6 pl.) was added with both samples to illustrate also the effect of air contamination on the chromatogram. I n comparing the microgasometer with the method of gas chromatography, the gas chromatograph has the advantage of convenience when assaying for oxygen. When carbon dioxide alone is required in the routine laboratory, the microgasometer yields results more
(1) Hamilton, L. H., Automated and Semi-ilutomated Systems in Clinical Chemistry, Ann. A;. Y . Acad. Sei. 102, 15 (1962). (2) Lukas, D. S., Avers, S. Rl., J . A p p l . Physiol. 16, 371 (1961). (3) Xatelson, S., Leigliton, D., Calas, C., Microchern. J. 6, 539 (1962). (4) Natelson, S., Rlenning, C., Clin. Chem. 1, 165 (1955). (5) Natelson, S., Sheid, B., Zbid., 5, 519 (19.59). ( 6 ) Ibid.,-7; '115 (1961). (7) Ramsey, 1,. H., Science 129, 900 (1959). (8) Sendroy, J., Jr., Dillon, R. T., Van Slyke, D. D., J . B i d . Chem. 105, 597 (1934). (9) Taylor, R. W., Preesau, J., Physiologist 2, 114 (1959). (10) Van Slyke, D. D., Dillon, R. T., hlargaria, R., J . Bid. Chem. 105, 571 (1934). RECEIVED for review December 13, 1962. Accepted March 18, 1963. Division of Biological Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962. Supported in part by National Institutes of Health Research Grant A-2829.
VOL 35, NO. 7 , JUNE 1963
851
