the height is proportionally less. However, if the peak is very sharp as in the case of valine, isoleucine, and leucine, the proportionality is maintained up to 2.0 absorbance units. The datz, from the shorter photometer cuvette can then be used with the nomogram in the following way to make the calculation. Peak absorbance and base line from the shorter cuvette are located on the nomogram, and the result in pmoles is multiplied by the appropriate factor that relates the response of the two cuvettes. Modification of the Nomogram. Certain properties of the nomogram in Figure 2 should be pointed out. If scales P , B , and H are left unchanged, then the pmoles and AA scales may be altered in inverse relation to each other. Suppose the 20-mm. cuvettes of Jones (4) were substituted for the 6-mm. cuvettes on which Figure 2 is based : the maximum absorbance for a given amount of amino acid would be about 3.33 times greater. The present pmoles scale can be used to graduate a scale for the longer cuvette on the opposite side. Thus, the response from the longer cuvette will be such that any quantity in micromoles p will lie op-
posite 3.33 p on the present scale. For example, 0.1 pmole will be opposite 0.333 pmole, 0.05 pmole opposite 0.167, etc. After this modification, the marks 1.5 and 6.5 on scale AA of Figure 2 would have the values of 5.0 and 21.6, respectively. Similar modification will make the nomogram applicable to the 15- and 150-cm. columns and the shorter cuvettes of the original automatic procedure (6). Such modifications as these, which permit the calculation of proline and regraduation for different dimensions of cuvettes, follow from the formulas that were used to construct the nomogram initially. In addition, it should be pointed out that all scales may be extended. For example, if the nomogram in Figure 2 were calibrated on scale AA with the data from another analyzer, the geometry of this analyzer might be such that some calibration marks would be above or below the margins of the nomogram: this will not nullify the results. Practical Considerations. Once a nomogram for specific analytical conditions has been devised, it has proved practical to duplicate some tens of copies of the nomogram without
any calibration of scale A A . After attachment to the plastic sheet for use with the indicator of Figure 3, scale AA can be calibrated. If some change in analyzer constants makes it necessary to recalibrate the nomogram or if usage mars it, a new copy can be mounted on the plastic sheet and calibrated. LITERATURE CITED
(1) Allcock, H. J., Jones, J. R., “The Nomogram,” 4th Ed., Sir Isaac Pitman and Sons, Ltd., London, 1952. (2) Benson, J. V., Jr., Patterson, J. A., ANAL.CHEM.37, 1108 (1965). (3) Hubbard, R. W., Kremen, D. M., Anal. Biochem. 12,593 (1965). (4) Jones, R. T., Weiss, G., Ibid., 9, 377 (1964). (5) Morris, C. J., Morris, P., “Separation Methods in Biochemistry,” Equation 4.48 on p. 62, Interscience, New York, 1964. (6) Spackman, D. H., Stein, W. H., Moore, S., ANAL. CHEM. 30, 1190 (1958). (7) Yonda, A,, Filmer, D. L., Pate, H., Alonzo, N., Hirs, C. H. W., Anal. Biochem. 10, 53 (1965). Investigation supported in part by the U. S. Public Health Service, National Institutes of Health, Grant HE-02558.
Cryoscopic Molecular Weight Determinations Using Dimethyl Sulfoxide as the Solvent R. S. George and R. K. Rohwer, University of California, Los Alamos Scientific Laboratory, P. 0. Box 1663, Los Alamos, N. M.
nitro comM pounds encountered in explosives research are so insoluble in the common ANY OF THE ORGANIC
cryoscopic solvents that their molecular weights cannot be determined with acceptable accuracy by the freezing point method. Since dimethylsulfoxide (DhfSO) appeared to be a reasonably good solvent for most organic explosives, we decided to determine whether it was otherwise suitable for cryoscopic work. DMSO has not commonly been used for this purpose in the past, although its convenient melting point (18.5’ C.) and its solvent properties (2) would appear to make it an obvious choice. While there are disadvantages also in its use, our conclusion from the work reported below is that DMSO is a useful and exceptionally versatile cryoscopic solvent. EXPERIMENTAL
Chemicals. Baker Analyzed Reagent DMSO and National Bureau of Standards naphthalene were used as received. Eastman Kodak phenanthrene and xanthone were recrystallized to a constant melting point. The nitro compounds were obtained from military sources. Octahydro1,3,5,7 - tetranitro - 1,3,5,7- tetrazocine (HMX) and hexahydro-l,3,5-trinitro-
s-triazine (RDX) were obtained in a purified form and have a minimum purity of 99.9%. The purity of the other nitro compounds was checked by thin layer chromatography. In several cases where further purification was indicated, the compounds were recrystallized until the desired purity was obtained. Apparatus. Measurements were made with a modified Aminco-Bowman Freezing Point Depression instrument (Xo. 5-2050). Although the mechanical features were retained, the electrical circuit was considerably modified to permit recording of the thermistor bridge output rather than visual observation of an electric eye deflection. The new circuit consists of an unbalanced Wheatstone bridge whose output is fed to a 0-100 millivolt recording potentiometer. Provision has been made for coarse adjustments of one arm of the bridge so that the pen of the recorder may be kept on scale as the thermistor cools. il line operated, Zener diode regulated power supply was used to operate the bridge. With this arrangement, the thermistor provided a readability of 0.004 degrees centigrade. The modified circuit diagram is shown in Figure l. Procedure. The freezing point depression measurements could not be made reproducibly if the solutions
were in contact with the atmosphere because of the highly hygroscopic nature of DMSO. Therefore, the molecular weight apparatus was enclosed in a dry box that was constantly purged with helium. The helium was monitored with a Consolidated Electrodynamics Corp. moisture monitor, and only helium tanks containing less than 10 p.p.m, water were used. It was not possible to monitor the dry box because the moisture monitor will also detect DMSO. All samples were placed in an entry port and purged overnight with helium before bringing them into the dry box. DMSO solutions should be handled cautiously as on contact DMSO is absorbed rapidly through the skin and can carry toxic materials with it. No attempt was made to measure the freezing point temperature directly with this apparatus. Instead, the recorded thermistor bridge output in millivolts corresponding to the freezing point of the solution was used. The values obtained for standard solutions of known concentrations were used to construct a calibration curve from which concentrations of unknown solutions could be interpolated. The thermistor bridge values were obtained by slightly supercooling the solution and then initiating freezing by scratching the side of the test tube with the VOL 38, NO. 9, AUGUST 1966
1285
IOV. D.C. SUPPLY
;
I/
100 K
IOOK
\
RANGE RANGE SW SW
115 V
P.C
3 3 .22 K each
THERMISTOR
Figure 1.
BRIDGE
Circuit diagram of thermistor bridge
130
Figure 3.
0
1
0
0.05
1
- a i 0
I
0.15
0.20
MOL-ARITV
Figure 2. Calibration curve for freezing point depression in DMSO
thermistor. The partly frozen solution will exhibit a constant temperature until all of the frozen material has melted. This constant temperature represented the freezing point of the solution and was observed as a straight line on the recorder. RDX and HMX, two commonly used explosives, and naphthalene, phenanthrene, and xanthone were used to construct the calibration curve. RESULTS AND DISCUSSION
The calibration curve for freezing point depression using a 330K thenn-
Table 1.
istor is shown in Figure 2. This curve represents a least squares fit of the data and has an average deviation of 0.1 millivolt. The differences between the reading for the pure solvent and the readings for the standard solutions were used for the ordinate of the calibration curve rather than the actual recorded values. In this way, small changes in the solvent due to slow decomposition and/or absorption of water were eliminated as the value for the pure solvent was determined immediately prior to dissolving the sample. It was found that the freezing point value of a freshly opened bottle of DMSO will decrease by 0.3 to 0.4 millivolts during several weeks storage in the dry box. Albers and Muller ( I ) have determined the molal freezing point depression constant of DMSO and found it to be 4.4 f 0.5’ C./moIal. By calibrating the wsistance of the thermistor through the temperature range 17’ to 19’ C. (Figure
Molecular Weight Determination of Known Compounds
Molecular weight Compound Theoretical Found Error (yo) 28.5 -0.7 N-methyl-N,2,4,&tetranitmaniline 287 440 -3.1 3,3’-Diamino-2,2’,4,4’,6,6’-hexanitrobiphenyl 454 250 $2.9 2,4Diammno-l,3,btrinitrobenzene 243 211 -1.0 1,3,&Trinitrobensene 213 277 +1.5 2,4Dimethoxy-l,3,btrinitrobenzene 273 225 -0.9 2,4,&Trinitrotoluene 227 313 -1.0 Pentaerythritol tetranitrate 316 384 -0.5 2,2,%Tnnitroethy1~4,4,4trinitrobutyrate 386 178 +1.1 N ,N‘-dinitro-l,4dlszacyclohexane 176 167 +3.1 N,N‘-dinitro-1,Bdiazacyclopentane 162
1286
ANALYTICAL CHEMISTRY
135 RESISTANCE (103 OHMS)
140
145
Calibration curve for 330K thermistor
3), we obtained a value of 4.0 f 0.1’ C./ molar from our data. The density of DMSO at 20’ C. is 1.10 grams/ml. (5), so the two values are in excellent agreement. This value is lower than, or a t best comparable to, the values for the common cryoscopic solvents. The results of molecular weight determinations on several organic explosives are shown in Table I. The reported values are averages of a t least four individual measurements. The great advantage of DMSO is that HMX, RDX, and the first three compounds in Table I are too insoluble for molecular weight determinations in any of the common cryoscopic solvents, and the generally excellent solvent properties of DMSO suggest that this advantage also may be important with other classes of compounds. The disadvantages of using this solvent are the requirement of operating in a dry atmosphere and its relatively low cryoscopic constant. However, for routine molecular weight determinations on various classes of organic compounds, DMSO is a valuable addition to the list of useful solvents. LITERATURE CITED
11’1 Albers. H.. Muller. E.. Nuturwissen~ ~ ~ schujlen’46, 75 (1959). ’ (2) “Dimethyl Sulfoxide,” Technical Bul\ - I
letin, p. 8, March 1963. Crown Zellerbach Corp., Chemical Products Divi(3)sion, Ibid., Camas, p. 2. Wash. WORKdone under the auspices of the U. S. Atomic Energy Commission.
