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Drying a t 180” C. does not entirely remove adsorbed water and hydrochloric acid and temperatures of more than 500” C. are therefore generally advocated. However, a maximum weight loss from the barium sulfate precipitate of only 2.8y0occurs on heating from 158” to 950” C. (4). Furthermore, as all samples were related to similarly prepared standards with identical selfabsorption corrections, it was felt that its greater convenience warranted the use of the lower drying temperature.
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ACKNOWLEDGMENT
finite thickness, were submitted to the analytical procedure. TKO milliliters of 0.1N sulfuric acid were added prior to precipitation with barium chloride. The deviation from the mean weight of barium sulfate recovered averaged approximately 0.3%. The variation in counting rate was of the same order. Losses in washing were negligible, as collected dried washes had an average of less than 0.4% of the activity of the radioactive barium sulfate itself. Difficulties reported by various investigators, such as incomplete oxidation of fecal matter and tissue, were not encountered with the sample sizes taken for analysis in this study. The use of Tween 80 prevented completely “creeping” of the barium sulfate precipitate. Adhesion of the finer particles t o the sides of the centrifuge tube mas adequately prevented by use of the dilute nitric acid wash solution. The design
Table 11. Determination o f Sulfur-35 in Replicate Blood Samples
Recovered Barium Sulfate“, hlg.
Specific Activity, C.P.M.
44.8 5340 45.0 5290 44.8 5325 44.6 5337 45.1 5324 Includes 23.4 mg. of carrier.
of the indented centrifuge tubes enhanced packing of the precipitate and facilitated removal of maximumamounts of the supernatant layer without disturbing the precipitate. !When ordinary conical centrifuge tubes were used, bumping was excessive and resuspension of the barium sulfate was difficult.
The authors wish to express their gratitude for the interest -shown by Joseph Seifter in this study. LITERATURE CITED
(1) Bo an, E. J., Moyer, H. V., ANAL. &EM. 28, 473 (1956). (2) Boursnell, J. C., Francis, G. E., Wormall, A., Biochem. J . 40, 743 (1946). 3) Carius, L., Ann. 116, 1 (1860). 4) Duval, C., “Inorganic Thermogravi-
metric Analysis,” . Elsevier, Amsterdam, 1953. (5) Erdey, L., Paulik, F., Magyar Tudo-
manyos Akad. 4, 73 (1954). (6) Folin, O., J . Biol. Chem. 1, 131 (1905). ( 7 ) Le Matte, L., Boinot, G., Kahane, E., Compt. rend. soc. biol. 96, 1211 (1923). (8) Pirie, N. W.,Biochem. J . 26, 2041 (1932). (9) Pregl, F., Grant, J., “Quantitative
Organic Microanalysis,” Blakiston, Philadelphia, Pa., 1946. RECEIVED for review November 30, 1956. Accepted March 15, 1957.
Microebulliometer for Determination of Molecular Weight MARTIN DIMBAT and F.
H. STROSS
Shell Developmenf Co., Emeryville, Calif.
b A microebulliometer has been developed which permits relatively convenient and precise determinations of molecular weight on as little as 10 mg. o f sample added to the ebulliometer in 1- to 2-mg. increments. The reduction in sample size over conventional methods was accomplished b y use o f a small solvent volume (5ml.) and a highly sensitive method of measuring temperature. By the use of thermistors, temperature differences can be measured to 6 X 1O P 5 O C. The ebulliometer design permits steadiness o f boiling within the limits of measurement for long periods of time. The sensitivity of temperature measurement also makes
possible extension of the range o f the determinations to molecular weights o f about 20,000.
T
of the small samples commonly obtained by modern separation techniques should often be known. To handle such small samples, ebulliometers have been developed (1, 4, 6-8, 11) which are capable of more sensitive temperature measurement and permit the use of smaller solvent volumes than before. Most recent developments are modifications of the Menzies-Wright ebulliometer (9) using liquid-filled differential thermometers. Ray (11) used a differential HE MOLECULAR WEIGHT
thermopile in a modified Menzies-11-right ebulliometer t o measure temperature difference precisely. Muller and Stolten (IO) reported using thermistors for the temperature observations in a HillBaldes molecular weight apparatus. Thermistors have been used in ebullionieters for service analyses in industry (3). The ebulliometer discussed here is a modification and refinement of this type of instrument. It differs from the hlenzies-Wright principle mainly in the method of measuring the pure solvent boiling point. I n the Menzies-m7right apparatus, sample and reference boiling points are observed by means of the characVOL. 29, NO.
10, OCTOBER 1957
1517
teristic differential thermometer in one ebulliometer. I n the thermistor ebulliometer, sample and reference solutions are measured in separate boilers as originally done by Kashburn and Read (13). One reference boiler can be used with any number of sample ebulliometers that employ the same solvent. This type of ebulliometer with its small vapor volume has small solvent and sample requirements.
Figure 1.
Microebulliometer
M
APPARATUS
Ebulliometer. The ebullionieter uses the Cottrell-type pump (5) to establish equilibrium between vapor and liquid (Figure 1). The reflux return line from the condenser is positioned so that the ebulliometer can be filled, emptied, and cleaned through the reflux condenser by use of small stainless steel tubing and vacuum. Thus, there are no joints or stopcocks in contact with boiling solution or vapor. To achieve adequate remixing, the solution, after having been pumped over the thermometric device, is mixed with the solvent stream from the reflux condenser by passing jointly through a drip tip before reaching the boiling solution. As a result, even fairly viscous solutions do not give rise to the visible concentration striations often observed in ebullionieters without this feature. Heat is supplied to the ebulliometer externally by use of the copper heater shown in Figure 1. The heater is made from a copper rod by drilling one end to fit over a Chromalox C-201C 50-watt cartridge heater. The other end of the rod is machined to make a snug fit in the ebulliometer heater well. I n order to promote smooth boiling, the inside of the heater well is coated with sharp, polvdered glass. This system gives smooth boiling which can be controlled easily. Much of the heat required by the ebulliometer is supplied by the vapor jacket (Figure 2 ) , which uses the same solvent as is used in the ebulliometer. Thus, the vapor jacket supplies heat to maintain the glassware temperature and only enough heat need be supplied by the ebulliometer heater to pump the solution up to the thermistor well. The ebulliometer is surrounded by a partially silvered vacuum jacket which reduces the effect of light and ambient temperature changes on the thermistor resistance. The vacuum jacket need not be silvered if the ebulliometer is housed in a lighttight box. Temperature Measuring Equipment. The thermometers used in the ebulliometers are Western Electric type 14A thermistors. These thermistors have a resistance of about 10,000 ohms a t 80' C. and have a negative temperature coefficient of about 4% per degree centigrade. The differential temperature between the reference and sample boilers is measured directly by having their respective thermistors in opposite arms of a Wheatstone bridge (Figure 3). A rotary selector srritch (Leeds &. Northrup 31-3-0-2) is included in the sample ebulliometer arm of the 1518 *
ANALYTICAL CHEMISTRY
("1 ,-4-mm. Hole
-Vacuum
Jacket
5-ml. Liquid
Level Sharp Fritted Glass
L
Heater
+ Thermistor
Leads
Heater Leads-
Ebulliometer Supported i n Jack:et on Glass Wool Figure 2. Microebulliometer assembly in vapor jacket bridge so that the temperature of up to 10 different ebulliometers can be compared to a single reference boiler. The smallest balancing decade of the Wheatstone bridge is a 25-ohm, 10-turn Helipot with a dial subdivided into 1000 divisions. Consequently, the bridge can be balanced to 0.025 ohm. I n order to make unnecessary the use of delicate, sensitive galvanometers, the output of the bridge is amplified. The alternating current signal produced by a 0.025ohm change in the sample ebulliometer thermistor is amplified and rectified so that a 100-pa. null indicator shows a 1-mm. deflection. Muller and Stolten (IO) reported that it was necessary to have a continuous current flowing through the thermistors when using a direct current bridge. This is unneces-
sary when using alternating current in the bridge. Ebulliometer Housing. Three ebulliometers one for reference and two for samples are housed in the metal cabinet shoir n i n Figure 4. The cabinet contains the water supply system for the condenser and the electrical supply for the jacket and boilerheateis. B potentionieter for each ebulliometer is supplied so that the curient t o each heater can he set t o the same value as used during calibration. The cabinet also contains a Moore Sullniatic pressure regulator Model 40-2 and a pressure manifold for keeping the ebulliometers under conitant pressure a3 discussed below. The bridge, amplifier. and null indicator are in a separate cabinet shonn on top of the ebullionieter housing in Figure 4. OPERATION
Pressurizing. The microebulliometers are operated a t slight positive pressure. This is done for tivo reasons: The boiling point elevations as measured by the differential thermistor readings are more stable when ebulliometers are under constant pressure; and it is difficult to find three or more thermistors lvith exactly the 3ame resistance and temperature coefficient of resistance. Therefore, change3 in temperature due to changes in atmospheric pressure may cause errors in differential temperature during prolonged deter-
minations. All possibilities of error due to changes in atmospheric pressure are eliminated by operating a t a pressure slightly above the highest atmospheric pressure observed at the laboratory. The pressure used during calibration
is used for all determinations of molecular weight. A Moore Nullmatic regulator (Model 40-2) is used to control the pressure on the ebulliometers. A manifold connects the ebulliometers with the regulator in such a manner that each ebulliometer can be disconnected separately without disturbing the rest of the system. The pressure of the system is measured to about 2 microns by measuring resistance of the reference boiler thermistor against a standard resistance in the bridge. The temperature response to small changes in pressure is rapid, so that disturbances following such adjustments last less than 30 seconds. Sampling and Weighing Techniques.
Figure 3. Bridge microebulliometer
for multiple-unit
I n applying molecular weight calculation methods to ebullioscopic data, the assumption is made that the weight of sample is accurate and that all of the error occurs in the temperature measurement. I n order t h a t this assumption be valid for the 1- to 2-mg. sample weights used in the microehulliometer, a microbalance must be used. A standard analytical balance could be used but in this case the sample additions would have to be larger. At least 0.02-gram additions would be required
to keep the weighing errors below 1%. Six sample additions of this size would make the concentration of the solution
Liquid samples call for special techniques because a drop ordinarily weighs 10 mg. or more; this is too large a sample for normal operation. Liquid samples are transferred from micro weighing bottles to the ebulliometer by dipping a small loop formed on the end of a wire, first into the sample, then through the condenser into the refluxing solvent. The loss in weight of the bottle is taken as the weight of sample. No measurable amount of solvent is removed from the ebulliometer if the wire loop is touched to the side of the condenser just above the reflux line prior to its removal. To show that no solvent is lost either in opening and closing the ehulliometer or in using the wire loop, the following experiment was performed. Twice the normal amount of solute was added to an ebulliometer, the clean wire loop was dipped into the refluxing solvent eight times over a 4hour period, the ebulliometer was pressurized, and the temperature was measured after each insertion of the wire. The temperature of the ebulliometer remained constant within the accuracy of measurement throughout the test. Any loss of solvent durine the test would have appeared as an increase in temperature of the ebulliometer. EXPERIMENTAL
Figure 4.
Table I.
Three-unit microebuliiometer assembly
Precision and Accuracy. The acaccuracy and precision of the ebulliometer can be derived from measurements on pure materials. The apparatus constant, which reflects a small empirical adjustment of the ebullioscopicconstant, RT? was calculated from duplicate determinations of pure diphenyl in benzene. The accuracy of the apparatus was studied by measuring a series of pure compounds of known molecular weight: dicetyl, anthracene, chrysene, phenanthrene, and cetane. Six separate sample additions were made for each compound. The total sample used varied from 8 to 15 mg. Thkse results are shown in Table I. The largest deviation from the true molecular weight
Molecular Weight of Pure Compounds Determined in Benzene in Microebulliometer
Sample Dicetyl Dicetyl Snthracene Chrysene Phenanthrene Cetane
Total Sample Used, Mg. 16.67 14.93 14.81 7.65 9.26 15.71
Molecular Weight Foundo Theoretical 454 451 177 227 178 227
451 451 178 228 178 226
% Error C0.7 0.0 -0.6 -0.4
Calculated by gradient method ( 3 ' ) . VOL. 29, NO. 10. OCTOBER 1957
1519
Table II.
P, Atmospheric Pressure, Mm.
R, Bridge Resistance Reading (Scale Divisions)
AI’
AR
760.1 761.3 762.0 764.5 768.1
1434 2090 2680 4338 6719
1.2 1.9 4.4 8.0
656 1246 2904 5285
was 0.7%; the standard deviation was 0.44%. These samples formed nearly ideal solutions and, therefore, represent the best possible accuracy of the apparatus. Larger errors are to be expected when dealing with higher molecular weight materials such as polymers. An estimation o’f the precision of the individual boiling point measurements can be made from these data. The molecular weights were calculated by fitting a least squares straight line through the six individual boiling point elevations for each sample. Assuming that all of the error in these points is in the temperature measurement, the deviations of the points from the fitted lines gives a measure of the precision of temperature measurement. The standard deviation for all of the determinations shown in Table I (36 temperature measurements) is equivalent to 12 x C. Prior to this application few attempts (4, 11) have been made to establish vapor-liquid equilibrium temperatures to this precision. Temperature Equivalent Determination. The temperature equivalent t o the smallest division of the bridge balancing decade has been determined by two independent methods: The change in boiling point of benzene with pressure is known to be 0.042’ C. per mm. The desired correlation can be made by measuring the resistance of one of the thermistors against a standard resistance with benzene boiling in the ebulliometer a t several different atmospheric pressures. The results (Table 11) indicate that the smallest scale division is equivalent to about 7 X loF5’C. Standard boiling point elevation tables for the benzene-biphenyl system (2) furnish a more accurate method of correlation between the temperature and the decade resistance. From these tables it was calculated that 0.1 mg. of biphenyl should give a boiling point C. in this elevation of 4.08 X ebulliometer. Calibrations of the ebulliometer with biphenyl in benzene showed that 0.1 mg. of biphenyl pro-
1520
Temperature Equivalent of Bridge Balancing Decade
ANALYTICAL CHEMISTRY
duced a change of 6.867 scale divisions on the bridge balancing decade. Therefore, one scale division (0.025 ohm) correC. The agreement sponds to 6 X between the two values is quite satisfactory. However, the figure is not used in the calculations and closer agreement is not needed. Effect of Design Improvements. The microebulliometer can be operated normally a t lower solute concentrations than conventional ebulliometers; therefore, samples which could not be measured previously because of insufficient solubility often offer no dficulty. Chrysene (Table I) is an example of such a compound. Its solubility in benzene is so slight that the molecular weight could not previously be measured in this solvent. However, in the microebulliometer six boiling point elevations could be measured. An additional advantage of operating in the low concentration range is the more nearly ideal behavior of the solutions, which simplifies the interpretation and computation of results (3). The increased sensitivity and stability of the temperature observation permits extension of the range of molecular weight measurements to a higher level. Only about 0.5 gram of sample having a molecular weight of 20,000 is required to obtain temperature differences of suitable magnitude. The applicability of the ebullioscopic method in this range is limited, however, by the stringent requirements for sample preparation and the difficulty of interpreting the data of the nonideal solutions obtained with higher molecular weight materials. Moreover, small amounts of low molecular weight materials seriously affect the number average molecular weight values. For instance, if only 0.1% toluene were inadvertently left in a polymer of molecular weight 20,000, the calculated average molecular weight would be only 16,440. The ebullioscopic molecular weight determined in benzene should approach this value closely. I n order to avoid this unfavorable averaging effect, the molecu-
AR -
C. per Scale Division X
547 656 660 587
7.7 6.4 6.4 7.2 Av. G.9
AP
lar weight is usually determined [in the last solvent used in the preparation of the polymer. The difficulty of interpreting ebullioscopic data from polymer samples limits the confidence which can be placed in the results. The behavior of polymer solutions is often far from ideal. Not only does the plot of boiling point elevation versus concentration have curvature, but the curve often fails to pass through the origin [zero point or constant error (S)]. Smith (la)attributes this zero error to the adsorption of the polymer on the surface of the bubbles in the boiling solutions. More work will be required before ebullioscopic data on polymeric materials can be interpreted with the confidence and accuracy now obtainable with lower molecular weight materials. The work is further complicated by the lack of samples of known molecular weight in the region of interest. LITERATURE CITED
(1) Barr, W. E., Anhorn, V. J., Znstrumenfs 20, 342 (1947). (21 Bonnar. R. U.. Shell Develoument CO., private communication. (3) Bonnar, R. U., Dimbat, hf., StrOEE, \
,
F. H., “Number Average Molecular meight,” in press, Interscience, Kew York. (4) Colson, A . F., Analyst 80,690 (1955). (5) Cottrell, F. G., J . Am. Chem. Soc.41,
721-9 11919). (6) Hill, F. g., Brown, A,, -4NAL. CHEM. 22, 562-4 (1950). (7) Ketchum, D., Zbid., 19, 504 (1947). (8) Kitson, R. E., Oemler, A. N., Mitchell. J.. Jr.. Zbid... 2 1 ,. 404-07 (1949).‘ ’
Menzies, A. W. C., Wright, S. L., .Tr.. J . Am. Chem. SOC.43. 2314
RECEIVEDfor review March 11, 1957. Bccepted May 20, 1957.
