Table 111.
Absorbance of Nitro Compounds in the Presence of Lithium Hydroxide (Read immediately at 300 m r ) Compound Concn., mM Absorbance E&. Nitromethanc 2. < 6 . 2 x 103 0.328 Nitroethane 177.0 0.161 0.91 1-Nitropropane 368.0 1.699 4.60 36.8 0.027 0.735 2-Nitropropane 188.0 0.032 0.17 Solutions prepared to indicated concentrations in 0.25M LiOH (pH 12.3).
+
Table IV.
EfFect of Dilution and Time of Reading for Nitroethane in Phosphate Buffers and Sodium Hydroxide Absorbance, 298-300 Mg Time of Dilution Time of Phosphate Buffers (1 : 10 H&) Reading NaOH PH 6 PH 7 PH 8 0 0 0.022 0.244 0.229 0.222 90 min. 90 min. 0.301 0.168 0.168 0.131 90 min. 20 hr. 0.409 0.102 0.097 0.086 20 hr. 20 hr. 2.000 0.137 0.181 0.097 Solutions prepared as 266 mM nitroethane in 0.15M phosphate buffers or in lnii NaOH and subsequently diluted 1: 10 with water a t times indicated.
the data the apparent extinction coefficients were calculated and are given for selected times. Since the apparent extinction coefficient expresses the relationship between absorbance and concentration, i t appears that at zero time proport~onate~y of the ab at the lowest sorbing material is present concentration.
Table V. Effect of Concentration and Time on Absorbance of Nitromethane in 1 .OM Sodium Hydroxide hpparent E&. X 103 4 Concn., 0 24 48 mM/L. time hr. hr. hr. 0.074 0 . 6 0 0 . 6 0 1.03 1.24 0.128 0 . 5 5 0 . 5 5 1.07 1.32 0.256 0 . 5 4 0 . 5 4 1.48 2.28
would not be fruitful as a method of analysis. LITERATURE CITED
T., Tapp, W. J.: Johnson, J. R., J . A m . Chem. SOC.67,
( 1 ) Blomquist, A.
1519 (1945). (2) Dalance, A., Healy, P. W., ANAL. CHEM.17,718-19 (1945). (3) Maron, S. H., LaMer, V. K., J . A m . Chem. SOC.60, 2588 (1938). (4) Schmidt, E., Rutz, G. G., Ber. 61, 2142 (1928).
JOSEPHH. GAST As i t has not been possible to find FRANCES L. ESTES Biochemistry Department conditions of complete or equilibrium Baylor University College of Medicine conversion, the absorbance does not Houston 25, Tex. follow B ~ law,~and ~the inJ ~ crease”of absorbance with time is not RECEIVEDfor review January 18, 1960. Accepted August 29, 1960. Work s u p a function of concentration, particularly ported by a research contract with the Air Pollution Medical Program, Public if dilution is required to permit a valid Health Service, U. S. Department of reading, it was concluded that this Health, Education and Welfare.
Spectrophotometric Determination of Sulfide End Groups and Number Average Molecular Weight in High Polymers SIP.: A number of methods have been devcloped for determining the number average molecular weight of polymers by end group analysis. A Ppectrophotometric method for the determination of number average molccular weight of polymers, with known sulfide end groups, is described. This procrdure can be applied directly for the qualitat,ive identification of the sulfide m d group in polymers if the number average molecular weight is known. The basis of this method is the complrs formrd between molecular iodine :tnd sulfidc sulfur. This complex absorbs intcnsc4y in the ultraviolet region a t 308 nip. In determining number average molwular wcight, the assumption is made that thrw is onc sulfide group per v h : i i r i : the fact that this method givcs ~ ~ c ~ ~ wiiii t e agreement with osmotic inol(~ i h r wcigl!ts is rvidcnce that the :isSuinption is v:ilid, ! (h]Jn!C’Ilt of this proccdure \vas I x i ~ t ~ 011 ~ 1 prc,vious iodine-sulfidc coni-
plex absorption studies concerned primarily with materials found in petroleum. The procedure as applied to polymers parallels that described by Hastings (3). EXPERIMENTAL
Identification of End Group. Before determining sulfide end groups, or number average molecular weights, i t is necessary t o “clean up” the polymer t o remove interfering contaminants. Various compounds, usually aromatic, introduced as additives in the preparation of polymeis, may also absorb strongly a t the same wave length as the complex. The rcmoval of such material is done preferably by column chromatography. The procedure consists of dissolving 5 t o 10 grams of polymer in mcthylene dic~liloritlrand passing the solution through a column of baiic duniina (Woelm). l‘lie column is i\arhcd two or thrt,cl tinics 111thmethyl(’ne divhloride, thcx rllripnt is c.ollcc*ted,
and the solvent completely evaporated, resulting in a clean sample of polymer. For our purposes, the interfering materials were sufficiently removed by column chromatography; however, this purification may be supplemented by countercurrent extraction using nitromethane-carbon disulfide as the solvent system.
A weighed portion of clean polymer is dissolved in 100 ml. of spectrograde methylene dichloride (approximately a 4% solution for molecular weights around l00,OOO). An aliquot of this solution is diluted with methylene dichloride, if necessary, to permit a reasonable absorbance reading of the iodine-sulfide complex. The iodine-sulfide complex is prepared by adding 5 ml. of the polymer-methylene dichloride solution to 5 ml. of the 0.374 iodine reagent (Irepared by dissolving 3 grams of iodine in methylene dichloride and diluting to 1 liter). The solution is agitatcd to obtain a uniform mixture. VOL. 32,
NO. 12, NOVEMBER 1960
1713
The product of a and the known molecular weight of the polymer results in the molar absorptivity ( E ) . The molar absorptivities of the reference compounds (model mercaptan dimers or model polymers) are found as described above. The molar absorptivities of the polymers are now compared directly to those of the model mercaptans and a qualitative identification of the sulfide end group determined. Molar absorptivity values and molecular weights of the model mercaptans (model polymers could also be used) used in this procedure are listed below:
w
z m
B
a 4
WAVE
LENGTH IN MILLIMICRONS
Molar Model Molecular AbsorptiMercaptans Weight vity X 10' Mercaptoethanol 278 2.26 Benzylmercaptan 310 3.40 Dodecylmercaptan 403 7.52
Figure 1. Ultraviolet spectra of typical polymer-iodine complex Pdymer, mercaptoethanol polyhethyl melhacMate) Temperature, 25' C.
The iodine complex solution is now placed in a 1-cm. silica absorption cell and the absorbance read a t 308 mp against a blank reference. The blank reference is prepared by combining 5 ml. of the iodine reagent with 5 ml. of methylene dichloride. By reading the blank reference against itself, a cell-difference blank absorbance is obtained. The iodine reagent absorbs a t 308 mp also. If the cells have different path lengths, there will be a resultant reagent contribution (either positive or negative). This can be taken care of by either determining a cell correction factor or by measuring the resultant absorbance difference and making a direct correction. Polymer absorbances are then read directly from the ultraviolet spectra, correcting for reagent blank readings. The absorptivities (a) are then calculated by Equation l : a = A/bc
(1)
where a = absorptivity A = absorbance (corrected) (polymer-iodine complex readingreagent blank reading) b = cell path length (1cm.) c = concentration in grams per liter The absorptivity of the iodine-sulfide complex is based on total sulfide content (gams/liter) rather than true concentration of the complex.
Number Average Molecular Weight Determination. Absorbances of polymers with known sulfide end groups are found using the procedure previously outlined. The number average molecular weight of the polymers is then calculated by Equation 2 :
n" = where
€C
x*= number average weight e =
c =
A
=
molecular
molar absorDtivitv of model mercaptan dimer having the same sulfide end group concentration of the polymer in grambiter absorbance of polymer (corrected) DISCUSSION
Absorbance readings throughout this procedure have been read a t a wave length of 308 mp. However, the maximum absorbance peaks of some polymer solutions do shift wave length z t 3 mp. The wave length of maximum absorption shifts slightly from 308 mp toward the lower wave-lepgth region with increasing concentrations; however, a t a regulated temperature of 25OC. this shift is not too significant and a maximum peak a t approximately 308 mp is maintained. An ultraviolet absorption spectrum of
- 0 . 0 280
WAVE
290
3W
320
308310
330
LENGTH IN MILLIMICRONS
Figure 2. Absorption spectra of polymer-iodine complex of poly(methy1 methacrylate) incorporating different sulfide end groups 1. Benzylmercaptan end group, 2% II. Mercaptoelhanol end group, 1.6% 111. Dodecylmercaptan end group, 0.2%
a typical polymer-iodine complex is illustrated in Figure 1. The methyl methacrylate polymer shown in Figure 1 has a mercaptoethanol end group. The spectra are taken a t a temperature of 25' C., a t varying concentrations. The absorbance of the sulfide-iodine complex follows Beer's law. Figure 2 illustrates the slight difference in maximum wave-length absorption for three different polymers. Absorptivity of the Polymer-Iodine Complex. The sulfide end groups of the three model sulfide dimers, a, b, and c, and the corresponding methyl methacrylate polymer, d, are different in structure. Mercaptoethanol dimer: HO-C HZ-CHz--S--CH2-C
H-C HzLOOCHs
Benzylmercaptan dimer : Cab--CH2-S-CHz-CH-CHzC~OOCHJ CH~--CH--CHJ &OOCH, Dodecylmercaptan dimer :
Table 1.
Comparison of Number Average Molecular Weights Obtained Spectrophotometrically and Osmotically
ic?,
a;
Methyl Methacrylate Model Polymers Mercaptoethanol
Osmotic
spectrophotometrica
66,200
70,300
Dodecylmercaptan
71,900
73,200
Benzylmercaptan
65,400
63,900
* Em value is an average of several determinations.
1714
e
ANALYTICAL CHEMISTRY
lo3 2.28 2.27 7.28 7.50 3.13 3.36
e X
C~~H~~MHZ--CH--CHZ-CH~COOCHI I
End Group Identification Mercaptoethanol Dodecylmercaptan Benzylmercaptan
Methyl methacrylate polymer:
Table 11.
Absorbance Readings of Mercaptoethanol Poly(rnethy1 Methacrylate)Iodine Complex Solutions
Sample
(% Concen- Temperature, tration)
ABSORBANCE
Figure 3. Effect of temperature on absorbance readings Mercaptoethanol dimer, 0.00% w./v.
The methyl methacrylate dimers do not have a linkage between monomer units that is the same as that for poly(methyl methacrylate) free radically polymerized. However, i t would not be expected that this type of structural difference would affect the absorptivity of the iodine-sulfide complex to any appreciable extent (1, 3). Assuming the molar absorptivity found for the sulfide group in the dimer is the same as that in the corresponding polymer, the number average molecular weight for the remaiping polymer chain may be calculated. The fact that the number average molecular weights obtained osmotically agree so well (Table I) indicates that the two assumptions made are true-namely, there is one sulfide group per chain and the molar absorptivity of the sulfide groups in the model compound is the same as that in the polymer. Osmotic Measurement of Number Average Molecular Weight. Osmotic
pressure measurements were made at 30' C. in a modified Schulz-Wagner osmometer (6). The membranes used were No. 300 grade wet generated cellulose which had been treated previously as described by Flory ( 2 ) . A minimum of three concentrations was used for each polymer and the data were extrapolated to infinite dilution using the square root method. Acetone was the solvcnt used. Effect of Temperature. During the experimental work, it was observed t h a t the absorbance readings of the polymer-iodine complexes changed whenexamined overa range of constant temperatures. This was not noticed, however, at a regulated temperature of 2 5 O c. These changing absorbances are not attributed to the effect of dark time, but rather to the time needed for the estaD!ishment of the sulfide-iodine complex equi!ibrium. There was no change in the absorbance of the reagent blank. Sbsorbance readings of various conxntrations of polymer-iodine complexes were then made a t 5-minu:e intervals
O
c.
5
Time in Minutes 10 15
20
0.8 1.6 3.2
0.830
0.378 1.170
...
... ,.. ...
...
15
0.8 1.6 3.2
20
0.259 0.556 1.145
0.270 0.575 1.165
0:590 1.185
0 590
0.8 1.6 3.2
25
0.230 0.490 0.945
0:500 0.936
0.965
0.963
0.8 1.6 3.2
30
0.251 0.475 0.833
0.251 0.460 0.805
: 0.815
...
0.338
...
at four regulated temperatures (Table 11). Table I1 illustrates that a t a temperature of 25" C., there is little fluctuation of absorbance readings with standing time; it is, therefore, unnecessary to establish a time limit in regard to such readings if the temperature is regulated (1, 4). The absorbance does show a large inverse temperature dependence and as noted by Drushell and Miller and Hastings and Johnson is attributed to the temperature dependence of the equilibrium constant (Figure 3).
REPRODUCIBILITY
OF
, . .
...
0 475
...
...
:
1.190
...
...
0 : 825
Table Ill. Accuracy as Function of Polymer Concentration
Polymer, mercaptoethanol poly( methyl methacrylate); temperature, 25" C. yo ConcenCalculated tration Number Polymer, Average Grams/ Absorbance Molecular 100 M1. of Solutions Weight 0.2 0.085 50,800 0.4 0.150 56,600 0.8 0.230 71,700 1.6 0.500 69,250 3.2 0.970 72,300
RESULTS
.4ccuracy as a function of polymer concentration is illustrated in Table 111; both calculated number average molecular weights and absorbance readings of the polymer are listed. The precision of the procedure as a whole is shown by the results given in the following table:
N o special precautions or screening of the results was made and the determinations were performed on different days. The standard deviation obtained was slightly less that loyo. The authors have data that indicate that replicate determiiintions made on a single day with some extra attention give a standard deviation of about 5%.
RESULTS
Polymer, mercaptoethanol polg(methy1 methacrylate) Solution concentration, 1.6% (w./v.) Temperature, 25" C. Absorbance Readings 0.550 0.580 0.581 0.543 0.500
0.536 0.480 0.534 0.457 Av. absorbance reading = 0.529 Std. dev.
=
0.05
Poly(methy1 methacrylate) samples were prepared by free radical polymerization using each of three mercaptans as chain transfer agents. The number average molecular weight of the polymers was determined osmotically and spectrophotometrically by the procedure given above. Table I summarizes the data which show an excellent agreement between the two methods. The absorbance data have been used to calculate molecular weights using the absorptivities of the model compounds and to calculate absorptivities using the osmotic molecular weights. A polymer containing various possible additives VOL 32, NO. 12, NOVEMBER 1960
1715
was analyzed by the above procedure as a test of the clean-up steps. The number average molecular weight obtained spectrophotometrically was 59,100, osmotically 57,000.
many ultraviolet spectra needed in developing this procedure.
try,” Pt. 42nd ed., P. 528, Interscience, New York-London, 1949. ISADORE ROSENTHAL GINOJ. FRISONE JEAN K. COBERQ
LITERATURE CITED
( 1 ) Drushel, H. V., Miller, J. F., ANAL. CHEW27,495 (1955). (2) Flory, P. J., J. Am. Chem. Soc. 6 5 , 372 (1943). The authors thank Marian Fegley and (3) Hastings, S. H., ANAL.CHEM.25, William Rakita for preparing the model 420 (1953). dimers and polymers, ~ l i ~ cohn ~ b ~ (4) ~ hHastings, s. H., Johnson, B. H., Zbid., 27,564 (1955). for obtaining the osmotic measurement% (5) Wagner, R. H., in A. Weissberger, ed.. and Carl Schmittinger for running the ‘‘Physical Methods of Organic Chemis-
Research Laboratories
ACKNOWLEDGMENT
~ $ ~ , & ~ ~ p ~ o ‘
RECEIVED for review June 2, 1959. Resubmitted July 11, 1960. Accepted -4ugust 11, 1960. Division of Analytical Chemistry, 136th Meeting, ACS, Atlantic City, N. J., September 1959.
Microdetermination of Active Hydrogen with Tritiated Ethyl Alcohol SIR: Active hydrogen in organic compounds is routinely determined by gasometric procedures using Grignard reagents or lithium aluminum hydride (7). As the molecular size or complexity of the sample increases, the accuracy of the gasometric proeedures is diminished because of incomplete reaction or the occurrence of side reactions. Thus the utility of these classical methods is often limited, and a more accurate method is required. Earlier papers (3, 6 ) have described good results based on hydrogen isotope exchange with deuterium oxide. Recently, Eastham and Raaen ( 2 ) reported a method involving the exchange of active hydrogen for tritium in excess tritiated isopropyl alcohol, and Chleck et al. ( 1 ) have demonstrated the use of lithium aluminum tritride in determining active hydrogen. The present method utilizing tritium exchange with excess tritiated ethyl alcohol requires only 0.05 to 0.10 meq. weight or 5 to 30 mg. of the compound. Tritium equilibration between the sample and ethyl alcohol a t room temperature is essentially instantaneous (4, 5 ) , so that a sample ran be run in about 30 minutes exclusive of cnlinting time. it was convenient I n this lab~rnt~ory, t o count the samples overnight using an automatic sample changer. METHOD
Reagents and Apparatus. Hydroxyltritiated Ethyl hlcohol. Dilute tritiated water (Sew England Nuclear Corp., 1.8 curies per mole) with sufficient absolute ethyl alcohol t o obtain an activity of about 0.6 gc. per mmole of alcohol. Standardize the tritiated 1716
ANALYTICAL CHEMISTRY
ethyl alcohol by adding 1.00 mi. to a mixture of 4.00 ml. of methanol and 5.00 ml. of ethyl alcohol, and diluting to 50.0 ml. with the toluene scintillation reagent. Count four 10-ml. aliquots from each of three such dilutions and calculate the activity of the tritiated ethyl alcohol. Alcoholic Scintillation Reagent. Dissolve 3.0 grams of 2,5diphenyloxazole (PPO) and 0.1 gram of 1,4bis[2-(5phenyloxazolyl) ]benzene (POPOP) in 1 liter of methanol-ethyl alcohol-toluene (10:15:75). Toluene Scintillation Reagent. Dissolve 3.0 grams of PPO and 0.1 gram of POPOP in 1 liter of toluene. Liquid Scintillation Spectrometer. Packard Tri-Carb, Model 314X. Counting Vials. Wheaton Glass Co., 20-ml., screw-cap vials made of low potassium glass. Distillation Column. Use a 15 X 1 cm., vacuum-jacketed, straight-through column with no dead spaces and a total take-off head. The column must facilitate rapid distillation and minimize mechanical transfer or fractionation of the hydrogen isotopes. Procedure. Accurately weigh 0.1 meq. of the sample into a 25-ml., 19/38 5 , round-bottomed flask and dissolve in 2.00 ml. of standard tritiated ethyl alcohol. Add 10 ml. of dry toluene. Connect the vacuumjacketed, microdistillation column and immerse the flask in a wax bath preheated to 170’ C. Collect 4 t o 5 ml. of distillate in 5 minutes, then remove the distillation column, allowing 15 to 30 seconds to vaporize any residual tritiated ethyl alcohol out of the 5 joint. Quickly cool the distillation residue and dilute to approximately 10 ml. with toluene, transfer this solution to a 50-ml. volumetric flask, and dilute to volume with the alcoholic scintillation reagent. Determine the activity of
the sample by counting four 10-ml. aliquots of the dilution with the liquid scintillation spectrometer. If the sample quenches the count, the degree of quench is determined by adding a known amount of tritiated ethyl alcohol and recounting the sample. CALCULATIONS
Because of the use of an overwhelming excess of tritiated ethyl alcohol, and because isotope effects in the exchange equilibria are small, the sample may be assumed to have approximately the same tritium activity, per equivalent, as the original ethyl alcohol. Thus: Eq. wt. =
(mg. sample) (sp. act. tritiated ethyl alcohol j (sp. act. sample)
1 008 eq. wt. mol. wt. NO. Act. H eq. wt.
70 Act. H
= -X 100
~
DISCUSSION
The determination of active hydrogen by tritium exchange can be applied to a wide variety of compounds, as shown in Table I. The method described here is applicable primarily to nonvolatile compounds since significant quantities of volatile materials would be lost during the distillation. Some difficulty is encountered in analyzing compounds, such as nicotinamide and indoleacetic acid, which are only slightly soluble in the alcoholic toluene scintillator solution. This has been overcome by allowing the sample to stand for 1 to 3 hours in contact with the scintillation reagent before making
