unstablc hvdroperoxide (1) which immediately undergoes rearrangement and degradation. The endothermal bands :it approximately 350' C. undoubtedly r c w i t from decomposition of the polymers, as the evolution of dense vapors, vigorous boiling, and, in many cases, ignition of volatile products were observed. The marked dissimilarity in the thermal behavior of Hetron 92, curve 33, from the other polymers is indicative 2f a difference in structure, constituents, and/or chemical bonding. I n this case, it i s known that the polymer, containing
,wtls,n-chlorinc bonitc,, iZt?., rs iron1 the ottif'r polyesters. F J x I T2595, , an epoxy resiii. is the or 13 poi) T which undergoes exotherrial decomuosition. Degradation of ai. oi the polymers is comFlete a t 450' C.,leavine; a carbonaceous residue. On the basis of Drevious work ( I ) , it is assumed that part of the residue reacts with oxygen, influencing the shape and slope of the ascending portion ofthe endotherm.
is) Freeman, E. S., CarroL
~ 3 . J,
'k,r
'.bm62,394 (1958). . A I ' ;ordon, S., Campbell, C., A N A L . 'iiEM. 29, 1102 (1955). (4)Hogan, V.,Gordon, S., Campbell, C., ibid., 29,306 (1957). (5) Morita, H., Ibid., 28,H (1956). (6)Murphy, C. B., Palm, J. A., Doyle, 9. C., Curtiss, E. M., J. Polymer Sa. 28,447 (1958). (7)Ibid., 28,453(1958). (8)Smothers, W.J., Chiang, Y., Wilson, A., Unw. rlrknnsas Inst. Sci. and Techml., Research Ser. No. 21 (1951).
LITERATURE CITED
(1)Anderson, D. A., Freeman, E. S., J. A p p l . Polymer Sei., in press.
RECEIVED for review November 28, 1958. Accept.edJune 12,1959.
Influence of Amino Acids upon the Anthrone Reaction of Uronic Acids j.
R. HELBERT
and K. D. SROWN
Veterans Administration Hospital, Downey, I//., and Marquette University School of Medicine, Milwaukee, Wis.
b The influence of 12 amino acids upon the anthrone color of the uronic acids has been investigated under various experimental conditions. Tryptophan alone produces a anthrone color and this color is additive with the glucuronic acid color and the mannurone color at 550 mp, but nonadditive with the idurone color at the same wave length. Methionine alone produces no anthrone color, but causes color enhancement with all uronic xids; cysteine alone likewise produces no anthrone color, but, depending upon experimental conditions, may cause either depression or enhancem e n t of uronic acid color. The remaining amino acids are without etfect. The magnitude of the observed effects varies with such exDerimental factors as tewperature, heating time, and concentration. ~~~REVIOU n-ork S
with a uronic acid polymer (3) indicated an anomaIUS coior reaction with anthrone. Chro- i ? ~ atuqraphic ana1ysc.s indicatcd the w-csmc" of small amounts of amino tc*itfs. In view of the reported behavior -G) of tryptophan with anthrone, ~ a r ~ i c i h rin~ vthe presence of carboI: drrttcs, tiir subject of the present incl>tig.:tticn scemcd pertinent. Morew c r , At is often desirable to determine (ironic :;rids or their polymers in the presence of amino acids or proteins. a
1700
ANALYTICAL CHEMISTRY
MATERIALS AND PROCEDURE
Most of the uronic acids used were obtained from noncommercial sources. The glucuronic, galacturonic, and amino acids were of best quality commercially available, and were used as received. Anthrone solutions were prepared by dissolving 0.160 gram in 100 ml. of 27.5 f 0.1N sulfuric acid, allowing about 60 minutes for complete solution. Uronic and amino acid solutions were prepared by dissolving solid sample in 27.5N sulfuric acid. About 30 minutes was allowed to effect complete solution of the uronic acids. All of the amino acids dissolved rapidly and were used within 30 minutes after solution. Basic Procedure. Four milliliters of anthrone solution were pipetted into uniform borosilicate glass test tubes, followed by uronic acid and/or amino arid solutions to make a final volume of 6 mi. For convenience, concentration has been expressed throughout as micrograms per 6 ml. As all reactants \$eredissolved in 27.5N sulfuric acid, no heat of mixing was evolwd. Test samples were then heated in a boiling-water bath or, if l o w r tenipcratures were desired, in a thermostatically controlled water bath. Aftm heating, samples were quickly transferred to a cold-water bath (4" f 1" C.)for 3 minutes. Heating and cooling time was measured to *2 seconds. The test tubes containing the reacting solutions were spaced in wire racks in the heating and cooling baths to facilitate uniform heat transfer. After removal from the coldwater bath, test wmples were held at
room temperature (23" f 2" C.) i i i a light-proof cabinet for 20 f 2 hours. Photometric readings were made with a Beckman DU spectrophotometer. EXPERIMENTAL RESULTS AND DISCUSSION
Uronic Acids. The spectra of cidurone, Lgulurone, and D-mannurone after reaction with anthrone are deliieated ;TI Figure 1 . Analogous spectra and other information about D-glucuronic and D-galacturonic acids have been published (2). Idurone was obtained and used as the 1,Z-O-isopropylidene derivative. The available quantity of gulurone permitted examination a t only one temperature. The color response of the idurone- or niannurone-anthrone complex to changes in heating temperature (Figure 1) is similar t o that previously observed (2) with galacturonic acid. -Vannurone is unique among the uronic acids in that its spectrum a t 100" has no maximum ?letween 500 and 600 mu. Solutions nith this kind of spectrum display no red coloration which can he readily drtected visually. On the other hand, the spectrum of mannurone reacted with snthrone a t 70" has a well-defined maximum a t 550 mp, and such solutions have a distinctly obsrrvablr red coior. The absorbances a t 550 mp for equal quantities of the various uronic acids relative to glucuronic acid are: Dglucuronic, 100; D-mannurone, 55; Lgulurone, 125; bidurone, 130; D-
0.2OOC
D-MANNURONE
L -1OURONE
L -CULURONE
0.0501
0.025
i
0 519
590
55C
510
550
590
.
. 510
.
.
.
550
. 590
Wave Length, mu
Figure 1.
Spectra of uronic acids
"C.
Indicated temperatures in
0.400
0.320
Concentration in ali cases, 100 7 / 6 mi.
l 3 Ai
B 3.200
-
3.160
$
" ,0.240-
L120
m
u " c
f? n Gl
0.160
-
0.080
I
1
o.o*,I-
0
3.040
1.
I
510
,
,
550
.
.
590
510
550
590
Wave Length, mu
Figure 2. A.
6.
Spectra of uronic and amino acids
Glucuronic acid and tryptophan Temperature, 100' C. Heating time, 10 minutes 1. Tryptophan alone, 200 7/6 ml. 2. Glucuronic acid alone. 200 y/6 ml. 3. Mixture of tryptophan and glucuronic acid, each a t 200 7/6 ml. Glucuronic acid and methionine Temperature, 70' C. Heating time, 20 minutes 1. Methionine alone, 160 7/6 ml. 2. Glucuronic acid alone, 100 7/6 ml. 3. Mixture of methionine (1 60 y / 6 ml.) and glucuronic acid ( I 00 y/6 ml.)
galacturonic, 150. Coior was developed by heating the reaction mixtures for 10 minutes a t 100' It is evident from Figure 1 that changes in experimental conditions may substantialiy alter the relative absorbances just noted. All the uronic acids examined followed Beer's law, a t least up to 200 y per 6 ml. Amino Acids with Uronic Acids. Tryptophan, cysteine, methionine, phenylalanine, tyrosine, proline, hydroxyproline, histidine, glutamic acid, lysine, valine, and arginine were each examined alone, in the presence of glu-
curonic acid, in the presence of glucosamine, and in the presence of glucuronic acid and glucosamine. Factorial designs (1) were employed for this purpose and for much of the work reported below. Glucosamine was included because of interest in heparin. Glucosamine was without effect either alone or in any of the various combinations, whether the color was developed a t 100" or a t 70". Among the 12 amino acids listed above, only tryptophan produces a color of its own with anthrone. This finding
ccnfirms the observstions of Sciftcr (4). The spectrum of the tryptophananthrone color at 100" differs somewhat from that of glucuronic acid (Figure 2,A) and this difference is visually dctectable. Likewise, the spcctrum of a mixture of tryptophan and glucuronic acid with anthrone differs from that derived by summing the componcnt spectra, except a t wave lengths greater than 550 mp where the two become congruent. Accordingly, one would expect the tnptophan and glucuronic acid coiors to be additive in mixtures when spectral readings are taken a t 550 to 560 mp- Dekrminations of color developed a t both 70" and 100" confirm this expectation. When spectral readings arc taken a t 530 mp (maximum for the mixture). the absorbanccs of tryptophan and glucuronic acid, respectively, are nonadditive. However, the magnitude of this effect is considerably iess than that observcd by Tuller and Keiding (6) for tryptophan with galactose and mannose, and markediy iess than that observed in this laboratory for tbptophan with glucose (unpublished data). Tryptophan has also been examined a t TO" C. with mannuronc and idurone, respectively. As was the case with glucuronic acid, thc mannurone and tryptophan absorbanccs a t 550 mp wcrc additive in mixtures. The idurone and tryptophan absorbances, however, were not; the absorbances of mixtures mere measurably less than the absorhances of the components. The influence of temperature on the development of the tryptophananthrone color is noteworthy. The allsorbance of tryptophan a t concentrations between 100 and 200 7 per 6 ml. is 5 to 6 times greater at 100' than a t 70". This behavior has analytical utility, in that tryptophan interference with uronic acid determinations may usually be effectively eliminated by simply reducing the temperature a t which color development is effected. Methionine and cysteine, although they themselves produce no color with anthrone, have a marked influence on the anthrone color of the uronic acids. As indicated by the spectra in Figure 2,B, methionine may enhance the color produced by glucuronic acid a t TO" by 30%,. Methionine exhibits this synergism with all of the uronic acids examined, though the magnitude of this effect for given experimental conditions varies with thc uronic acid. Also, the magnitude of this effect for a given uronic acid is in general a nonlinear function of the concentrations of methionine and the uronic: acid. For example, the behavior of methionine and glucuronic acid is given in Figure 3 , where the data from a 3' factorial design (1) are depicted graphically. In view of the findings described here, it seems reasonable to suppose that methionine was the unknown factor enVOL. 3!, NO. 10, OCTOBER 1 9 5 9
* 1701
countcred by Tuller and Xeiding (6) in their work with carbohydrates in the presence of proteins. Cysteine rcduces the intensity of the uronic acid color d t ~ e l o p e dby heating for 20 minutes at i o o . In the case of glucuronic, acid, this reduction amounted to 10% when the concentration of both reactants was between 100 and 200 y per 0 ml. Two experiments conducted with ryst.eine and glucuronic acid at 100" indicated that at this temperature cyst,eine caused a measurable amount of color cnharmment rather than color !oss . As a spot check for significant higher order interactions when more than one :imino acid is present, tryptophan, methionine, and cysteine in the presence of glucuronic acid, mannurone, and idiirone, respectively, have been ex:imined at 70" employing 2' factorial tlcsigns (1). Analyses of variance of t h r data indicate that none of the inter:ictions above first order is signifitaantl\. large in ternis of the residual or clsptxriiiwiital crror. :\'one of the amino acids stutlicd other than tryptophan, mc.thionint1, :ind cystc>inecithrr protluws an niithi.onc cdor of its own or has a nieasurablt. influencat. on the anthrone color of the uronic acids. The present findings indicate that the vstiniation of uronic acids with anthrone i n the presence of amino acids or proteins 1 1 1 : ~be ~ tlifficult,. part,icularly for sniall :imounts of :t uronic acid in the pres( w e of larg(. :tniounts of amino acids.
.
with tlcw-casing concentram Consequentlv, by using a htiatiug temperature near 70" and by -uitat)iy reducing the concentration of the sample, aniino acid interference in some cases can be effectivc.l\- eliminated, ACKNOWLEDGMENT
0.080
0
80 Conc. Methionine,
160 J
'6ml.
Tlic authors are indebted to 11. L. IYolfroni for specimens of D-niannuronr and 12-0-isopropylidrne-L-idrii ono-ylactolie, to Philip Hoffman for a specimen of ~)-niannurone,and to F. G. Fischcr for a specimeli of L-guluronrL. The!. gratcfully acknowledge the assistr a w e of I,. C. Massopust in preparing the dranings and photographs and of Waldcnier Rosenthal in rarrying out part of thc esperimriital work.
Figure 3. Absorbance of various concentrations of methionine and glucuronic acid G A 200 = glucuronic acid, 200 7/6 ml.
However, when the aniount.e of a uronic acid and amino acids are more nearly alike, the follov ing observations may be utilized to reduce t.he interference of the latter. First. the rfftvts of t'ryptophan. methionine, :tiid (.ystcinr ( i i i terms of absorbance) d o c w : i s c L siil)rtantially as the heat'ing tcmpcraturt, is rtduccd from 100" to i o o ; under the siimc conditions the absorbances of all the uronic acids increase. Second, the nonadrMive effects of the methionine and (,!.stvine diminish
Ana IyticaI Study of Aminoethyl Vinyl Ethe r System Based on Counte rcurre nt Extracti o n lSADORE ROSENTHAL, FRANCIS JACKSON, and WARREN WATANABE Rohm & Haas Co., Philadelphia, Pa.
b Conventional methods of analysis applied to aminomethyl vinyl ether {vinyl and base) give a misleading impression as to the purity of the material. It is possible for this compound to disproportionate to form impurities that have the same ratio of vinyl ether to base as the starting material. The system was investigated by countercurrent extraction on a Craig machine and the impurities were isolated. Schiff bases and ethanolamine were identified. A short analytical method based on extraction was developed. The results obtained with a combination of iodine-hydroxylamine-base analysis are discussed. 1702
ANALYTICAL CHEMISTRY
I
s >EPARATIO~-S ~ O R K on nniinoethyl vinyl ether it was found that aminoethyl vinyl ether, H?C=CH-O-CH:CH2-SH2 (AEI'E) (I1 or Rand 11) prepared by certain nwthods contains two principal inipuritirs: cthanolamine, HO-CH2--CHz-SH2 (E.4) (I or Band I), and the Schiff base of AEVE, HzC=CH-O-CH?-CHz-S=CHCH, (I11or Band 111). Chemical analysis of samples by vinyl analysis Imodified Siggia hydroxylamine method (2))jand/or titration of amine groups is of no value in this case, because the impurities are often produced in such proportions that anaiytical values on mixtures are always close to the theoretical values for pure AEVE. For
(~siinipl(~, typical anaiyses on three s ~ n i -
I) did not agree with the \xlu(~sohtaincd by countercurrent cstrwtion (Figurc 1). 011tlw basis of amine and vinyl dott~niiii:ttion done, the samples appear to 1 i a . i thc ~ correct anal~.sis. Moreover, the wtio of amine to Tiny1 is closely 1 to 1, as expected. Tet examination of the results obtained by countercurrent cstractions shows a wide variarinn in the actual coniposition of the samples, Countercurrent estractions of the order of 30 to 40 transfers are tedious, and a simpler analytical method is desirable. The method described here consists of extracting the ethanolamine ~ ) i t h s (Table
