maximum intensity even after heating for 100 minutes, and developed less color than the other uronic acids. D-galacturonic acid showed more intense color a t 55" than at 100' C. When the absorbance at one designated time of heating (30 minutes) was plotted against temperature, a linear relation was exhibited by D-glucuronic and L-iduronic acids between 65" and 85" C. The results obtained when D-glucuronic and L-iduronic acids were heated for 30 minutes a t 65", 'ioo, 75", 80", and 85" C. are shown in Figure 2. In addition, mixtures of various proportions of these tivo uronic acids are illustrated with theoretical results. The close agreement of expected and actual observations indicates that the carbazole reaction following the digestion with given concentration of sulfuric acid a t various temperatures offers a method of assessing the relative proportions of two uronic acids in a mixture. Employing the method described above, additional evidence Jvas presented for the presence of L-iduronic acid among the products of hydrolysis of heparin by formic acid (7). The carbazole reaction of LIPS containing D-glucuronic and L-iduronic acids was studied over the same format of experimental conditions. The polysaccharides resulted in somewhat less color than the corresponding constituent uronic acids, possibly because of impurities or state of hydration. The color responses exhibited by chondroitin sulfate3 X and B, hyaluronic acid, heparitin sulfate. and heparin when
heated a t 65", 70", 75", 80", and 85" C. for 30 minutes are shown in Figure 3. It can be noted from the slopes of the curve that chondroitin sulfate A, hyaluronic acid, and heparitin sulfate, all known to contain D-glucuronic acid, behaved similarly, and chondroitin sulfate B, known to contain L-iduronic acid, demonstrated less change over the range of temperatures. Since variations in heating techniques with the carbazole assay may be of value in determining certain mixtures of hIPS, various proportions of chondroitin sulfate A and B were studied. The results are illustrated in Figure 4. Although not shown, the experimental and theoretical curves Rere observed in close agreement. These results indicate that the carbazole reaction nhen employed as described above offers a method to estimate relative amounts of n-glucuronic and L-iduronic acids as part of polymers in mixtures of MPS. Vsing this method, several mixtures of I P S isolated from normal and pathological urines lvere studied in this laboratory for the relative proportion. of chondroitin sulfate B and the D-glucuronic acid containing polysaccharides. The results were verified by hydrolyzing the NPS v i t h formic acid and comparing the intensities of the spots of D-glucuronic and L-iduronic acids of the hydrolyzate on paper chromatograms. I n other studies of MPS fractions from aortas, reasonable agreement was observed by estimating mixtures of hyaluronic acid and chondroitin sulfate B by a method employing testicular hyaluronidase.
At all of the temperatures studied, heparin w3.q observed to give a relatively more intense color than the other hIPS (Figure 3), yet the slope of the curve was slightly less than those of the polysaccharides containing D-glucuronic acid. Although the total greater intensity of heparin is unexplained, this behavior of heparin suggebts that it contains. in addition to o-glucuronic acid, another uronic acid. L-Iduronic acid ha. recently been found in heparin ( I ) , and the observation confirmed in the laboratory (7'). Estimates from the slope (if based on those obtained with chondroitin sulfate. ;1 and B as shown in Figure 4) suggest that the uronic acid.; of this preparation of heparin contained allproainiately 307, of L-iduronic acid; howeyer, the validity of this estimate n-ill require further observation. on other heparin preparations. LITERATURE CITED
(1) Cifonelli, J. .4,> Dorfman, .4., Bioche7n. B i o p h y s . Res. Cornmiins. 7, 41 (1962). ( 2 ) Dische, Z., J . Bioi. Chem. 167, 189 (1947). ( 3 ) Dische, Z., Ibid., 183, 489 (1950). (4) Gregory, J. D., Arch. Biochein. Bioph?ls. 89, 157 11960).
(5) Helbert, J. R . , Brown, K. D., A s A I . . CHEM.33, 1610 (1961). (6) Linker, A , , Hoffman, P., Meyer, I17 2 64
6 79 6 76 6.77 6.76 6.77 6.77 6.80 6 83 6 80
boil for 7 minutes. For oils, use 20 ml. of 0.05.V nitric acid :ind boil for 2 minutes. Cool, transfer to a 250-ml. beaker, and dilute to 100 ml. Because there is a small blank, a sample cup must be burned and thr: absorber must be carried through the entire procedure. Metals Determinatia'n. Seutralize the solution of metal t:ifluoroacetates or nitrates t o pH 4 t o 5 with sodium or potassium hydroxide. *Idd 20 i d . of pH 10 buffer (8.25 grams of ammonium chloride and :.13 nil. of concentrated animoniuni hydroside per liter). For ext,racts from acidic decomposition, use standard 0.0LU magnesium and 0.05Jf DTPA solutions. For the alxorber solutions from Schoniger oxidation, use 0.01.11 solutioiis for additives and 0.0025f solutions for oils. The titration sequenct, is given for the acid extracts; the absorher solutions are titrated similarly escepl; for the use of less of the masking agents and only 25 nil. of ethyl alcohol. To determine total metals, add 2 ml. of standard niagnesiuni solution, Eriochrome Black T indicator (XaC1 currier), and add about 2 nil. excess of DI'P.4. Stir for 1 minute, add excess niagnesiuni solution to a bright pink, and finally titrate with DTPA to the pure blue end point. For zinc, add 2 granis of potassium cj-anide after the deterniination of total metals and stir for 30 seconds. Add escess magnesium solution to a bright link. stir for 30 seconds. and titrate ;\.it h' D T PA\. For barium. add 2 m a n s of ootassium sulfate arid 50 ml. f t ethyl alcohol to the solution after the determination of zinc. ildd excess magnesium solution t o a bright pink, stir for 30 seconds, and titrate n ith DTPA. Calcium is usually determined by tlillference. Ilon-ever, f the calcium content is very low compared n i t h the other metals, it may he necessary to determine it on a separate sample after precipitating barium sulfate and complexing the zinc. RESULTS
Precision and accuracy ivere determined by replicate analyies on barium phosphonate, calcium sulfonate, and
Table II.
hlixture No.
1 2
3 4 5
6
7 8
9 10 11
12 13 14 15 16 17
Accuracy for Mixtures of Additives
Ba, % Calcd. Found 7.22 7.18 6.49 6 42 3.68 3.64 2 87 2.88 2.24 2.26 1.23 1.24 8.68 8.7.5 5.32 5.37 3.72 3 75 6.55 6.61 4.66 4.66
5.42
7.09 8.05
ACIDICDECOMPOSITION Ca, 70 Calcd. Found 0.183 0.180 0.526 0.522 0.88 0.88 1.19 1.20 1.18 1.20 0.52 0.51 0.528 0 534 1.34 1.33 1.73 1.70
Zn, 75 Calcd. Found 1.81 1.83 1.41 1.40 2.22 2.25 1.44 1.43 2.22 2.28 4.66 4.64
1.40 1.29 SCHONIGER OXIDATIOS 5.46 0.81 0.78 7.10 0.92 0.93 8.02 1.11 1.08
Table 111.
1.36 1.28
0.56 0.123 0.43 0.065 0.92 0,113 0.090
0.156 0.T2
0.024 0.111
2.59 3.95 3.27 3.47
1.32
1.38
1.77 3.91
1.75 3.84
Analyses of Commercial Oils
Metal, 70 Acidic decomposition Ba Zn Ba Zn Ba Zn Ca Zn Ba Ca Zn
2.63 4.01 3.34 3.50
Schoniger oxidation 0.51 0.123 0.40
0.91 0.111 0.090 0.157 0.43 0.026 0.109
0.068 0.88 0.111 0.084 0.165
0.91 0.112 0.091 0.155 0.42
0.026 0.108
0.43 0.024 0.107
0.52 0.120 0.42 0.073 0.89 0.114 0.083 0.1.60
0.43 0.022 0.107
zinc dialkyldithiophosphate, each of which had been exhaustively analyzed by ASTM procedures to obtain a known value. The relative error of these results, ahoivn in Table I, is less than 0.570. Analyses of mixtures of the same additives, covering a wide range of concentrations and ratios of metals, are shown in Table I1 The relative error is about 1% for acidic decomposition, and nearly 2% for Schoniger oxidation.
but somewhat less accurate analpis, Schoniger oxidation may be preferred: although for use with oils each metal concentration must be greater than 0.05%, and an uncertainty in the result of up to about 1OYo relative must be tolerable.
mas. reasonably be-considered just as accurate as the results for additives in Table 11 because the same quantities of metals are taken for analysis. However, in the Schoniger oxidation of oils, where the sample size is limited to 100 mg., the titration volumes of D T P X are only a few milliliters and the method is less accurate for oils than for additives; the relative error is about 6%.
(3) Bertolacini, R. J., Petrol. Refiner 37, S o . 2, 147 (1958). (4) Davis, E. s.9 r-an Sordstrand, R. -4.j A 4 ~ CHEM. a ~ . 26,973 (1954). (5) G ~E, L., ~ l b z~d , 32,, 1449 (1960). (6) Pagliasotti, J. P., Porsche, F. W., Ibzd., 23, 1820 (1951). ( 7 ) whisman IT., Eccleston, B. H., Ibzd., 27, 1861 ( l & ) .
LITERATURE CITED
(1) iim. Soc. Testing Materials, .'ASTM Standards on Petroleum Products and Lubricants." D. 331. Philadelahia. Pa..
\A"-V,.
CONCLUSION
The combination of acidic decomposition and DTPA titration is accurate and to serve as a referee precise method. AIcomplete analysis takes less than 3 hours. For a rapid ( l / ~hour)
A. L. HENSLEY J. G. BERGMANS Research and Development Depart. American Oil Co. Whiting, Ind.
Division of Analytical Chemistry, 137th Meeting, ACS, Cleveland, Ohio, April 1960. Pittsburgh Conference on Analytical Chemistry and Applied Spectrescopy, Pittsburgh, Pa., March 1961. VOL. 35, NO. 9, AUGUST 1963
1319
