Color Reaction of Anthrone with Monosaccharide Mixtures and Oligo- and Polysaccharides Containing Hexuronic Acids J. R. HELBERT and K. D. BROWN Department of Biochemistry, Marquette University School of Medicine, Milwaukee 3, Wis.
b Synthetic mixtures of galacturonic acid, galactose, and rhamnose have been quantitatively resolved. Overall concentrations as low as 21 y per ml. were used. The color produced by the reaction of di-, tri-, or tetragalacturonic acid with anthrone obeys Beer's law, at least up to 200 y per 6 ml.; the quantity of color i s proportional to the anhydro units present. Conditionssuitable for estimating monogalacturonic acid are suitable also for the above oligouronides. Sodium heparinate reacts with anthrone to yield the characteristic uronic acid color. The absorbance of glucosamine is zero, and 1 to 1 mixtures of glucosamine and glucuronic acid are additive. Nevertheless sodium heparinate produces a substantially greater quantity of color than would be anticipated from the proportion of uronic acid present.
I
earlier study (6) it was learned that monouronic acids react with anthrone in 27.5.V sulfuric acid to produce a red color with maximum absorbance a t 540 mp. Under suitable experimental conditions, microgram quantities of monouronic acids could be estimated with a precision equal to or better than that reported in the literature for aldohexoses. This inquiry has now been extended t o multicomponent mixtures of monomers and to oligo- and polysaccharides containing uronic acids. The results obtained are the subject of this report. " N AN
quired for complete solution. These solutions were always freshly prepared. Four milliliters of anthrone solution were pipetted into uniform borosilicate glass test tubes, followed by 2 ml. of carbohydrate solution. Because 27.5N sulfuric acid was the solvent for both reactants, no heat of mixing was evolved. Test solutions were heated in a thermostatically controlled water bath. After heating, samples were quickly transferred to a cold-water bath (4' =t 1' C.) for 3 minutes. Heating and cooling time was measured to + 2 seconds, and the test tubes containing the reacting solutions were spaced in wire racks in the heating and cooling baths to facilitate uniform heat transfer. Photometric readings were taken after 20 hours at room temperature (23" + 2' C.), except as indicated in particular instances. Test samples were always protected from direct sunlight. Although solid samples were used in all of the work discussed here, the foregoing procedure can be satisfactorily modified to accommodate aqueous solutions. However, precision in the latter
:I r"-
case always tends to be less than in the former. EXPERIMENTAL RESULTS AND DISCUSSION
Monomeric Mixtures. The relationship between heating time and absorbance for rhamnose, galactose, and galacturonic acid at two wave lengths is given in Figure 1. The conditions under which two absorbing species can be most accurately estimated are those which make t h e difference between the ratios of their absorbance indices a maximum (1, '7). This principle was extended to three components, and heating times of 13 and 28 minutes were chosen. Of the ,even temperatures investigated, 70" C. proved the most suitable. The three sets of experimental conditions used to set u p three equations in three unknoms were as follows: K1. Heat 13 minutes, read after 30 minutes a t 625 mp KP. Heat 28 minutes, read after 20 hours at 540 mu
Galacturonic Acid
B
3.350
: 0.600 L .7
3.300
r
z 0.250
E
0.700
2 Y
n Ig
0.200
% .a yl
0
PROCEDURE
0.150
The materials and apparatus used were similar to those described in previous publications (4, 6).
-2-
3.100
3
Anthrone solutions were prepared by dissolving 0.160 gram of the reagent in 100 ml. of 27.5 =k 0.1N sulfuric acid; about 60 minutes were allowed to effect complete solution. Such solutions were usually freshly prepared, but were never more than 24 hours old. Carbohydrate solutions mere prepared by dissolving solid sample in 27.5N sulfuric acid; about 30 minutes for monomers and u p to 60 minutes for polymers were re1464
0
ANALYTICAL CHEMISTRY
n
0 050
I
0
5
IO
15
20
25
30
5
H e a t i n g T i m e [min. ) a t
Figure 1.
10
I
15
20
I
25
30
70°C
Heating time vs. absorbance at two wave lengths
A. Photometric readings taken after 30 minutes B. Photometric readings taken after 20 hours Concentration in all cases, 100 7/6 ml.
540 and 625 mfi. I n the present study it was found that mixtures of these carbohydrates also follow Beer’s law a t both wave lengths; the range of total concentrations examined was 125 to
KI. Heat 28 minutes, read after 30 minutes at 626 mp It has been shown (4, 5 ) that rhamnose, galactose, and galacturonic acid individually follow Beer’s law a t both
250 y per 6 ml. At several points in this range the following relative proportions of galacturonic acid to galactose to rhamnose were used: 3 to 2 to 1, 10 to 10 to 4, and 12 to 12 to 1. I n each case absorbances were additive. It is valid, therefore, to substitute the indices and coefficients in Table I, A, into the equations below: ca(as)am
ccr(as)ax2
+ + +
ca(al)axs
CP(US)BK,
+ + +
Cp(Ue)pfl, CP(Us)Pn*
CB(09)PKI
(1)
= (kS)PKl
CP(~S)P= X~
(ks)WZ
(2)
cp(as)pna =
( M W 3
(3)
where CY
p p
= = = =
as =
k, c
galacturonic acid, 10 y per ml. galactose, 10 y per ml. rhamnose, 4 y per ml. CY p p , 24 y per ml. k A absorbance index = = -!
+ +
c
cb
absorbance coefficient, em.-’ concentration, grams per liter A , = absorbance b = cell thickness, em.
450
I
I
I
500
550
600
Wave Length, mir.
Figure 2.
Absorbance spectra of oligouronides
A. Digalacturonic acid, HzO, 114 y/6 ml. B. Trigalacturonic acid, HzO, 80 y/6 ml. C. Tetragalacturonic acid, 3H20, 66 y/6 ml.
D. Monogalacturonic acid, 42 y/6 ml.
0 SODIUM HEPARINATE.400 2’/6 ML
ANTHRONE BLANK
Figure. 3. Absorbance spectrum of sodium heparinate
0.375
0.30C
5U
-
;0 . 2 2 : z 4
fL Eo
2 m 0.15C
0.075
0 450
500 WAVE
550
LENGTH',^"
600
= =
The concentrations reported in Table I, B, were obtained from the foregoing equations by setting up appropriate determinants and applying Cramer’s rule. Absorbance indices should be constant, a t least for a given reference material. Hence, once accurately determined, they need not be redetermined in subsequent experiments. Three further trial mixtures \\-ere quantitatively resolved, using the absorbance indices in Table I, A. The largest observed deviation from the known or nominal value was 107’. I n this work controls were always included as a check on experimental conditions; blanks were also included with each run. A study of the precision of the weighing and pipetting operations indicated that the major part of any observed deviation arose from the random variation inherent in preparing and manipulating solutions. Consequently, a great deal of care must be expended on these operations. Furthermore, greater accuracy and precision should be obtainable by refinements in this part of the procedure. Oligouronides. Phaff and coworkers (2, 8 ) have isolated and purified a number of galacturonides during an investigation of the action of yeast polygalacturonase on pectic acid. Milligram quantities of di-, tri-, and tetragalacturonic acids were obtained from this source. Phaff found these compounds to be chromatographically distinct and, taken separately, homogeneous ; their molecular weights, elemental analyses, carboxyl to aldehyde ratios, etc., characterized them as the oligouronides just mentioned. Although the mode in which the indicated water (cf. Figure 2) is present in these molecules is not VOL. 29, NO. 10, OCTOBER 1957
1465
known, i t is not removed by drying under high vacuum at 35” C. (9). I n preparing the absorbance spectra in Figure 2, different quantities of each uronide were used in order to separate the curves for purposes of clarity. The shapes of these curves as well as the wave length of maximum absorbance correspond very well with those observed for monogalacturonic acid. The absorbance-concentration relationship is linear for the three oligouronides considered here. Furthermore, when each oligouronide is calculated to a n equivalent amount of monogalacturonic acid, for concentrations up to at least 200 y per 6 ml , the absorbance-concentration plot coincides with that of the monomer. From this fact and from the information in Figure 2 it seems evident that the color reactions of these oligo forms are essentially the same as that of the monomer. These results are analogous to those found by Koehler (6) and others for oligoaldohexoses. Sodium Heparinate. 1 sample of this material, having a hiological activity of 117 units per mg., was obtained from a commercial laboratory. Figure 3 s h o n s t h e absorbance spect r u m of this material after reaction with anthrone at 70’ C. The v a v e length of maximum absorbance is in excellent agreement n i t h t h a t of monoglucuronic acid, and the absorbance-concentration relationship conforms to Beer’s law, a t least to concentrations of 400 y per 6 ml. Honever, the amount of color obtained with sodium heparinate is abnormally high, being
Table 1.
Data on Three-Component Mixture“
A. Typical Data K2
K1
a,
2.3089 13.8773 59.8654
Ly
P
I* a
ae
k,
11.6025 8.3350 22.8496
K3
ka
ar
0,3745 0.2896 Each entry is mean of four replicates.
more than four times that anticipated from the amount of glucuronic acid present. The latter amount was estimated on the basis of the repeating unit proposed by Rolfrom (9). Moreover, the glucosamine in the polymer appears not to explain the anomalous results obtained. Glucosamine does not enhance the glucuronic acid-anthrone color. The absorbance of glucosamine hydrochloride-anthrone has been directly determined to be zero; and the absorbance-concentration plot of 1 to 1 mixtures of glucuronic acid and glucosamine hydrochloride with anthrone is congruent with that of glucuronic acid alone. The high results observed for sodium heparinate with anthrone are analogous to the findings of Dische (3) for hyaluronic and chondroitinsulfuric acids with carbazole. The reason for the unusual behavior of heparinate is not known. This phenomenon, however, is currently under investigation in this laboratory. ACKNOWLEDGMENT
The authors are indebted to H. J. Phaff, University of California. Davis,
2.4486 23.7949 53.1187
B._ _ Typical Results Concentration, y k, Known Found 10.0 10.9 10.0 9.9 4.0 3.6 0.4600
Calif., for specimens of the oligouronides, to L. L. Coleman, Upjohn Laboratories, for the sample of sodium heparinate, and to L. C. hIassopust and F. TV. Faust for the preparation of dran-ings and photographs. LITERATURE CITED
(1) Berry, C. E., Ann. Math. Stat. 16, 398 (1945). (2) Demain, A. L., Phaff, H. J., Arch. Biochem. Biophys. 51, 114 (1954). (3) Dische, Z., J. Biol. Chem. 183, 489 (1950). Hilbert,’ J. R., Brown, K. D., A N ~ L . CHEW27, 1791 (1955). Ibid., 28, 1098 (1956). Koehler, L. H., Ibid., 24, 1576 (1952). Mellon, M. G., ed., “Analytical Absorption Spectroscopy,” p. 370 ff, Wiley, New York, 1950. Phaff, H. J., Luh, B. S., Arch. Biochem. Biophys. 36, 231 (1952). Whistler, R. L., Smart, C. L., “Polysaccharide Chemistry,” p. 415, Academic Press, New York, 1953. RECEIVED for review December 31, 1956. Accepted May 11, 1957. Division of Carbohydrate Chemistry, 130th Meeting, ACS, Atlantic City, N. J., September 1956. Work supported by grant-in-aid from Lakeside Laboratories, Inc., Milwaukee, Wis.
Acidimetric Determination of Fluorine after Ion Exchange Application to Aluminum Fluoride, Cryolite, and Fluorspar HENRI SHEHYN Aluminium laboratories, Ltd., Arvida, Quebec, Canada
b An accurate and precise method for the determination of fluorine in aluminum fluoride, cryolite, and fluorspar is presented. The fluorine is solubilized b y fusion with mixed alkali carbonates and silica, followed by a water leach. Subsequent treatment with a cation exchanger removes the cations, and titration of the effluent with standard alkali gives an accurate measurement of the amount of fluorine present. For sulfate-bearing materials a correction based on the sulfate content is applied. The pro1466
ANALYTICAL CHEMISTRY
cedure is simple and it permits handling larger amounts of fluorine than the steam distillation-thorium nitrate titration method.
T
are currently used for the determination of large amounts of fluorine such as encountered in aluminum fluoride, cryolite, and fluorspar. The oldest of these involves fusion of the sample with mixed alkali carbonates and silica, removal of silica and other interfering elements, and eventual precipitation of the fluorine as HREE METHODS
calcium fluoride. This procedure is too difficult and time-consuming to be of any value in routine industrial control. The second is similar, except that fluorine is precipitated as lead chlorofluoride, which has the advantage of a larger equivalent weight than calcium fluoride and also permits an argentometric finish. This method is capable of giving good results with a shorter time expenditure, but because of the properties of the precipitate i t requires very careful control of the operating conditions.
