A Micromethod for the Determination of Uronic Acid

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A MICROMETHOD FOR THE DETERMINATION OFURONIC ACIDS*

B Y BERNARD BURKHART, LORENZ BAUR, ANDKARL PAUL LINK(From the Biochem istry Research Laboratory, Department of Agricultural

Chemistry, University of W iscon sin, Madison)

(Received for publication, October 9, 1933)The original macromethod of Tollens and Lefevre (1) for thedetermination of uranic acids has undergone various modificationsin the hands of different investigators (2--g). All of t,he modifica-tions are based on the principle that the simple uranic acids andthe complex polyuronide substances yield carbon dioxide quant.i-tatively when distilled with 12 per cent hydrochloric acid. Insome the carbon dioxide is determined gravimetrically, in others

by tit,rimetric methods. The size of the sample used variesbetween the limits of 0.2 to 1.0 gm. and the period of heating from4 to 8 hours.Recently Buston (9) described a micromet.hod embodying ti-trimetric technique for the determination of uranic acid anhydridegroups in pectic subst,ances. In conjunction with t,he studies onthe carbonyl sugar acids in progress in this laboratory, one of us(K. P. L.) and a collaborator1 were engaged with the developmentof a satisfactory micromethod for their determination prior t,o thetime of the appearance of Bustons method. The details had notbeen completely worked out, consequently it appeared advisableto evaluate the merits a.nd defects of Bustons method in conjunc-tion with our work.Bustons method is open to criticism. The apparatus is very

* Published with the permission of the Director of the Wisconsin Agri-cultural Experiment Station.Supported in part by grants from the University Research Fund.1 The assistance of Dr. Eugene Schoeffel in the preliminary work isacknowledged. We are also indebted to Mr. Sam Morel1 for various helpful

suggestions contributed to the titrimetric modification.171

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172 Microdetermination of Uranic Acidsfragile and difficult to clean. We were also impressed by thefact that the method appeared to be supported by rather meageranalytical data. The data were confined entirely to such com-plex substances as calcium pectate (Ca5H4eO&a2), sodium pectate(CasH46033Na4), and pectin, whose exact composition might still beconsidered an open question. With substances of such complexitythe substitution of one or more residues of a pentose sugar byhexose residues or the loss of a methoxyl or acetyl residue, wouldalter but slightly the elementary composition. Thus the galac-turonic acid anhydride figures obtained might approximate thevalues expected on the basis of the empirical formula presented,even though the complex molecules are actually not identical withthese formulz Furthermore, within the limits of the analyticalfigures reported, considerable quantities of impurities might bepresent.

In the method described herewith the desirable features ofBustons procedure are employed along with certain parts of theexcellent apparatus described by Clark (10) for the microdeter-mination of methoxyl groups.2 With authentic preparations ofd-glucuronic and d-galacturonic acid, various methoxyl derivativesof d-galacturonic acid, and highly purified polyuronide substances,the optimum temperature and the duration of the heating periodnecessary to effect complete decarboxylation were ascertained.It was found that when the decarboxylation is performed at abath temperature of 133-136, results in agreement with thetheoretical values were obtained, provided the hydrolysis and de-carboxylation period was conducted for a period of not less than120 minutes. With authentic polygalacturonic acid anhydridepreparations, (CsHS06)n, he duration of the reaction had to be ex-tended to 24 hours. Highly purified specimens of alginic acid,(CeHsOs),, required 33 hours.3It is well known that the accurate measurement of small quanti-

2 The apparatus designed by Clark is a modification of the original micro-Zeisel apparatus developed by Pregl ((11) p. 199).8 This is in agreement with the experiences of Nelson and Cretcher (12),and those of Schoeffel and Link (13), who observed that in the macrodeter-mination of the uranic anhydride content of alginic acid the time requiredfor hydrolysis and decarboxylation had to be increased considerably inorder to obtain consistent results.

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Burkhart, Baur, and Link 173ties of carbon dioxide by either gravimetric or titrimetric methodpresents formidable difficulties. The numerous factors involvedhave been thoroughly presented in the various treatises dealingwith organic microanalysis (11, 14, 15), hence they need not berestated here.

We have explored both the gravimetric and titrimetric methodsusing the same conditions for the decarboxylation. Although wehave been able to obtain consistent results with the titrimetricmethod presented below, we prefer the gravimetric method sinceit appears to be more accurate. It is also less tedious and morerapid. It should be emphasized that the titrimetric procedure ispreferable when the temperature and atmospheric conditions aresuch as to make it difficult to obtain accurate weighings of theabsorption tubes by the gravimetric method.

Since the gravimetric technique employed for the estimationof the carbon dioxide is identical with that developed by the lateProfessor Fritz Pregl for the determination of carbon in organicsubstances, the details need not be given. It should, however, bepointed out that accurate and reliable results cannot be realizedunless the conditions originally prescribed by Pregl (11) and laterby others (14-16) for the manipulation of the absorption tubes andthe microbalance be followed rigidly.

EXPERIMENTALDescription of Apparatus-The apparatus consists of a reactionflask, A, with a capacity of 25 cc., which is connected to an air

condenser, B, through a standard ground joint No. 6. The aircondenser is 25 cm. long and terminates in a trap, C, containingsilver sulfate dissolved in concentrated sulfuric acid, to retain anyhydrogen chloride gas that might pass the condenser from theflask A. The joint in trap C is also standard No. 6. From trapC a side arm leads to the absorption tubes, or bottles D and E,through a a-way stop-cock, S. The absorption system in thetitrimetric method consists of small Jena gas wash bottles of 20cc. total capacity, equipped with interchangeable ground glassstoppers carrying the inlet and outlet tubes, The inlet tube ter-minates in a Jena sintered glass disk (porosity No. 0) which breaksthe gas stream into a spray of fine bubbles to insure efficient ab-sorption. The bottles are connected to a filter pump through a

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174 Microdetermination of Uranic Acidsneedle valve, F, which enables the rate of aspiration to be con-trolled accurately. A safety bottle, G (2.5 liter capacity equippedwith a stop-cock), is shunted in to serve as a reservoir to equalizethe flow of gas through the system. The aspirated air is freedfrom carbon dioxide by a train consisting of a small tower, H,filled with ascarite or soda-lime and a gas bottle, I, of the typedescribed above, filled with saturated barium hydroxide solution.A small mercury trap, J, prevents the liberated CO, from backingup into the purification train. The reaction flask A is placed inan oi l bath which is heated with a microburner. The air condenseris protected from the heat of the bath with a piece of sheet asbestos,KK, which is placed over the oi l bath and around the condenser.The reaction flask should be immersed in the oi l bath to such adepth that the levels of the reaction mixture in flask A and theoutside oil are the same. The thermometer should not touch thesides of the bath. In order to promote smooth boiling a smallboiling rod is placed in the flask. It consists of a piece of glasstubing, approximately 2 mm. internal diameter and 5 cm. long,sealed at one end and also about 5 mm. from the other end. Theopen end is fire-polished. The rod is introduced open end down.All the rubber connections should consist of properly treated andaged thick walled tubing of the type used in the Pregl carbon andhydrogen microdeterminations.

Analytical Procedure for Titrimetric Method-A sample of 9 to11 mg. is weighed out accurately in a boat made from aboutone-third of a cigarette paper. The paper boat is folded carefullyand introduced into the reaction flask.4 7 cc. of 12 per cent HClsaturated with sodium chloride are then added, the boiling rodintroduced, and the flask connected to the apparatus. The trapC is previously fi lled with a few drops of concentrated sulfuric acidcontaining silver sulfate. The 2-way stop-cock is set to establisha direct connection to the needle valve. The air in the apparatusis then removed by drawing a small but steady stream of CC+free air through the system. This requires about 10 minutes.Meanwhile 20 cc. of 0.02 N Ba(OH)2 are added to each of the gasbottles D and E which are then connected to the apparatus with

* Blank determinations on the cigarette paper used showed that no appre-ciable quan tities of CO:! are produced by the action of the hydrochloric acid.

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176 Microdetermination of Uranic Acidsthick walled tubing as shown in Fig. 1. The heating is now startedwithout altering the init ial rate of aspiration. (A small burner isconvenient for this purpose.) When the temperature of the oi lbath reaches 100 the stop-cock S is turned so that the air streampasses through the gas bottles. As the temperature approaches120 care must be taken to maintain sufficient aspiration to pre-vent excessive back pressure on mercury trap J. The contents ofthe flask begin to boil at approximately 120 and equilibrium issoon reached. The burner is adjusted so that the temperature ofthe oi l bath is maintained between the limits of 135-137. Theaspiration is adjusted so that a small, steady stream of bubblespasses through the gas bottles. At the end of 2 hours or a longerperiod, depending on the substance, the heating is discontinuedand the stop-cock S is turned to its former position. The gasbottles D and E are disconnected, the ground glass heads replacedby rubber stoppers, and the barium carbonate allowed to settle.

The titration of the barium hydroxide solution from the gaswash bottles should always be preceded by the following method ofstandardizing the relative strength of the acid and the alkali solu-tions. Approximately 20 cc. of the standard barium hydroxideare withdrawn from the burette into a small flask (25 cc.). Bymeans of a standardized 5 cc. Ostwald pipette, two aliquots arethen rapidly pipetted into 125 cc. Erlenmeyer flasks containing 20cc. of carbon dioxide-free water and 3 drops of phenolphthalein.The aliquots are then titrated with 0.01 N sulfuric acid. Not morethan two aliquots should be pipetted at one time. The aliquotsshould agree within 0.04 cc. of 0.01 N acid. These titrations givethe acid-alkali ratio which must be determined each time a sampleis analyzed, since it may vary slightly from time to time. Inexactly the same manner two 5 cc. aliquots from each of the gasbottles D and E are then titrated.The barium hydroxide solution is accurately standardized onceeach week with analytically pure potassium acid phthalate.The calculations are made as follows:Let A equal cc. 0.01 N acid equivalent to 5 cc. alkaliL B L 0.01 5 I aliquots of bottle DI c I I< 0.01 I I 5 I I jjJEach gas bottle contains 20 cc. of standard alkali. 1 cc. of 0.01 N HzSOn isequivalent to 0.00022 gm. of COz; therefore,

4 ((A-B) + (A-C))(O.O0022)(acid factor)(lOO)Weight of sample = per cent CO*

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Burkhart, Baur, and Link 177The following representative calculation is based on figures ob-tained in a determination of the uranic acid content of methyl-d-galacturonide dihydrate, C~HEO,.~H~O.

Acid-Alkali Ratio-5 cc. of 0.02 N Ba(OH)2 (factor = 1.005) re-quired 9.45 cc. of 0.01 N sulfuric acid. Therefore the 0.01 N HzS04has a factor of 1.050.Titration of Aliquots from Bottles D and E

Two 5 cc. aliquots from bottle D = 7.387.387.38 cc., averageTwo 5 cc. aliquots from bottle E = 9.359.349.345 cc., average

Calculation4 ((9.45-7.38) + (9.45-9.34))(0.00022)(1.05)(100)0.01119 gm. = 18.00 70 CO?

The theoretical for the methylgalacturonide dihydrate is 18.03per cent.Achromatic indicators described by Smith (17) may be used inplace of phenolphthalein as suggested in Bustons article (9).Such indicators were tried but we feel that they offer only slight,if any, advantages over phenolphthalein.Analytical Procedure for Gravimetric Modijication--In the gravi-metric method the standard Pregl soda-lime absorption tubes ((11)p. 45) are used to collect the carbon dioxide liberated. A SchwartzU-tube equipped with ground glass stoppers, containing concen-trated sulfuric acid and a few glass beads is connected directly tothe side arm of the decarboxylation apparatus in place of the 2-waystop-cock S. The other outlet of the U-tube is connected to asecond Schwartz U-tube containing dehydrite (18) or porous an-hydrous calcium chloride (groat size) as prescribed by Pregl (( 11)p. 54). The first U-tube contains just enough concentrated sul-furic acid to cover the beads in the bottom of the tube. The flowof the gas bubbles through this tube should be about 18 to 24 perminute.The 2-way stop-cock X is next, being joined to the side arm ofthe secondU-tube containing the dehydrite or the calcium chloride.The Pregl soda-lime tubes follow; they are connected to one of thelimbs of the stop-cock S. The Pregl tubes are connected with the

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178 Microdetermination of Uranic Acidsregular thick walled rubber tubing (8 mm. outer diameter, 2 mm.bore) treated as prescribed for the carbon and hydrogen micro-determination ((11) pp. 54-57). A Pregl calcium chloride tubewith two connecting tubes bent at right angles ((11) p. 54) is placedbetween the second soda-lime absorption tube and one limb of theY-tube that leads to needle valve F. The other l imb of the Y-tube is connected to the remaining limb of stop-cock S. This tubeprevents the second Pregl soda-lime tube from absorbing waterfrom the aspiration syst,em. The other aspects of the procedureused in the gravimetric method are comparable to the detailsgiven above for the titrimetric method. It should be reemphasizedthat the soda-lime tubes should be manipulated as prescribed forthe Pregl carbon and hydrogen microdetermination (( 11) pp. 43-53, 8047).

Sources of Errors-The micromethod presented is recommendedonly for pure uranic acids or their derivatives. Other carbohydratesubstances like the sugars, st,arch, cell wall polysaccharides, andcert,ain organic acids yield small quantities of carbon dioxide whenboiled wit,h 12 per cent hydrochloric acid. While the quantityof carbon dioxide liberated from these substances frequently doesnot interfere with the accuracy of the macromethods (7), the sameis not necessarily true in the microdetermination. Consequentlyt,he method is not recommended for uranic acid determinations onplant ext,racts or crude polysaccharide preparations containingsmall quantities of uranic acids. The most important sources oferror in the uranic acid microdetermination are the rate of aspira-tion and the length of the heating period. Low results are in-variably obtained when the rate of aspiration is too rapid and whenthe heating period is too brief. A very slow aspiration accom-panied with the existence of an occasional back pressure in thecourse of the determination can likewise produce low results.

The titrimetric modification is obviously subject to the varioussources of error that accompany the handling of dilut,e standardsolutions. Consequently the standard precautions recommendedfor titrimetric work must be rigidly observed. With the gravi-metric modification, the commonest source of error is invariablydue either to the improper handling of the absorption tubes or toadverse temperature and atmospheric conditions that influencethe constancy of the absorption tubes and the microbalance. The

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180 Microdetermination of Uranic Acidssources of these errors have been fully described in the handbooksdealing with the carbon and hydrogen microdetermination (11,14-16). The various substances used in the absorption and gaswashing trains must obviously be changed at frequent intervals inorder that fresh effective reagents are always at hand. The firstPregl soda-lime tube can be used for seven to eight determinations-the second tube can be safely used for twelve to fifteen det,er-minations.

SUMMARY1. A micromethod with either a volumetric or gravimetric

modification is given for the accurate determination of uranicacids and uranic acid derivatives by decarboxylation with hydro-chloric acid (sp. gr. 1.06).

2. The results obtained with authentic specimens of d-galac-turonic acid, d-glucuronic acid, methyl-d-galacturonide dihydrate,methyl ester methyl-d-galacturonide monohydrate, a methylglyco-side methyl ester polygalacturonide, CsHs05COOCH3 (CsH70,-COOCHS), GH703 (OCH,) COOCHS, a pure polygalacturonide,(CeHS06)+,, and alginic acid, (GJH~OJ,,, are presented (Table I).

3. In al l cases, the results obtained by either the volumetricor the gravimetric modification approximate the theoretical values.

4. The micromethod presented is capable of giving results ofthe same order of accuracy as the best macromodifications.

BIBLIOGRAPHY1. To llen s, B., and Lefevre, K. V., Ber. them. Ges., 40, 4153 (1907).2. van der Haar, A. W., Anleitung zum Nachw eis, zur Trennung und Bes-

timmung der Monosaccharide und Aldehydesiiuren, Berlin, 71 (1920).3. Nanji, D. R., Paton , F. T., and Ling, A. R., J. Sot. Chem. Znd., 44,253 T

(1925).4. Dore, W. H., J. Am. Chem. Sot., 48, 232 (1926).5. McKinnis, R. B., J. Am. Chem . Sot., 60, 1911 (1928).6. Eh rlich, F., and Schubert, F., Ber. &em. Ges., 62, 1974 (1929).7. Dickso n, A. D., Otterson, H., and Link, K. P., J. Am. Chem. So t., 62,

775 (1930).8. Klein, G., Handbuch der Pflanzenanalyse, Vienna, 3, 121 (1932).9. Buston, H. W., Analyst, 67, 207 (1932).

10. Clark, E. P., J. Assn. Of. Agric . Chem., 16, 136 (1932).11. Pregl, F., Die quan titative organische Mikroanalyse, Berlin, 3rd edi-

tion (1930).12. Nelso n, W. L., and Cretcher, L. H., J. Am. Chem. Sot., 61, 1914 (1929).

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Burkhart, Baur, and Link 18113. Schoeffel, E., and Link, K. P., J. Biol. Chem., 96, 213 (1932).14. Weygand, C., Quantitative analytische Mikromethoden der organischenChemie, Leipsic (1931).16. Friedrich, A., Die Praxis der quantitativen organischem Mikroanalyse,

Vienna (1933).16. Boetius, M., Ueber die Fehlerquellen bei der mikroanalytischen Bestim-mung des Kohlen und Wasserstoffes, Berlin (1931).17. Smith, L., Quarl. J. Pharm., 3, 499 (1930).18. Smith, G. F., Znd. and Eng. Chem., 16, 20 (1924).

19. Morell, S., and Link, K. P., J. Biol. Chem., 100, 385 (1933).

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A Micromethod for the Determination of Uronic Acid