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ReductorTable I. Reduction of Thallium(lll) to Thallium(1)

KO. fDetns .Cadmium 557aBismuth 4

444Silver 4325

T1, Mg.Taken Found, av .3 2 . 0 3 1 . 953.1 5 '2.91 0 6 . 4 1 0 6 , 3212.8 212.73 2 . 0 3 2 . 05 3 . 1 5 3 . 1106 . 5 106 . 4212.8 213.132 . 0 32 . 053 . 1 53 . 0106.5 106.5212.8 213.1

Std.Dev.0 . 2 20 . 3 30 . 2 00 . 4 00.100 . 170 . 2 50 . 3 90 . 1 10 . 1 00 . 1 40 . 4 4

mium rod (Fisher Scientific Co.) were was add ed 40 nil. of 6-Y hydrochloricwashed with lil' hydrochloric acid and acid. Th e thallium(1) lyas t i t ra tedthen w ith 1N sulfuric acid. potentiometrically with 0.1000A7 potas-Bismuth. The chunk metal (Fisher sci- sium bro mat e using th e Fisher Titrim-eter with a platinum-calomel electrodentific Co.) was crushed and the coarsepieces were sorted ou t.Silver. Granu lar silver (G . Frederick pair*Smith Chemical Co.). Blanks were run on the reductorswith the above procedure , but sub s t i tut-The columns were washed ni th 1K in g 50 nil, of l,oLv ulfuric acid fo r thesulfuric acid an d kep t f i lled ni th 0.01N thallium solution. Th e correction, insulfuric acid when no t in use. ter ms of milliliters of 0.1s potass iumbrom ate , was 0.02 ml. for the bismuth,0.03 ml. for the silver, and 0.05 ml. forROCEDUREMeasured a l iquots of the s tandardthallium solution were treated to givesulfuric acid concentrations from 0.1 t ol.O*V an d to ta l volu mes of 50 t o 100ml.Each solution was uassed th rough th e

the cadmium reductor.After the blank correction was de-ducted, the resul ts shown in Table Iwere obtained.DISCUSSION

\Tit11 jo-ml. portions of 0.1N sulfuric bismuth, cadmium, or silver. Theacid. To the solution and washings blank corrections for these metals are

Use of DinitrosaIicyIic Acid Reagentfor Determination of Reducing SugarGAIL LORENZ MILLERPioneering Research Division, Quarfermasfer Research and Engineering Center, Natick, Mass.

sm a l l bu t m ay va ry f rom lo t to lo t ,depending upon the pur i ty .The results obtained with silver com-pared favorably n th those obtaineda i th b i sm uth o r cadm ium . Upon ad-dition of hydrochloric acid, a precipi-ta te of silv er chloride is obta ined . Al -though the resul ts do not indicate anyloss of tha llium chlorid e by coprecipi-tation with the silver chloride, the useof t he bism uth or cadmium reductorsis considered more convenient. K h enbismuth or cadmium is used, thallium(1)chloride, formed upon addition of hy-drochloric acid, disappears as the t i t ra -tion proceeds. I n the ease of silver,the silver chloride remains, and there isno vis ible rr idence that the thal l iumchloride has been completely oxidized.This is not a criticism of the reduct ionby s i lver , but ra ther a limit ation of thebromate method, which requires theaddition of hydrochloric acid.Other reductors s tudied were lead,aluminum, zinc, amalgams of zinc,cadmium, lead, and bismuth. So ne ofthese proved sat is factory. S ick el re-duced the tha ll ium quan t i t a t ive ly bu tthe b lanks ne re h igh and e r ra t i c . Abet ter g rade of nickel m etal noul dprobably serve satisfactorily.

LITERATURE CITED(1) Anderson, J. R. A , , rlx.4~. HEBI . 5 ,108 (1953).(2) Zintl, E., Rienacker, G., 2 . anorgChem. 153, 276 (1926).RECEIVEDor revieTY January 17, 1958.Accepted October 27, 1958.

b Rochelle salt, normally present inthe dinitrosalicylic acid reagent forreducing sugar, interferes with theprotective action of the sulfite, but isessential to color stability. The diffi-culty may be resolved either byeliminating Rochelle salt from the rea-gent and adding it to the mixture ofreducing sugar and reagent after thecolor is developed, or by addingknown amounts of glucose to thesamples of reducing sugar to com-pensate for the losses sustained in thepresence of the Rochelle salt. Theoptimal composition of a modifieddinitrosalicylic acid reagent is given.

HE D I N T R O S A L I C Y L I C A C I D REAGENT,*Teveloped by Sumner and co-worker ( 1 1 - 1 4 for the determinat ionof reducing su gar, is composed of di-nitrosalicylic acid, Rochelle salt, phenol,sodium bisulfite, an d sodiu m hydroxide.According t o the au thor s of th e tes t ,Rochel le sa l t is introduced to preventthe reag ent fro m dissolving oxygen ( 1 2 ) ;phenol, to increase th e am ou nt of colorproduced and to balance the effect ofphenol encountered in urine ( I S ) ; andbisulfite, to stabilize the color obtainedin the prescnce of the phenol ( I S ) .The alkali is required for the reducingactio n of glucosc on dinitrosalicylic acid.

The m ajor defect in the tes t is in theloss of p ar t of th e reducing sugar beinganalyzed. This was pointed out bySumner (12 , 1 4 , was referred to byBrodersen and Ricket ts (Z), and hasbeen observed repeatedly in this lab-o r a t o r y (6, 8, 9). Evidence of loss ofsugar is a lso given by the data of Hos-tettler, Borel, and Deuel (4 ) and ofBell , hlanners , and Palmer ( 1 ) . Asthis defect appears never to have beenful ly remedied, the present s tudy wascarried out to inves t igate the differentfactors which might cause i t . I n thecourse of the investigation , the effectsof vary ing the conce ntrations of th e426 ANALYTICAL CHEMISTRY

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different components of the reagentalso were determined. Th e findingswhich resulted have led to the develop-m e n t of a modified reagent and pro-cedure.METHOD

The color tests were made with 3-nil .al iquots of reagent added to 3-ml .al iqu ots of glucose solution in 14-mm.tubes. Th e mixtures were heated for5 minutes in a boi l ing water bath andthen cooled under running tap n-ateradjusted to ambien t tempera ture. Cool -i ng t o ambi en t t empera t u re was madenecessary by the effect of temperatureon the absorbance of the colored reac-t ion product (w), n effect confirmedby the present s tudies . Th e color in-tensi t ies nere measured in a Beckmanl l o d e l D L spect rophotometer at 575nip n th a sl i t n dth of 0.06 m m .T he reagent of Suniner an d Sisler ( 14)and a modif ied reagent w r e used inthe test s . Th e former contained ap-proximately 0.63Yc d i n i t rod i cy l i c ac id ,18.2% Rochelle salts, 0.57, phenol,0.5% sodium biqulfitc, and 2.14%sodium hydrouide; the modified rea-gent coiitained 1% dinitrosalicylic acid,0.2% phenol, 0.057c sodium sulfi te,and 1% sodium hydroxide.For cer tain te st s the modified reagentincluded varying concentrations of Ro-chelle sal t . Th e composit ion chosenfor the modified reagent n-as based onthe resultq of preliminary tests n hichindicated that such a reagent wasop t i mal and ~ o u l d e n e b e s t a s t h ebasis of rrference for testing effects ofvariat ion 111 composit ion. In the ab-qcnce of Rochelle salt , the color ob-t ained n th the modified reagent n sunstable. T o stabil ize the color unde rthese condi tions, 1 ml . of a 40 % solutionof the sal t was adde d to th e mixture ofreactants subsequrnt to the develop-nicnt of the color and prior to cooling.The modified reagent n as preparedby placing all solid components in acontainer and dissolving them simul-taneously by s t i r r ing wi th th e requi redvolume of sodium hydroxide solution.T h i s w as m u c h s i m d e r t h a n o t h e r m o -cedures (3 , 1 4 ) .The modified reagent produced thesame color wi th g lucose f rom day today, thus proving more s table in th isrespect th en t he reage nt of Brodersenand R i cke t t s ( 2 ) . Modified reagent towhich Rochelle salt vias added also didno t change f rom day t o day i n t h i srespect . Depending upon stora ge con-dit ions, however, the modified reagenttended eventual ly to deter iorate f romatmospheric oxidation of the sulfitepresent . Deter iorated reagent was re-juveiiated b y the add it ion of freshsul f i te . The danger at tendant uponoxidation of sulfite could be avoided byprepar ing the reagent in large batcheswithout sulfi te, the sulfi te being addedt o al iquots just pr ior to th e t ime whenthe reagent \vas to be used.

STUDY OF VARIABLESRem oval of Di sso l ved Oxygen .W I T HS T R E A MF K I T R O G E N . u m n e r

TRATD

0 0.1 0.2

0 0.3 0.6

fbl

0 03 06

63

0 0.3 06

I ROCH . SALTS^ I

63

0 0.3 06fc ) 0 03 06fdJ

0 03 06

03

CITRATE

0 0.3 06(e / f f J f g l fh lMILLIGRAMS GLUCOSE

Figure 1 .ac id reagen tEf fect o f var iab les on co lor produced with glucose and dini trosal icyl ica .b.d .e .f.9.h.

C.

a n d

Stream of nitrogen passed through mixture; Sumners reagentSodium sulfite treatment piior to addition of Suniners reage ntRochelle salt concentration; modified reagentSulfite concentration; modified reagentPodium hydroxide concentration; modified reagentPhenol concentration; modified reagentDinitrosalicylic acid concentration; modified reagentCarbox) me thl ]cellulose, citrate, and miiture s of both prese nt; modified reagent

Sisler ( 1 4 ) i n d i c a t e d t h a t t h eloss of glucose with the dinitrosalicylicac i d r eagen t was due t o des t ruc t i onby ox i da t i on , and based t he i r s t a t e -me nt on unrepor ted resul t s of ex-per im ents in which a s t re am of n i t ro-ge n w as passed t h rough t he r eac t an t s .An a t tem pt to conf irm this observationin the present nork indicated thatpassing a st ream of ni t rogen throug h amixture of Sumners reagent and glu-cose for 2 minutes pr ior to the develop-ment of color largely eliminated thedestruction of glucose (Figure 1, a ) .WITH STLFITE. As sulfi te had pre-viously been used successfully for re-moving dissolved oxygen from aqueoussolutions ( b ) , it was surpr is ing that thesulfi te present in Sumner and Sislersreagent fai led to accomplish this pur-pose. T o test whether this fai lure of thesulfite ma y have been due to interfereiiceby other components of the reagent ,sulfite a t a level of 0.1% n-as added t oglucose samples prior to mixing themwith the reagent. This procedure re-duced the destruction of glucose bya b o u t 707,. The rewl ts , shown inFigure 1, b, thus provided strongevidence for the suspected interferenceby th e other components of th e reagentunder the usual condit ions of the test .IN TER F ER EW EF ROCHELLESALT.Comparat ive test s were next carr iedout with the modified dinitrosalicylicacid reagent to M hich varying amounts

of Rochelle salt were ad de d. Th eresu lt s, s ho rn i n F i gu re 1, c, clearlyimplicated Rochelle salt as t he maj o rfactor involved in the interference withthe removal of oxygen by sulfi te,because, in the absence of t he sa lt , thesulfi te appeared able to remove thedissolved oxygen and thereby to pro-tect the glucose. i iside from con-trib utin g to the loss of a por tion of th eglucose, the Rochelle salt caused anenhancement of the color due to theremaining glucose.Sul f i te Conce nt rat ion. T he effectof different co nce ntra tion s of sulfi tein the modif ied reagent (Figure I , d )i n d i c a t e d t h a t a maxi mum co l o r i n -t e n s it y n a s o b t a in e d at 0.057, ulfi te.I n exper i men t s no t shown , essen t ia l l yt he same r esu l t s were ob t a i ned a t0.025 a n d 0.1% s u lf it e a s a t t h e0.057, level . Lo w concen t r a t i onscaused a lack of l inea r i ty , nh i leboth high and low Concentrationscaused a depression in color intensityand a loss of glucose.Sod i um Hydrox i de Concen t r a t i on .T he effect of d i f ferent conce nt rat ion sof sod i um hydrox i de i s shown i nF i gu re 1, e . High concent rat ions ofsod i um hydrox i de l ed t o enhancedc o l o r d e v e l o p m e n t , b u t a t t h e s a m et i me con t r i bu t ed t o a l o ss of glucose.The level of 1% sod i um hydrox i dea p p e a r e d t o b e t h e m o s t s u i t a b l e , asit produced t he max i mum co l o r i n -

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tensity possible without concomitantloss of glucose.Phe no l Concen t r a t ion . Max i mumco l o r deve l opmen t was a p p r o a c h e da t a concent rat ion of about 0.2%pheno l (F i gu re 1, f ) . In exper i -men t s no t shown i n t he f i gu re , t hesam e r esu l ts were ob t a i ned wi t h 0.5q.,pheno l as at th e 0.2% level . Lo wconcent rat ions resul ted in a l ack ofl i near i t y . Th e i n t ens i t y ob t a i ned i nthe presence of 0.2% pheno l wasa b o u t 5 t imes that obtained in the ab-scence of phenol. Over the rangetested, the phenol had no effect on theloss of glucose.Dinitrosalicylic Acid Concentration.Wh en th e amou nts of d ini t rosal icyl icacid Fyere var ied , the color in tensi tya p p r o a c h e d a m a x i m u m at a concen-t rat ion of 1% (F i gu re 1, 9). T h edini t rosal icyl ic acid , l ike the phenol ,had no effect on the loss of glucoseover t he r ange t es t ed .Ot he r Subs t ances . Th e r esu l t s ofthe preceding test s suggested the pos-s i b i l i t y t ha t o t her subs t ances mi gh taffect the d ini t rosal icyl ic acid test .For example, i t was of in terest toasce i t a i n whet her , i n u s i ng t he t es tf o r t h e m e a s u r e m e n t of cellulaseact iv i ty (8, IO), the presence of car-boxymethylcellulose and citrate buffera t p H 5 might cause interference.K i t h am ounts of carboxymethylcellu-lose and citra te buffer corresponding tothose used in the cellulase measure-ment, the effects shown in Figure 1, h,were produced. Th e carboxym ethyl-cellulose caused an enhancement incolor, whereas the ci trate caused adepression. A mixture of the sub-stances approximately neutralized thet n o opposite effects.To determine whether the effect ofthe ci t rate may have been a conse-quence of i ts buffering action, testsere made wi th acetate buffer of p H 5a t an equivalent concent rat ion. Th eacetate did not, however, affect thetest .

FINAL METHODKhen pure reducing sugar solut ionsare involved or when an y contam inantswhich may be present are knorvn notto affect the color development or to

cause any loss of reducing sugar, themodified reagent in the absence ofRochelle salt is used. Fo r stabil izationof the color produced under such con-dit ions, Rochelle salt is added to themixture immediately after the develop-me nt of the color and before th e mixtureis cooled. Th e time of heating is in-creased to 15 minutes because the 5-

minute per iod, adeq uate for the or ig inalprocedure, does no t produce completecolor development in the modifiedprocedure. B y this method l ineari ty ofdata, protection of glucose, and sta-bility of color are realized.If interfering substances occur inunknown samples, special controls arerun. Such controls consist of s t andardreducing sugar solutions to n hich ap-propr iate amounts of the interferingsubstances are nddrd. K he n the in-terfering substanctis bring about a lossof reducing sugar, and part icularlywhen the amounts of reducing sugarto be measured in unknown samplesare equal to or smaller than the am ountlost, known amounts of glucose areadded to both the unknown samplesand t he s t andards .Th e procedu re of add ing glucose canalso be applied advantageously to com-pensate for the loss of reducing sugarincurred nhen Rochelle salt is incor-porated in t he dinitrosalicylic acid rea-gent. Fo r example, i t is convenientin the cellulase test (IO) o introduceglucose into the carboxymethyl-cellu-lose-citrate subst rate an d to use modifieddinitrosalicylic acid reagent containing20% Rochelle salt (8). Under thesecondit ions the separate addit ion ofRochelle salt to the reaction mixtureafter color dcrelopment is omitted.Th e controls for such tests consist of ablank and standard glucose solutions,each c ontaining carboxymethylcellulose,ci trate, and compensatory glucose.

DISCUSSIONThe chemistrv of the dinitrosalicylicacid test for reducing sugar has beenclarified previously, a t least in pa rt .The 3,5-dinitrosalicylic acid is reducedto 3-amino-5-nitrosalicylic acid while,in the simplest instances, the aldehydegroups appear to be oxidized t o car-boxyl groups ( 4 ) . The facts, however,that the equivalence between amino-nitrosalicylic acid produced and sugaris not e xact (4)and tha t d i f ferent sugarsyield different amo un ts of color ( 1 , 4 , 7) ,suggest tha t the ch emistry of th e testmay actually hc appreciably more com-plicated.Such complications could conceiv-

ably be associated with the variousdecomposition reactions of sugars inalkaline solution ( 3 ) . If this explana-tion is corre ct, the reaction of th e sugaraldehyde grouping with dinitrosalicylicacid could be viewed as competing withside reactions involving decomposit ionof the suga r. Th e effects of differentconcentrations of the v arious consti tu-

ents of th e dinitrosalicylic acid reag ent,a n d also of extraneous substances suchas carboxymethylcellulose or ci tratebuffer, upon the amount of color pro-duced and upon the dest ruct ion ofglucose, as shown in the present s tudy,could similarly be interpreted as effectsupon th e natu re and degree of sidereactions.The dinitrosalicylic acid reagent, ina form consisting only of dinitrosalicylicacid dissolved in strong alkali, has beenused with a ppa rent success for molecularn-eight measurement of starch break-down products (?). This method de-pends upon the assumpt ion that al lhighe r oligosaccharides of t he homol-ogous series start ing with maltosewould produce equivalent amounts ofcolor with the reagent. Actual studiesof the reactions of members of homolo-gous series with the dinitrosalicylicacid reagent, start ing with the disac-charide, have not been reported, butu-ould be of considerable interest inthe present connection.

The principal virtue of the dinitro-salicylic acid test for reducing sugarlies in i ts great convenience comparedto most other sugar tests, part icularlyu-hen large numbers of tests must becarried out . However, the factors dis-cussed above must be given due con-sideration to avoid misinterpretationof results.LITERATURE CITED

(1) Bell, D. J. , Manners, D . J. , Palmer, A ,,J . Chem. Soc. 1952, 3760.(2) Brodersen, R., Ricketts, H. T., J .Lab. Clzn. Med. 34, 1447 (1949).(3 ) Gilman, H., Organic Chemistry,Advanced Trratise, 2nd ed., Vol. 2 ,p. 1640, Wiley, Xew York, 1943.(4) Hostettler, F., Borel, E., Deuel, H.,Helv. Chznz. d c t a 34, 2132 (1951).(5 ) Kolthoff, I. PII., Lingane, J. J. ,Polarography, Interscience, KewYork, 1946.(6) hlandels, G. R , Quartermaster Research and Engineering Center, Natick,Mass., private communication.( 7 ) bleyer, K . H., van der Wyk, A. J. A.,Peng, C., Helv. Chznz. A c t a 37, 1619(1954).(8) XIiller, G. L., Blum, R., Glennon,W. E., Quartermaster Research andEngineering Center, Xatick, Mass.,unpublished data.(9) Reese, E. T., Quartermaster Researchand Engineering Center, S ati ck , illass.,private communication.H. S., J . Bacter id. 59 , 485 (1950).5 (1931).

(10) Reese, E. T., Siu, R. G . H., Levinson,(11) Sumner, J. B., J . B i d . C hem . 47 ,(12) Zbid., 2, 287 (1924-25).(13) Ibzd . , 65 , 393 (1925).(14) Sumner. J. B.. Sisler. E. B.. Arch. Bzochem. 4: 333 (1944) .RECEIVEDor review Sovember 4, 1957.Accepted September 23, 1958.

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