Bismuth, silver and cadmium reductors were used. Thallium lyas were titrated potentiometrically with 0.1000A7 potassium bromate. Results varied from lot to lot, depending upon the purity.
Descrição original:
Título original
Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar
Bismuth, silver and cadmium reductors were used. Thallium lyas were titrated potentiometrically with 0.1000A7 potassium bromate. Results varied from lot to lot, depending upon the purity.
Direitos autorais:
Attribution Non-Commercial (BY-NC)
Formatos disponíveis
Baixe no formato PDF, TXT ou leia online no Scribd
Bismuth, silver and cadmium reductors were used. Thallium lyas were titrated potentiometrically with 0.1000A7 potassium bromate. Results varied from lot to lot, depending upon the purity.
Direitos autorais:
Attribution Non-Commercial (BY-NC)
Formatos disponíveis
Baixe no formato PDF, TXT ou leia online no Scribd
Table I. Reduction of Thallium(lll) to Thallium(1) depending upon the purity.
T1, Mg. The results obtained with silver com- K O . of Std. Reductor Detns. Taken Found, av. Dev. pared favorably n ith those obtained a i t h bismuth or cadmium. Upon ad- Cadmium 5 32.0 31.9 0.22 5 53.1 5'2.9 0.33 dition of hydrochloric acid, a precipi- 7 106.4 106,3 0.20 tate of silver chloride is obtained. Al- a 212.8 212.7 0.40 though the results do not indicate any Bismuth 4 32.0 32.0 0.10 loss of thallium chloride by coprecipi- 4 53.1 53.1 0.17 tation with the silver chloride, the use 4 106.5 106.4 0.25 of the bismuth or cadmium reductors 4 212.8 213.1 0.39 is considered more convenient. K h e n Silver 4 32.0 32.0 0.11 bismuth or cadmium is used, thallium(1) 3 53.1 53.0 0.10 2 106.5 106.5 0.14 chloride, formed upon addition of hy- 5 212.8 213.1 0.44 drochloric acid, disappears as the titra- tion proceeds. I n the ease of silver, the silver chloride remains, and there is no visible rridence that the thallium mium rod (Fisher Scientific Co.) were was added 40 nil. of 6-Y hydrochloric chloride has been completely oxidized. washed with lil' hydrochloric acid and acid. The thallium(1) lyas titrated This is not a criticism of the reduction then with 1N sulfuric acid. potentiometrically with 0.1000A7 potas- by silver, but rather a limitation of the Bismuth. The chunk metal (Fisher sci- sium bromate using the Fisher Titrim- bromate method, which requires the entific Co.) was crushed and the coarse eter with a platinum-calomel electrode addition of hydrochloric acid. pieces were sorted out. Silver. Granular silver (G. Frederick pair* Other reductors studied were lead, Smith Chemical Co.). Blanks were run on the reductors aluminum, zinc, amalgams of zinc, with the above procedure, but substitut- cadmium, lead, and bismuth. S o n e of The columns were washed nith 1K ing 50 nil, of l,oLvsulfuric acid for the sulfuric acid and kept filled n i t h 0.01N thallium solution. The correction, in these proved satisfactory. Sickel re- sulfuric acid when not in use. terms of milliliters of 0.1s potassium duced the thallium quantitatively but bromate, was 0.02 ml. for the bismuth, the blanks nere high and erratic. A PROCEDURE 0.03 ml. for the silver, and 0.05 ml. for better grade of nickel metal nould the cadmium reductor. probably serve satisfactorily. Measured aliquots of the standard After the blank correction was de- thallium solution were treated to give ducted, the results shown in Table I sulfuric acid concentrations from 0.1 to were obtained. LITERATURE CITED l.O*V and total volumes of 50 to 100 ml. Each solution was uassed through the DISCUSSION (1) Anderson, J. R. A,, rlx.4~.CHEBI.2 5 , 108 (1953). (2) Zintl, E., Rienacker, G., 2. anorg Chem. 153, 276 (1926). \Tit11 jo-ml. portions of 0.1N sulfuric bismuth, cadmium, or silver. The RECEIVEDfor revieTY January 17, 1958. acid. To the solution and washings blank corrections for these metals are Accepted October 27, 1958.
Use of DinitrosaIicyIic Acid Reagent
for Determination of Reducing Sugar GAIL LORENZ MILLER Pioneering Research Division, Quarfermasfer Research and Engineering Center, Natick, Mass.
b Rochelle salt, normally present in
the dinitrosalicylic acid reagent for reducing sugar, interferes with the *T HE D I N T R O S A L I C Y L I C ACID REAGENT, developed b y Sumner and co- worker (11-14 for the determination The major defect in the test is in the loss of part of the reducing sugar being analyzed. This was pointed out by protective action of the sulfite, but i s of reducing sugar, is composed of di- Sumner (12, 1 4 , was referred to by essential to color stability. The diffi- nitrosalicylic acid, Rochelle salt, phenol, Brodersen and Ricketts (Z), and has culty may be resolved either b y sodium bisulfite, and sodium hydroxide. been observed repeatedly in this lab- eliminating Rochelle salt from the rea- According to the authors of the test, oratory (6, 8, 9). Evidence of loss of gent and adding it to the mixture of Rochelle salt is introduced to prevent sugar is also given by the data of Hos- reducing sugar and reagent after the the reagent from dissolving oxygen (12); tettler, Borel, and Deuel (4) and of color i s developed, or b y adding phenol, to increase the amount of color Bell, hlanners, and Palmer ( 1 ) . As known amounts of glucose to the produced and to balance the effect of this defect appears never to have been samples of reducing sugar to com- phenol encountered in urine ( I S ) ; and fully remedied, the present study was pensate for the losses sustained in the bisulfite, to stabilize the color obtained carried out to investigate the different presence of the Rochelle salt. The in the prescnce of the phenol ( I S ) . factors which might cause it. I n the optimal composition of a modified The alkali is required for the reducing course of the investigation, the effects dinitrosalicylic acid reagent is given. action of glucosc on dinitrosalicylic acid. of varying the concentrations of the
426 ANALYTICAL CHEMISTRY
different components of the reagent also were determined. The findings which resulted have led to the develop- TR€AT€D IROCH. SALTS^ I ment of a modified reagent and pro- cedure. 63 METHOD The color tests were made with 3-nil. aliquots of reagent added to 3-ml. aliquots of glucose solution in 14-mm. 0 0.1 0.2 0 0.3 06 0 03 06 tubes. The mixtures were heated for fbl fc) fdJ 5 minutes in a boiling water bath and then cooled under running t a p n-ater adjusted to ambient temperature. Cool- ing to ambient temperature was made necessary by the effect of temperature on the absorbance of the colored reac- tion product (w), a n effect confirmed 03 by the present studies. The color in- tensities nere measured in a Beckman CITRATE llodel DL spectrophotometer a t 575 nip n ith a slit n idth of 0.06 mm. The reagent of Suniner and Sisler (14) 0 0.3 0.6 0 03 06 0 03 06 0 0.3 06 and a modified reagent w r e used in (e/ ffJ fgl fhl the tests. The former contained ap- MILLIGRAMS GLUCOSE proximately 0.63Yc dinitrodicylic acid, 18.2% Rochelle salts, 0.57, phenol, Figure 1. Effect of variables on color produced with glucose and dinitrosalicylic 0.5% sodium biqulfitc, and 2.14% acid reagent sodium hydrouide; the modified rea- gent coiitained 1% dinitrosalicylic acid, a. Stream of nitrogen passed through mixture; Sumner’s reagent 0.2% phenol, 0.057c sodium sulfite, b. Sodium sulfite treatment piior to addition of Suniner’s reagent and 1% sodium hydroxide. C. Rochelle salt concentration; modified reagent For certain tests the modified reagent d. Sulfite concentration; modified reagent included varying concentrations of Ro- e. Podium hydroxide concentration; modified reagent chelle salt. The composition chosen f. Phenol concentration; modified reagent 9. Dinitrosalicylic acid concentration; modified reagent for the modified reagent n-as based on h. Carbox) methl ]cellulose, citrate, and miitures of both present; modified reagent the resultq of preliminary tests n hich indicated that such a reagent was optimal and ~ o u l ds e n e best as the basis of rrference for testing effects of and Sisler (14) indicated t h a t t h e of Rochelle salt were added. The variation 111 composition. I n the ab- loss of glucose with t h e dinitrosalicylic results, s h o r n in Figure 1, c, clearly qcnce of Rochelle salt, the color ob- tained n ith the modified reagent n as acid reagent was due t o destruction implicated Rochelle salt as the major unstable. T o stabilize the color under b y oxidation, and based their state- factor involved in the interference with these conditions, 1 ml. of a 40% solution ment on unreported results of ex- the removal of oxygen by sulfite, of the salt was added to the mixture of periments in which a stream of nitro- because, in the absence of the salt, the reactants subsequrnt to the develop- gen was passed through t h e reactants. sulfite appeared able to remove the nicnt of the color and prior to cooling. An attempt to confirm this observation dissolved oxygen and thereby to pro- The modified reagent n as prepared in the present nork indicated that tect the glucose. iiside from con- by placing all solid components in a passing a stream of nitrogen through a tributing to the loss of a portion of the container and dissolving them simul- taneously by stirring with the required mixture of Sumner‘s reagent and glu- glucose, the Rochelle salt caused an volume of sodium hydroxide solution. cose for 2 minutes prior to the develop- enhancement of the color due to the This was much simder than other mo- ment of color largely eliminated the remaining glucose. cedures (3, 14). destruction of glucose (Figure 1, a ) . Sulfite Concentration. T h e effect The modified reagent produced the WITH STLFITE. As sulfite had pre- of different concentrations of sulfite same color with glucose from day to viously been used successfully for re- in the modified reagent (Figure I , d ) day, thus proving more stable in this moving dissolved oxygen from aqueous indicated t h a t a maximum color in- respect then the reagent of Brodersen solutions (b), it was surprising that the tensity n a s obtained at 0.057,sulfite. and Ricketts ( 2 ) . Modified reagent to sulfite present in Sumner and Sisler’s I n experiments not shown, essentially which Rochelle salt vias added also did not change from day to day in this reagent failed to accomplish this pur- t h e same results were obtained a t respect. Depending upon storage con- pose. T o test whether this failure of the 0.025 and 0.1% sulfite as a t t h e ditions, however, the modified reagent sulfite may have been due to interfereiice 0.057, level. Low concentrations tended eventually to deteriorate from by other components of the reagent, caused a lack of linearity, nhile atmospheric oxidation of the sulfite sulfite a t a level of 0.1% n-as added to both high and low Concentrations present. Deteriorated reagent was re- glucose samples prior to mixing them caused a depression in color intensity juveiiated by the addition of fresh with the reagent. This procedure re- and a loss of glucose. sulfite. The danger attendant upon duced the destruction of glucose by Sodium Hydroxide Concentration. oxidation of sulfite could be avoided by about 707,. The rewlts, shown in T h e effect of different concentrations preparing the reagent in large batches without sulfite, the sulfite being added Figure 1, b, thus provided strong of sodium hydroxide is shown in to aliquots just prior to the time when evidence for the suspected interference Figure 1, e . High concentrations of the reagent \vas to be used. by the other components of the reagent sodium hydroxide led t o enhanced under the usual conditions of the test. color development, b u t a t t h e same INTERFEREWE OF ROCHELLESALT. time contributed t o a loss of glucose. STUDY OF VARIABLES Comparative tests were next carried T h e level of 1% sodium hydroxide Removal of Dissolved Oxygen. out with the modified dinitrosalicylic appeared t o be t h e most suitable, as WITHSTREAMOF KITROGEN.Sumner acid reagent to M hich varying amounts it produced t h e maximum color in-
VOL. 31, NO. 3, MARCH 1959 * 427
tensity possible without concomitant minute period, adequate for the original ents of the dinitrosalicylic acid reagent, loss of glucose. procedure, does not produce complete and also of extraneous substances such Phenol Concentration. Maximum color development in the modified as carboxymethylcellulose or citrate color development was approached procedure. By this method linearity of buffer, upon the amount of color pro- a t a concentration of about 0.2% data, protection of glucose, and sta- duced and upon the destruction of phenol (Figure 1, f ) . I n experi- bility of color are realized. glucose, as shown in the present study, ments not shown in t h e figure, the If interfering substances occur in could similarly be interpreted as effects same results were obtained with 0.5q.’, unknown samples, special controls are upon the nature and degree of side phenol as at t h e 0.2% level. Low run. Such controls consist of standard reactions. concentrations resulted in a lack of reducing sugar solutions to n hich ap- The dinitrosalicylic acid reagent, in linearity. T h e intensity obtained in propriate amounts of the interfering a form consisting only of dinitrosalicylic t h e presence of 0.2% phenol was substances are nddrd. K h e n the in- acid dissolved in strong alkali, has been about 5 times that obtained in the ab- terfering substanctis bring about a loss used with apparent success for molecular scence of phenol. Over the range of reducing sugar, and particularly n-eight measurement of starch break- tested, the phenol had no effect on the when the amounts of reducing sugar down products (?‘). This method de- loss of glucose. to be measured in unknown samples pends upon the assumption that all Dinitrosalicylic Acid Concentration. are equal to or smaller than the amount higher oligosaccharides of the homol- When t h e amounts of dinitrosalicylic lost, known amounts of glucose are ogous series starting with maltose acid Fyere varied, t h e color intensity added to both the unknown samples would produce equivalent amounts of approached a maximum at a concen- and the standards. color with the reagent. Actual studies tration of 1% (Figure 1, 9). T h e The procedure of adding glucose can of the reactions of members of homolo- dinitrosalicylic acid, like t h e phenol, also be applied advantageously to com- gous series with the dinitrosalicylic had no effect on t h e loss of glucose pensate for the loss of reducing sugar acid reagent, starting with the disac- over t h e range tested. incurred nhen Rochelle salt is incor- charide, have not been reported, but Other Substances. T h e results of porated in the dinitrosalicylic acid rea- u-ould be of considerable interest in the preceding tests suggested the pos- gent. For example, it is convenient the present connection. sibility t h a t other substances might in the cellulase test (IO) to introduce The principal virtue of the dinitro- affect t h e dinitrosalicylic acid test. glucose into the carboxymethyl-cellu- salicylic acid test for reducing sugar For example, it was of interest t o lose-citrate substrate and to use modified lies in its great convenience compared asceitain whether, in using t h e test dinitrosalicylic acid reagent containing to most other sugar tests, particularly for the measurement of cellulase 20% Rochelle salt (8). Under these u-hen large numbers of tests must be activity (8, IO), the presence of car- conditions the separate addition of carried out. However, the factors dis- boxymethylcellulose and citrate buffer Rochelle salt to the reaction mixture cussed above must be given due con- a t p H 5 might cause interference. after color dcrelopment is omitted. sideration to avoid misinterpretation K i t h amounts of carboxymethylcellu- The controls for such tests consist of a of results. lose and citrate buffer corresponding to blank and standard glucose solutions, those used in the cellulase measure- each containing carboxymethylcellulose, LITERATURE CITED ment, the effects shown in Figure 1, h, citrate, and compensatory glucose. (1) Bell, D. J., Manners, D. J., Palmer, A,, were produced. The carboxymethyl- J . Chem. Soc. 1952, 3760. cellulose caused an enhancement in (2) Brodersen, R., Ricketts, H. T., J . DISCUSSION Lab. Clzn. Med. 34, 1447 (1949). color, whereas the citrate caused a depression. A mixture of the sub- (3) Gilman, H., “Organic Chemistry, The chemistrv of the dinitrosalicylic Advanced Trratise,” 2nd ed., Vol. 2 , stances approximately neutralized the acid test for reducing sugar has been p. 1640, Wiley, Xew York, 1943. t n o opposite effects. clarified previously, a t least in part. (4) Hostettler, F., Borel, E., Deuel, H., To determine whether the effect of The 3,5-dinitrosalicylic acid is reduced Helv. Chznz. d c t a 34, 2132 (1951). the citrate may have been a conse- (5) Kolthoff, I. PII., Lingane, J. J., to 3-amino-5-nitrosalicylic acid while, “Polarography,” Interscience, Kew quence of its buffering action, tests in the simplest instances, the aldehyde York, 1946. ere made with acetate buffer of p H 5 groups appear to be oxidized to car- (6) hlandels, G. R , Quartermaster Re a t an equivalent concentration. The boxyl groups (4). The facts, however, search and Engineering Center, Natick, acetate did not, however, affect the Mass., private communication. that the equivalence between amino- ( 7 ) bleyer, K. H., van der Wyk, A. J. A., test. nitrosalicylic acid produced and sugar Peng, C., Helv. Chznz. Acta 37, 1619 FINAL M E T H O D is not exact (4) and that different sugars (1954). yield different amounts of color ( 1 , 4 , 7), (8) XIiller, G. L., Blum, R., Glennon, W. E., Quartermaster Research and Khen pure reducing sugar solutions suggest that the chemistry of the test Engineering Center, Xatick, Mass., are involved or when any contaminants may actually hc appreciably more com- unpublished data. which may be present are knorvn not plicated. (9) Reese, E. T., Quartermaster Research to affect the color development or to Such complications could conceiv- and Engineering Center, Satick, illass., private communication. cause any loss of reducing sugar, the ably be associated with the various (10) Reese, E. T., Siu, R. G. H., Levinson, modified reagent in the absence of decomposition reactions of sugars in H. S., J . Bacterid. 59, 485 (1950). Rochelle salt is used. For stabilization alkaline solution ( 3 ) . If this explana- (11) Sumner, J. B., J . B i d . Chem. 47, of the color produced under such con- tion is correct, the reaction of the sugar 5 (1931). (12) Zbid.,62, 287 (1924-25). ditions, Rochelle salt is added to the aldehyde grouping with dinitrosalicylic (13) Ibzd., 65, 393 (1925). mixture immediately after the develop- acid could be viewed as competing with (14) Sumner. J. B.. Sisler.’ E. B.. Arch. ment of the color and before the mixture side reactions involving decomposition ‘ Bzochem. 4: 333 (1944). is cooled. The time of heating is in- of the sugar. The effects of different RECEIVED for review Sovember 4, 1957. creased to 15 minutes because the 5- concentrations of the various constitu- Accepted September 23, 1958.