Bretz, Elizabeth S.

The Ohio Journal of Science

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COPPER SOLUTIONS FOR MICROSCOPICALDETECTION OF GLUCOSE ANDFRUCTOSE*ELIZABETH S. BRETZPROBLEMA number of copper reagents have been proposed for dis-tinguishing glucose from fructose. Since the distinction dependsupon the relative rate of oxidation of the two sugars, the testswill necessarily be influenced by concentration of sugar, time,temperature, and concentration of reagent. For a micro-scopical detection of these sugars, the problem therefore is tofind the temperature and time of reaction at which thesesugars may be most readily distinguished when they occur atdifferent concentrations. Methyl fructosazone may be usedto distinguish glucose from fructose; but because the coppertests are much more sensitive for microscopical work, it seemsimportant to have more definite knowledge about their possi-bilities and limitations.REAGENTSThe copper reagents proposed by Ost, Pieraerts, and Benedict formacroscopical tests and the one proposed by Fliickiger for microscopicaltests for sugars in plant cells were employed. A brief account of thecomposition and of the results reported for these reagents follows.1. Fliickiger''s copper tartrate reagent (2, p. 272). Copper tartrateis prepared by mixing a solution of 30 gm. copper sulfate in 300 cc. hotwater with a solution of 70 gm. potassium sodium tartrate in 200 cc.hot water. The precipitate is filtered, dried in a desiccator, and keptin a brown bottle. For each separate test a few crystals of coppertartrate are placed on a slide in a drop of 20 per cent sodium hydroxide.When the copper tartrate has dissolved, a drop or small section of thematerial to be tested is added. According to Fliickiger (2, p. 272),reduction by fructose occurs immediately at room temperature and byglucose very soon with slight warming.In order to have a uniform reagent for comparative tests the pro-cedure may be modified by preparing standard solutions. If thelatter procedure is followed, the solution should be renewed daily, asautoreduction slowly occurs in the solution. Since autoreduction'increases with an increase in concentration of copper tartrate in thereagent, the amount of copper tartrate present should not greatlyexceed the amount necessary to oxidize the sugar in the test object."Papers from the Department of Botany, The Ohio State University, No. 389.84No. 2 DETECTION OF GLUCOSE AND FRUCTOSE 85Lampe (6, p. 347), by using a "sky-blue" solution, obtained themaximum amount of cuprous oxide with fructose in 7 minutes at roomtemperature and with glucose in 3 minutes at 45° C. The solutionsused in the present tests were 0.05 gm. copper tartrate in 10 cc. of 20per cent sodium hydroxide and 0.05 gm. copper tartrate in 20 cc. of 20per cent sodium hydroxide.2. Ost's copper carbonate reagent (7, p. 840, footnote 2). To 700 cc.boiling water 250 gm. potassium carbonate is gradually added. Tothat solution 100 gm. potassium bicarbonate is slowly added until itcompletely dissolves. A solution of 17.5 gm. copper sulfate in 100 to150 cc. water is then added under vigorous agitation. After cooling,the solution is made up to 1000 cc. and filtered. Klein (4, p. 779)says this reagent reduces with gentle warming only ketoses. Zerbanand Sattler (8, p. 307) made macrochemical tests using Nijns' modifica-tion of the above solution. This contained 15 gm. copper sulfateinstead of the 17.5 gm. used by Ost. They placed 20 cc. sugar solution—containing not over 0.3 per cent fructose—in 50 cc. of the copper reagentpreviously warmed in a small Erlenmeyer flask to 48.5° C. to 49° C.After 2.5 hours at that temperature the cuprous oxide obtained wasweighed. Zerban and Sattler (8, p. 308) found that with about equalquantities of glucose and fructose, 1 mg. of glucose reduces about one-thirteenth the amount of copper reduced by 1 mg. of fructose. Thereducing effect per mg. of glucose decreases as the concentration ofglucose increases, and also as the concentration of fructose in themixture increases.3. Pieraerts' modification of Ost's reagent (7, p. 840). The solutionis prepared in the same way as Ost's reagent, but the amounts of thesalts are changed to 140 gm. potassium carbonate, 100 gm. potassiumbicarbonate, and 15 gm. copper sulfate. The reagent deposits basiccopper carbonate after 24 hours. It must be renewed weekly (7,p. 851). Tests made by Pieraerts in the cold (13° C. to 16° C.) wereexamined every 15 minutes during the first 6 hours, thereafter on theninth, twelfth, and twenty-fourth hours (7, p. 837). According toPieraerts, in the cold glucose does not reduce the reagent in 24 hours,while 4 to 5 per cent fructose reduces it in 1 hour, 3 per cent in 1.5hours, 2 per cent in 2 hours, and 1 per cent in 2.25 hours. Pentosescause reduction in 4 hours in the cold (7, p. 842). Tests made byPieraerts on a water bath were examined as reduction became evident.At the end of 1 hour the tests were concluded (7, p. 837). He statesthat glucose gives no precipitate of cuprous oxide at 30° C, 35° C,40° C, or 45° C. Above 5.0° C. there is reduction (7, p. 842). At30° C. 1 to 5 per cent fructose reduces the reagent by the end of 1 hour.Pentoses cause no reduction at this temperature in 1 hour, but at 35° C.they do reduce the reagent in 1 hour (7, p. 843). Pieraerts summarizes(7, pp. 846-847) the use of his modification of Ost's solution by sayingthat it can distinguish 1 to 5 per cent free fructose in the presence ofother natural sugars, pentoses excepted, by heating 1 hour at 35° C.If pentoses are present, it is still possible to identify free fructose in2.5 hours in the cold, or in 1 hour at 30° C. One is surprised, then,to read Pieraerts' statement (7, p. 847) that the diagnosis of fructose is86 ELIZABETH S. BRETZ Vol. XXXVIIIimpossible if less than 20 per cent of that hexose is present, especiallyif pentoses are also in the mixture.4. Benedict's copper citrate reagent (1, p. 486). A solution is madeof 173 gm. sodium citrate and 100 gm. anhydrous sodium carbonate byheating in 600 cc. water. This is filtered if necessary and made up to850 cc. A solution of 17.3 gm. copper sulfate in 100 cc. water is dilutedto 150 cc. The copper sulfate solution is poured slowly with constantstirring into the carbonate-citrate solution. The mixture is ready foruse and does not deteriorate on standing. It is not caustic and maybe kept in cork or glass stoppered bottles. Upon reduction the solutionis apt to yield green or yellow hydrated cuprous oxide, rather than thered anhydrous oxide.Kolthoff (5, p. 888), working with 1 cc. sugar solution in 5 cc. ofBenedict's reagent, could distinguish 1 per cent fructose from glucoseand lactose after heating 0.5 hour at 37° C. to 40° C, but with 0.5 percent the distinction was doubtful. At room temperature for 24 hours0.5 per cent fructose was distinctly evident, 0.2 per cent gave a veryslight reaction, and 0.1 per cent gave no apparent test. According toKlein (4, p. 780), copper solutions containing citric or tartaric acid areextremely photosensitive. Before beginning a test it should be deter-mined whether autoreduction has occurred.5. Pieraerts' copper-glycocol reagent (7, p. 841). To a solutionof 12 gm. glycocol in hot water one adds little by little 6 gm. freshlyprepared cupric hydroxide, which dissolves almost completely after15 minutes on a steam bath. The solution, cooled to 60° C, is addedto 50 gm. potassium carbonate, made up of 1000 cc, and filtered. Theliquid keeps very well. If the potassium carbonate is added at atemperature higher than 60° C. there will be a precipitate of carbonicanhydride, which greatly diminishes the sensitivity of the reaction.In the preparation of cupric hydroxide for the above reagent certainprecautions are necessary to prevent its immediate oxidation. Add5 per cent of ammonium chloride to 5 per cent copper sulfate solution.Pour this into 20 per cent sodium hydroxide. Wash the precipitate on aBuchner funnel with cold water. Keep the funnel covered to preventoxidation. Drying the precipitate in an oven will hasten oxidation.It is best to dry the precipitate on a suction filter, weigh, and use itimmediately.Pieraerts (7, p. 845) says that free fructose forms an abundantprecipitate of cuprous oxide in the cold after 24 hours and is the onlynatural sugar which does so. The results are uncertain with heat.One cannot trust the indications at 30° C. for an hour, if more than 3per cent fructose is present.METHODSThe concentrations of glucose and fructose used were 0.1 per cent,0.5 per cent, 1 per cent, 5 per cent, and 10 per cent. The threetemperatures used were room temperature, 40° C., and that of a steambath. Slides at room temperature were kept in a moist chamber toprevent drying. One drop of sugar solution was thoroughly mixedwith two drops of copper reagent on a microscopic slide.No. 2 DETECTION OF GLUCOSE AND FRUCTOSE 87Accurate counts were made of the cuprous oxide crystals obtainedin each test. A description of the method of counting is not includedin this paper, since the objective is chiefly to indicate with whichreagents differentiation between glucose and fructose can be obtained.In Fliickiger's reagent it was not known what proportions of coppertartrate and sodium hydroxide would give the best results. Twosolutions were tried, 0.05 gm. copper tartrate in 10 cc. of 20 per centsodium hydroxide solution, and 0.05 gm. copper tartrate in 20 cc. of20 per cent sodium hydroxide solution. The first solution did not givedifferentiation between 10 per cent glucose and fructose. The solutionof 0.05 gm. in 20 cc. gave differentiation at all concentrations at roomtemperature.DISCUSSIONThe following paragraphs point out discrepancies in theresults of the present microscopic tests and the results of themacroscopic tests mentioned in the section on reagents.Ost's copper carbonate reagent was not found to reduce"only ketoses" with gentle warming, as reported by Klein,although at 40° C. there was good differentiation between thesugars at all concentrations.Pieraerts' modification of Ost's reagent was found to bereduced by glucose to some extent at room temperature andat 40° C, contrary to the macroscopical observations ofPieraerts. It should be noted, however, that Pieraerts' roomtemperature was 13° C. to 16° C, while that of the presenttests was about 25° C.Benedict's copper citrate reagent gave differentiation betweenglucose and fructose at 0.1 per cent and 0.5 per cent at roomtemperature and at 40° C. when used in microscopical mounts,whereas Kolthoff, working macroscopically, failed to finddifferentiation at these low concentrations, except in the caseof 0.5 per cent at room temperature.Pieraerts' copper-glycocol reagent, recommended by Pier-aerts for use at room temperature in distinguishing fructosefrom glucose, failed to do so in the present tests at 0.5 per centand at 1 per cent. Even more cuprous oxide was reducedby glucose than by fructose in some cases. Differences in thesize of the crystals obtained made it difficult to compare theamount of precipitate. 'SUMMARY OF RESULTS1. Five copper reagents were tested at the temperatureof a steam bath, at 40° C, and at room temperature as totheir use in distinguishing between 0.1 per cent, 0.5 per cent,ELIZABETH S. BRETZ Vol. XXXVIII1 per cent, 5 per cent, and 10 per cent glucose and fructose inmicroscopical mounts.2. None of the reagents gave positive tests only for fructose.Positive tests for glucose varied from zero to a copious precipi-tate, depending upon the temperature and concentration ofthe sugar.3. Each of the reagents may be used for distinguishingglucose from fructose under certain conditions of temperatureand concentration of the sugars.4. All except Pieraerts' copper-glycocol reagent gave differ-entiation between glucose and fructose at room temperatureat all concentrations. None of the reagents gave differentiationwith steam at all concentrations. Fltickiger's copper tartratereagent and Pieraerts' copper-glycocol reagent failed to givedifferentiation at 40° C. at all concentrations.5. Table I indicates the time interval in relation to differenttemperatures at which the reagents are useful in distinguishingbetween glucose and fructose at different concentrations,TABLE IDIFFERENTIATION BETWEEN GLUCOSE AND FRUCTOSEREAGENT AND TEMPERATUREFluckiger's Room TempOst's Copper CarbonateSteam bath40° CRoom TempPieraerts' Modification of Ost's.Steam bath40° CRoom Temp ,Benedict's Copper Citrate.40° CRoom TempPieraerts' Copper-Glycocol.40° CRoom TempTIME IN MINUTES AT WHICH DIFFERENTIATIONAPPEARSCONCENTRATION OF SUGAR SOLUTION0.1 percent15-60..*K-2..30-60120H-l . .30-60..30-120..10-60..60..1440..0.5 percent4-15H-2..30-60. 1440..H15-60..15-120..5-60..60..601 percent4-60..H-2,.15-60120-1440..10-60..15-120..5-60..60..30-605 percent4-60..5-1440..60-1440..5-60..15-120..5-60..60..10-606010 percent4-60..5-1440..60-1440..2-60..7-120..5-60..30-60..10-6010-60*Two periods („) after a figure indicate that no tests were made for a longertime interval. Where there are no periods the last figure given is the longest timeinterval in which there was good differentiation, as shown by further tests.88No. 2 DETECTION OF GLUCOSE AND FRUCTOSE 89The writer wishes to express her appreciation to Dr. H. C.Sampson, of the Department of Botany of The Ohio StateUniversity, for his assistance in the direction of the experimentalwork and in the preparation of this paper.LITERATURE CITED(1) Benedict, Stanley R. A Reagent for the Detection of Reducing Sugars. Journalof Biological Chemistry, V: 485-487. 1908709.(2) Fliickiger, F. A. and Tschitch, A. The Principles of Pharmacognosy. Power,F. B., tr. New York: William Wood and Co. 1887.(3) Haar, A. W. van der. Anleitung zum Nachweis, zur Trennung und Bestimmungder reinen und aus Glukosiden usw. erhaltenen Monosaccharide und Alde-hydsauren. Berlin: Gebruder Borntraeger. 1920.(4) Klein, G. Handbuch der Pfianzenanalyse II. Berlin: Julius Springer. 1932.(5) Kolthoff, I. M. De reduceerende werking der suikers. Chemisch WeekbladXIII: 887-895. 1916.(6) Lampe, L. A Microchemical and Morphological Study of the DevelopingEndosperm of Maize. Botanical Gazette, XCI: 337-376. 1931.(7) Pieraerts, M. J. Diagnose du d-fructose en presence d'autres sucres naturels.Bulletin de l'Association des Chimistes de Sucrerie et de Distillerie de Franceet des Colonies. July, 1907-June, 1908: 830-848.(8) Zerban, B. W. and Sattler, Louis. Effect of Dextrose and Sucrose on theDetermination of Levulose by Nijns' Method. Industrial and EngineeringChemistry, Analytical Edition II: 307-309. 1930.

Copper Solutions for Microscopical Detection of Glucose and Fructose

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Copper Solutions for Microscopical Detection of Glucose and Fructose

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