Improvement in Anthrone Method for Determination of Carbohydrates u3 :I qualitative ( 2 ) and quantitative tietermination of' carbo1iydr;iteh has beer1 considerably iinpet1t.d by the darkening n-it11time oi the anthro~ie reagent, iv1iic.h is usually 0.1 to 0.2% anthrone in 'JEi70sulfuric acid (21. The tIar:;ening of the reagent has been shown to be iiiversely related 10 the purity of the sulfuric acid employed. \Vheti thcs reqyiit j, used quantitatively, this darkening pheiionienoii creates :L iwed either fur careful staridarization with wspect to the age oi the reagent used or for preparation of a i l e \ ~ s t a n d u d curve iur td:ich set of carbohydrate deterniinations. .I Iiioililicatioii of lforris' method (4) has bee11 devised which :ivuids tlie necessity of dissolving the anthrone in sulfuric acid prior to the determination, thus eliininating color variations :irking ironi this sourcc~and :illowing a single standardieatioii curve Lo suficc .
h plot oi the optical deiisitj. a g a i i ~ tweiglit of glucuse in the siiiiple gives a a~ruiglit-liiierelationship froni 0 to 80 micrograms. 'The acetic acid arid ethyl alcohol produced by the hydrolysis oi the ethyl acetate do riot interfere v i t h the reaction in any way. 'The ethyl acetate solutiori of anthrone will keep several R-eeks when stored in :in umber glass-stoppered bottle. Thus, the troublesonie developinent of color in the sulfuric wid-nnthronc rragent eniployed in ~ a r l i e rprocedures is avoided. Using the values reported by Morris (4)as conversion factors, :I single standard curve prepared from glucose uill suffice for the quantitative determination of a11 carbolij.drates listed. 13). deteriiiining the conversion factors, inuny other hexoses and hesoseyielding conipounds, including those listed by Dreywood ( 1 ), Viles and Silverman ( 5 ) , and Ko~valdand r\IcC:orinack (3),cn11 be quantitatively estiniated in terms of glucose.
To 2 nil. of tlie carboliydwte solutiori (containing ail amount of carbohydr:tte that will give a color intensity in the rauge given by 0 and 80 microgranis of glucose) in a 19 X 150 111111. tcst tube is added 0.5 nil. of a solution of 2% anthrone (recryst:illized from benzene nnd light, petroleum et'her) in reagent grade or e.?. ethyl :tcetate. Then 5 nil. of concentrated sulfuric acid are carefully layered into the tube. The tube is gently swirled until the ethyl scetate has hydrolyzed, as indicated by the floc of anthrone ivhich appe:trs. More rapid swirling then thorou hly mises the contents of the tube and dissolves the anthrone. t h e developed color may be read after 10 minutes a t 620 me in a spectrophotometer against distilled water and corrected for the absorption of a blank containing onlv the reagents and water.
Errors in Volumetric Analysis Arising from Adsorption yOS\VAY ( 1 ) in his study of errors in voluinelric andysis made no inention of adsorption as a possible source of crro1'. However, the import:ince of adsorption in one nnalytical method WLS strikingly denioustrated by Schoonover's (6) studies of :I colorimetric niethotl Cor the determination of silver. Further-
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Figure 1.
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Adsorption on Kirk Pipet
niorc, r:idioclieniists have long liceii aivare of the fact that C U I I siderable losses may occur hen very dilute solutio~isare stored i n glass contaiuers. .Igeneral study of the adsorption of certaiil ions onto soft gl:iss, borosilicate gl , and iiietallic surfaces, carried out by Hensley, Long, and \Villard (5), iiidicatcd that it \vas necessary, for ivork IJeing done in this laboratory, to deterniine the mignitude of the losses to be expected froni adsorption iii pipeting very dilute solutions of silver perchlorate in 0.1 .Y perchloric acid. In view of the sniall aiiiount of silver involved, and because radioactivity offered a sensitive and convenient method of measurement, radioactive Xg111 obtained by neutron irradiation of palladiuni in the Oak Ridge pile was used for this study. Carrieri'ree Ag111 of high specific activity was recovered froni the palladium by electroseparatioii froni cyanide solution according to the inethotl of Griess and Rogers ( d ) , following which the silver activitj. \vas dissolved b>, electrolysis into a few niilliliters of 0.1 -11 percliloric acid for use in tlie adsorption studies. The concentration of silver in thc resulting solution could be calculated lis less than 1 0 - b J I (C), but as especially purified reagents were not used, the concentration was probably greater. Standard solutions containing higher concentratioiis of silver were prepared by adding portions of this trace1 solutiori to kno\\n aniounts of 2.00 Jf silver perchlorate o r dilutions thereof and bringing to volunit: \vith 0.1 III perchloric acid. Solutions \rere .stord in glassstoppered bottles coated with ceresine \vas i n orclei, to niininiize changes in concentratioiis \\-it11tirne. 219
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