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-
(J
c
0
p(Ag*i
I
I
I
!
1
2
3
4
Figure 1.
5
6
I
I
I
7
8
9
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
I
ANALYTICAL CHEMISTRY
220 The following procedure was used to deterniinc the extent of adsorption onto a single 5 0 4 . Kirk pipet. The pipet was cleaned with concentrated nitric acid, followed by several rinses with distilled nTater. It was then filled with 1 ill potassium thiocyanate and again rinsed three times with distilled water. The pipet was then filled with a particular solution of silver and the experiment was completed in one of the following ways : The total activity in a given aliquot was determined by counting one pipetful plus three washes with 1 M potassium thiocyanate by means of an end-window counter, using the technique of Freedman and Hume ( 2 )as modified by Goddu and Rogers ( 3 ) . The losses due to adsorption were determined after expelling and discarding the solution together with three washings of distilled water by combining and counting three washes with 1 -21 potassium thiocyanate. Five replicates were taken for each point, a t least four of which were usually suitable for counting. The ratios betyeen the counts for the thiocyanate washings and total number of counts obtained from the active solution were taken as the measure ot' the amount of adsorption which had occurred. The results are shown in Figwe 1. One may judge the reli:tbilit!, of each point from the data for the .I1 solution 01' silver. The total activity in 50 PI. was found to be 7727 counts per minute \vith a coefficient of variation of 2.8y0. The activity of the bhiocyanate washings \vas 129 counts per minute with a coefficient of variation of l.OyG, The percentage of adsorbed silver removed by the thiocyanate \vas therefore 1.67y0. Eve11
in the case of the 1 M solution of silver, where the fractioii ot silver adsorbed was very small, the coefficient of variation lor the washings was only 14%. Whether or iiot there is a plateau a t very low concentrations of silver ion is not certain, inasmuch as the concentration in the "trace" solution ohtained by the electroseparation is unknown. If the concentration of silver in the trace solution was as high as 10-6 ?.I, the values for the two solutions serve as an excellent check upon each other. The results obtained in this work indicate that the volunietric error due to adsorption can be important in handling solutiolis that are only moderately dilute. A similar study with an ordinary 1-ml. pipet having nearly the same surface-to-volume ratio as the mici,opipet used in the above study gave values which agreed, n-ithin the limits with which the ratio could be determined, rvith those given above. L r r E m w m CITED , I 1 Conway, E. J., "blicrodiffusion Analysis and Volunictric Error." London, Crosby Lockwood and Son, 1947. (2) Freedman, -1.J., andHume, D. N.,Science, 112,461 (19501. (3) Goddu, R. F., and Rogers, L. B., Ibid., 114,99 (1951). ( 4 ) Griess, J. C.. J r . . and Rogers. L. B . , J . Electrochorn. Soc., 95, 129 (1949). ( 5 ) Hensley, J . J\-., Long. -1.O . , and JVillard, J. E., I n d . Eng. Chem., 41,1415 (1949). (6) Schoonover. I. C..J Researcii Satl. Bur. Standards. 15, 377 (1935). KEC'L'IVED
\I.&Y
18. 1 Y S l
Determination of 1,1,1-Trichloro -2,2-bis( p-methoxyphenyl)ethane in Milk and Fatty Materials H. k . CLABORN U D HEHlIAN F. BECKMAN Bureau of Entomology and Plant Quarantine, U . C. Department of Agriculture, Kerrcille, Tex.
'HE proposed use of methoxychlor [l,l,l-trichloro-2,%bis(pmethoxyphenyl) ethane] for the control of livestock pests ( I , 6 ) made further studies necessary to determine more accurately than has previously been possible the amount of methoxychlor deposited in fat and excreted in the milk of animals treated for livestock pest control. The methods for determining methoxychlor which were previously available were n o t satisfactory. The Fairing niethod (3), as published, cannot be used satisfact'orilp for the determination of methoxychlor in fats, but if modified by introducing the Laug-noble step wherein the dehydrohalogenated i,esidue is taken up in Skellysolve and passed through a column containing a 50-50 mixture of heavy magnesia and Celite to effect c*hromatographicseparation prior to the color development treatu e n t with 85% sulfuric acid, very little interfewncc will l x i.ncountered because of unsaponified fat residuw. Prickett and coworkers ( 7 ) have descritied a method that is comprised of three stepa: saponification and removal of fat, nitration of the tloii~-drocliloii~iated derivative of methoxvchlor \rit,h a 1 t,o 1 mixture of fuming nitric and sulfuric acids at a teinperaturc helow 100" C',, and measurement of the red color produced when the nitrated product is treated with sodium inet,hylate. The disadvantage of this method is that a yellow color produced by interfering substances in fatty tissue and butterfat is not constant and therefore interferes with the determination of small amounts of methoxychlor. Both methods make use of saponification for the removal of the fat and a3 methoxychlor is readily dehydrochlorinated, it is separated as its ethylene derivative. The method described by Schechter el al. (8) for separating D D T from tats by extracting a chloroform solution of the fat (>on-
taining the insect,icide with a sulfuric acid-funiiiig sulfuric acid mixture could not be used because methoxychlor is destroyed by the strong sulfuric acid. Harris (5) has suggested that methoxychlor could be separated from fat by dissolving the fat containing the insecticide in n-hexane and extracting successively with nitromethane. The authors found this methodsatisfactory for separating t,he insecticide completely from the fat). The nitromethane extracts the methoxychlor and leaves the fat in the hexane. .%II added advantage of this method is that the insecticide is separated as unchanged niethoxychlor and is not dehydrochlorinated. A mlor reaction suitable for measuring methoxychlor without dehydrochlorination has not been described. The color reactioii used by Schechter et al. (8) for the colorimetric measurement of DDT could not be used, because the nitrating 1 to 1 mixtureof iuniing nitric-sulfuric acid destroyed the niet,hoxychlor when heated to 100' C. From experience in trying to separate niethoxychlor from fat the authors had learned that the methoxychlor \vas destroyed by the strong sulfuric acid mixture. Therefore, they suspected t,hat the dest,ruction by the nitrating mixture was due to the action of the sulfuric acid. When fuming nitric acid \va6 used alone, the nitration prooeeded without apparent decomposition at 100" C. The reaction was complete in 15 minutes, but heating for 1 hour caused no decomposition of the nitrated product. The nit.rated product gave the characteristic blue color when treated with sodium methylate. The blue color is not distinguishable from that produced bi the tetranitro derivativeR of p,p'-DDT and TDE [l,l-dichloro-2,2-bis(p-chloropheny1)ethane 1 (8). However, the niethod of nitration used makes the method specific for methoxychlor, as nitration with fuming nitric acid does not produce the tetranitro derivatives of
