Determination of Sugars in Plant Materials Use of Decolorizing Carbon in the Ferricyonide Method AKTHUK BEVENUE, Western Regional Research Laboratory, Albany, Calif. Experimental data are presented on the sugar adsorption propertiesof some commercially available charcoals, as they may be used in the clarification process of plant extracts for quantitative sugar analyses. Percentage of sugar adsorption by charcoals varies with different sugars, type of carbon, and concentrations of sugar and carbon.
I
PROCEUC R E
K T H E preparation of some plant extracts for sugar analysis,
clarification is necessary for the removal of nonsugar-reducing substances. In many cases, the use of neutral lead acetate, and removal of excess lead with disodium phosphate, will not wholly accomplish this purpose. Unless the extract is water-clear, the organic coloring matter present may be oxidized, giving high sugar values. Hassid (3) using Carboraffin, a charcoal of European origin, and Forsee (g), using the charcoal Norite, obtained 100% recovery of dextrose but presented no data on the recovery of levulose or sucrose from carbon-treated pure sugar solutions. Hiscox ( 5 ) stated that charcoal treatment lowered the reducing-sugar content of silage and root crops but gave no data. Lott ( 6 ) and Morris and Wesp (‘7) treated plant extracts with Baker & Adanison Code 1551 decolorizing carbon and found no measurable loss of dextrose. In the course of experiments on methods for the determination of sugars in plant materials, it was necessary to find the sugaradsorptive properties of some commercially available decolorizing carbons. Solutions of dextrose, sucrose (both obtained from the National Bureau of Standards), and levulose (Eastman Kodak Company) were used separately with and without the lead clarification process. The selection of the carbons was based primarily upon a study of the origin and the properties of decolorizing carbons as outlined by Dritz (1).
Twenty-five milliliters of a dextrose solution, containing 0.2 gram of dextrose, were pipetted into a 100-ml. volumetric flask, 50 ml. of distilled water were added to dilute the sugar solution, and 0.5 gram of decolorizing carbon was added. The volume was made to 100 ml. with distilled water, the contents of the flask were mixed thoroughly, and the mixture was allowed to stand a t room temperature for 10 minutes with occasional shaking. (Prevlous experiments indicated that adsorption was no greater if time of standing was 30 or 60 minutes.) The mixture was filtered through a fluted filter paper of analytiral quality. After the first 25 ml. of filtrate were discarded, 25 ml. of the filtrate were diluted to 100 ml. and used for analysis. The same procedure was followed with levulose solutions. Sucrose solutions (0.1 gram per 100 ml.) were treated with carbon as described for dextrose and levulose. After the carbon treatment, the solutions were adjusted to pH 4.8 to 5.1 with a 10% solution of acetic acid. Invertase was added and hydrolysis was allowed to take place a t 25’ C. overnight. Similar experiments were conducted in which the amount of carbon was varied. I n another series, the sugar concentration was varied. The sugar analyses !?ere determined by the Hassid method (4), and the values given in the tables are the averages of several determinations, with a maximum variation of *2?7~. RESULTS
RBAGESTS
Saturated solution of neutral lead acetate. Saturated solution of disodium phosphate. Ceric sulfate, alkaline potassium ferricyanide, and setopaline C indicator solutions prepared as directed by Hassid (3). Dextrose, National Bureau of Standards sample 41. Sucrose, National Bureau of Standards sample 17. Levulose (Eastman Kodak Company). Acetic acid, 10% solution. Invertase. Carbons as listed in Table I.
The data in Table I show that there was no adsorption of dextrose or levulose by ten of the twelve decolorizing carbons studied. The five animal charcoals did not adsorb sucrose. However, sucrose recovery from the other charcoals varied from 43 to 87%.
To simulate conditions of actual analysis, neutral lead acetate was added to dextrose, levulose, and sucrose solutions; the lead was removed with a slight excess of disodium phosphate and the solutions were filtered. The filtrates were treated wlth carbon under the conditions of procedure described above. Six of the twelve carbons described in Table I were used in this experimentthr three Darco varieties, Baker & Adamson Code 1551, animal charcoals 325-mesh and KO,2. The absorption of sugars by the carbons from these filtrates was the ~ _ _ _ _ _ ~ ~ _ _ same, within the limits of error (*27,), as from t,he distilled-water solut,ions. Table I. Recovery of Dextrose and Levulose from 0.29’0 Solutions and of Sucrose from a 0.1% Solution In the experiments in \vhich the carbon con(After treatment of 100 ml. of .sugar aolution with 500 mn. of decolorizing carbon) D ~~ ~ ~ ~s ,~ ~~ ~l ~cent,ratiorl ~ ~ ~~ ~was ~~ , varied ~ (Table ~, 11), ~ Baker , & ;idamCarbon % 55 % son Code 1551 carbon adsorbed no dextrose or Norit SG (American S o r i t Co., Jacksonville, Fla.) 93.6 levulose a t the lower carbon concentrations. 9 2 ., 0 43 1 4 ,. 21 Norit A (Pfanstiehl Chemical Co.. Waukegan, Ill.) 94.0 97.2 71.1 However, a t the higher carbon concentrations a Nuchar (Eastman Kodak Co., Rochester, N . Y.) 99.6 100.8 80.4 Darco G 6 0 iDarco Corp., New York, N. Y.) 99.2 slight adsorption of these two sugars was indicated. 98.3 Darco SSl(Darco Corp S e w York, ;V T.) 102.0 82.6 Darco KB (Darco Corp” Yew York \.‘Y.) 99.1 75.4 The adsorption of sucrose increased with added 99.2 Baker & Adamson Co& 15.51 (G&eral Chemical Co., Xew York, iY.Y.) 100.0 100.2 86.6 increments of B. & A . Code 1551 carbon. Animal 100.8 99.8 Animal charcoal 8/2r-mesh} (Conpolidated, Chenlical Indua- 1 0 0 . 4 charcoal, 325-niesh, did not, adsorb dextrose, 100.8 98.8 Animal charcoal: 325-mesh tries, Inc.. San Francisco, Calif.) 101 . 0 Animal charcoal, S o . 1 (American Agricultural Chemical levulose, nor sucrose a t the carbon concentrations Co., Detroit, bIich.) 101.2 loa, studied. Animal charcoal, No. 2 (American .Igricultural Chemical Co. Detroit l l i c h . ) 1 0 1 . 8 101.2 99.6 When the sugar concentration was varied (Table 101.6 102.0 Bone Alack. ~i (Baugh & Sons Cu., PhiladellJilia, Pa.) 101.2 111), none of the carbons tested adsorbed a mrasur-
586’
587
V O L U M E 21, NO. 5, M A Y 1 9 4 9 Table 11. Recovery of Dextrose and Levulose from 0.2qc Solutions and of Sucrose from 0.1% Solution (After treatment of 100 ml. of sugar solution with varied amounts of decolorizing carbon) car- B. & A. Code 1.551 Carbon Animal Charcoal, 325-Mesh bon, Dextrose. Levulose, Sucrose, Dextrose, Levulose Sucrose. r-/r hlg. % 7c % % % 100 99.6 98.5 98.3 100.8 99.0 100.8 200 99.6 99.3 96.0 100.1 100.0 100.8 300 99.6 98.5 92.8 98.2 100.0 100.0 500 98.8 98.4 88.8 100.0 99.0 100.3 700 98.4 97.3 84.4 100.8 98.0 99.6 1000 96.4 97.3 77.6 100.4 98.4 98,4
Table I l l . Recovery of Dextrose, Le\-ulose, and .Suerow from Solutions of Varied Sugar Concentration (After treatment of 100 nil. of sugar solution x\-ith 500 nip. of decnlorizinE carbon) B. & A . Code 1551 Carbon ..\nirnal Charcoal, 323-Xlesh Sugar., Dextrose, Leviilose, Sucrose. Dextrose, Levulose. Sucrose,
53
7c
5%
$4
%
5%
C‘
0.05
98.4
9G.O
100.0
99.6
98.8
98.9
80.0 88.0 92.4 93.2 95.6 97.2
98.8
100:!4
l00:O 99.6
68:l 98.6
96.0 98.4 98.4 99.2 98.4 48 8
0.10 0.20
0.30 0.50 1.00
,..
98.8 98.8
...
98.2 99.5
...
selected,for the decolorization of plant extracts for sugar analysis. Sugar concentration and the quantity of carbon must also be taken into consideration. As solutions of dextrose and levulose can be treated with selected carbons without appreciable loss, it is advisable to carry out hydrolysis after lead clarification but prior t o treatment Tyith carbon and thereby avoid the adsorption of sucrose. SUMMARY
The percentages of recovery of dextrose, levulose, and sucrose from ivater solutions after treatment Kith commercially available decolorizing carbons have been determined. Animal charcoals did not adsorb any measurable quantity of the three sugars, except sucrose a t the lowest &gar concentration studied. Sucrose recovery from the other carbons varied from 43 tu 87%. Within certain useful limits of carbon and sugar concentrations, ten of the tqelve carbons studied did not absorb levulose or de\trose. Sugar solutions were also treated with neutral lead acetate and dibasic sodium phosphate prior to the carbon treatment, and it was found that the adsorption of the sugars by the carbons was the same as from the water solutions. LITERATURE CITED
(1) Deitz, 1’. R., “Biblioglaphy of Solid Adsorbents,” p. lxxi, U. 5.
able quantity of dextrose. Only a t the lowest sugar concentration was there evidence of levulose adsorption by B. & A. Code 1551 carbon. The adsorption of sucrose by this carbon was appreciable a t all concentrations. Animal charcoal, 325-mesh, adsorbed 110sucrose except a t the lowest sugar concentration studied. The data show that i t is necessary to determine the adsorption of sucrose as well as dextrose and levulose when a carbon is
Cane Sugar Refiners and Bone Char Mfrs. and National Bureau of Standards, Washington, D. C., 1944. (2) Forsee, W.T.,Jr., IND. EXGCHEM., ANAL.ED.,10, 411 (1938). (3) Hassid, W. Z., Ibid., 8, 138 (1936). (4) Ibid., 9,228 (1937).
( 5 ) Hiscox, D. J., Can. Chem. Process Ind., 26,496 (1942). (6) Lott, R. V., Proc. Am. SOC.Hort. Sci., 46, 149 (1945). (7) Morris, V. H., and Wesp, E. F., Plant Phusiol., 7, 47 (1932).
RECEIVED Jul) 20, 1948.
STUDIES ON RESINS Specijic Color Reaction f o r Dehydroabietic Acid and Its Application i n Analysis of Technical Resins WlLHELJI SANDERJIANN Zentralinstitut f u r Forst-und Holzwirtrchaft, Hamburg-Reinbek, G e r m a n y (British Zone)
This paper describes a new specific intensive blue-violet color reaction which has been used to detect dehydroabietic acid in various resin products. A resin containing dehydroabietic acid is sulfonated in the cold, and after standing 12 hours at room temperature is neutralized with concentrated sodium hydroxide or potassium hydroxide. A blue-violet color appears; the intensity varies with the amount of dehydroabietic acid in the sample.
T
HE simplest test for resinic acids is the color reactiou with acetic anhydride and sulfuric acid according to Storch and
Morawski ( 1 , 3 ) . However, this reaction is not specific because other compounds such as terpenoids give the same color. The Halphen-Grimaldi reaction with phenol and bromine ( 4 )is knoiyn to have the same disadvantages. Both of t,heseidentification tests show good results with I-pimaric acid, abietic acid, and proabietic acid, but not with hydrogenated resinic acids, dehydroabietic acid, d-pimaric acid, and the compound of I-pimaric acid with maleic anhydride. 4 new specific color reaction for dehydroabietic acid has bren found. PROCEDURE
In an ice-cooled test tube 0.05 to 0.1 gram of fine powdered resin is added in small portions to 2 ml. of sulfuric acid (density
1.84). After standing for about 12 hours a t room temperature the test tube is placed in a test tube clamp, and 3 ml. of water are added. Then slowly with a pipet a 50% solution of sodium hydroxide in water is added until the mixture shows an alkaline reaction. The reaction is extremely violent and every precaution must be taken. If the sample contains dehydroabietic acid a blueviolet color appears, which is stable for days and often for weeks. If the sample is acidified the color disappears but will reappear to a lesser degree if sodium hydroxide is added. If ammonia or amines are substituted for alkali hydroxide no color is produced, nor does any color appear if the neutralization with sodium hydroxide takes place in the cold.
.Spparently a sulfonic acid is transformed into a phenolic compound under the influence of heat and concentrated alkali hydroxide. If the pure sulfonic acid I1 is neutralized with concentrated sodium hydroxide there is no color reaction, but a very intensive one if I1 is first treated with sulfuric acid in the same
