hydrocarbon of such a structural configuration that it could not enter the pores of the adsorbent. The hydrocarbon, iso-octane, used as an eluent in this investigation, can enter the pores of both the 1OX and 13X adsorbents. Its use is possible with aromatic hydrocarbons because the latter are very strongly adsorbed and can enter the pores even in the presence of iso-octane. For the separation of mixtures of branched paraffins and cycloparaffins. the separation obtained with isooctane as the eluent is poor. In this case competition between the molecules of the original charge and those of
the eluent for the pores of the adsorbent is keen and the process is not effective. As an eluent for the separation of mixtures of branched paraffins or cycloparaffins with Molecular Sieve adsorbent lox, 1,3,5-triethylcyclohexanemay prove satisfactory. It can be removed from the components of any higher boiling charge by azeotropic distillation with diethylene glycol monomethyl ether (methyl carbitol). LITERATURE CITED
(1) Barrer, R. M., J . SOC. Chem. Znd.
(London) 64, 133 (1945).
( 2 ) Barrer, R. M., Belchetz, L., J. Soc. Chem. Ind. (London) 64, 131
(1945).
(3) Barrer, R. M., Ibbitson, D. A., Trans. Faraday SOC.40, 195 (1944). (4) Barrer, R. M., Riley, D. W., J . Chem. Sac., 1948, 133. (5) Mair, B. J., Marculaitis W. J., Rossini, F. D., ANAL. HEM. 29,
92 (1937).
(6) Nelson, K. H., Grimes, hl. D., Heinrich, B. J., Ibid., 29, 1026
(1957). (7) Schwartz, R. D., Brasseaux, D. J., Ibid., 29, 1022 (1957). RECEIVEDfor reviem June Accepted October 18, 1957. tion performed under the Petroleum Institute Research
10, 1957.
Investigahmerican Project 6.
Determination of Dihydroquercetin in Douglas Fir and Western Larch Wood G. M. BARTON and J. A. F. GARDNER Vancouver laboratory, Forest Products laboratories of Canada, University o f British Columbia, Vancouver, British Columbia
b A rapid colorimetric method suitable for the routine determination of dihydroquercetin (3,3',4',5,7-pentahydroxyflavanone) in the wood of Douglas fir and western larch has been developed. The flavanone in an aqueous extract of the wood is reduced with zinc and acetic acid under standardized conditions prior to the production of a characteristic reddish purple color b y the additon of hydrochloric acid. A leucoanthocyanin-like substance present in Douglas fir which produces a similar color is not extracted from the wood with water. Other 3-hydroxyflavanones which would give the same color reaction do not occur in Douglas fir and occur only in trace amounts in western larch wood.
D
(taxifolin) is a 3-hydroxyflavanone (3,3',4', 5,7-pentahydroxyflavanone) m-hich occurs in the heartwood of Douglas fir, Pseudotsuga tazifolia (Poir.) Britt. (IS), Japanese larch, Larix kaempferi Sarg. ( l a ) , Lariz leptoteptis (Sieb. and Zucc.) Gord. ( 2 ) , western larch, Larix occidentalis Nutt., and Prunus companulata Naxim. (4). Douglas fir bark is a rich source of this substance, yielding as much as 7% (9). Because of its flavonoid character and ready conversion to quercetin (8), the aglycone of rutin, dihydroquercetin is potentially useful in medicinals. It is an effective antioxidant for the prevention of rancidity in fats, oils, and dairy products (10). Proper-
ties of dihydroquercetin which have an important bearing on the industrial usefulness of the wood in which it occurs include inhibition of calcium base sulfite pulping (IS), the ability to corrode digester steel in the presence of kraft alkaline pulping liquors (11), and fungicidal activity which suggest that it is the dominant factor in Douglas fir durability (6). It is therefore of considerable importance to be able to analyze wood samples for dihydroquercetin and to determine its distribution in relation to growth conditions, sample position in the tree, age of the tree, and other factors. A simple colorimetric method was used to plot the dihydroquercetin content in Douglas fir and western larch stems APPARATUS AND REAGENTS
IHPDROQUERCETIN
Fisher electrophotometer with 525 green filter. Intermediate Wiley mill. Dihydroquercetin, melting point 2402" C., prepared from Douglas fir bark by the method of Kurth and Chan (9). Zinc, granular, 30 to 40 mesh, ACS reagent grade. PROCEDURE
Sufficient wood material to provide duplicate 2.0-gram samples for hot water (100" C.) extraction and moisture determination is ground in an Intermediate Wiley mill to pass a 40-mesh screen. The sawdust (2.0 grams) is weighed
accurately into a 125-ml. Erlenmeyer flask to which is added 50 ml. of distilled water. The flask is then fitted with an air condenser and placed in a boiling water bath for 1 hour. The extract is decanted through a filter into a 100-ml. volumetric flask, 25 nil. of fresh distilled water is added to the partially extracted sawdust , and the procedure is repeated for 1 hour. After extraction with two additional 15-ml. portions of distilled water for 30 minutes each, the spent sawdust which retains some water is washed with water and the extracts and washings are made up to 100 ml. After mixing. a 1.0-ml. aliauot of this extract is pipgtted into a teit tube (13 x 100 mm.) to which has been added 0.,50 gram of screened, graded zinc. To this mixture 2.0 ml. of acetone is added, followed by 1.0 ml. of glacial acetic acid. Immediately upon the addition of the acetic acid the mixture is stirred and maintained a t 65" h 0.1 O C. for 40 minutes. The reduced solution is decanted into a 25-ml. volumetric flask, and the residual zinc washed twice with a total of 10 ml. of acetone. After mixing, 1 ml. of concentrated hydrochloric acid is added and the solution again thoroughly mixed. The volume is adjusted to 25 ml. Tvith acetone. The color, which forms immediately, reaches a maximum intensity in 5 minutes and remains constant for the next 10 t o 15 minutes. The density is determined with the electrophotometer. The concentration of dihydroquercetin in the wood on a moisturefree basis is calculated from the moisture content results and the color calibration curve prepared by reduction of standard dihydroquercetin solutions with the same grade of zinc. I
VOL. 30, NO. 2, FEBRUARY 1958
279
4 0 (3
z r
-80 3 0
*7 0
[L
c ul :2
0
r6 0
c
0
a r
W
210 c
0
W W >
K
2 *50
0
0
m
0
~ 4 0
Figure 1. Effeft of time on color produced by reduction of dihydroquercetin
-3 0
---Reduction with zinc and hydrochloric acid -Reduction with zinc and acetic acid followed by hydrochloric acid
*2 0
DEVELOPMENT
Pew (13) and Kurth and Chan (9) estimated the dihydroquercetin content in Douglas fir heartwood by isolation experiments. These isolation methods require large samples of raw material and involve lengthy purification steps. For routine use a relatively rapid and simple method applicable to 1- or 2-gram samples of heartwood was desirable. Pew pointed out that the production of a purple-red color by reduction with metallic zinc in hydrochloric acid eolution was a useful qualitative test for the presence of 3-hydroxyflavanones. Geissman and Clinton (3) showed that the purple-red color is due not to the simple formation of flavylium salts but to formation of a complex mixture of flavones and pinacoh. The color differs from true anthocyanidins in stability, fading rather rapidly. Other possible reduction media including stannous chloride, sodium hydrosulfite, titanous chloride, hypophosphorous acid, and aluminum isopropoxide solutions, were tested in attempts to produce the color without using metallic zinc, None were successful. Passage of an acidified taxifolin solution through a column containing amalgamated zinc (Jones reductor) was very effective in producing the color. However, the reduction reaction with amalgamated zinc rapidly goes beyond the color stage, and hence the residual color in a treated solution is a function of the original concentration and the column size used. Concurrently, Kennedy and Wilson (6),attempted to determine dihydroquercetin in Douglas fir extracts by measuring the color produced by treatment with standardized quantities of zinc and hydrochloric acid. They found that although accurate results were obtainable with pure solutions, with fir extracts the method accounted for roughly half of the dihydroquercetin 280
ANALYTICAL CHEMISTRY
L
400
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