V O L U M E 26, NO. 9, S E P T E M B E R 1 9 5 4 Table 11.
1423
Spectrophotometric Determination of Phenolic Hydroxyl Groups in Lignin Preparations
Sample Hemlock native lignin Pine alkali lignin Pine kraft lianinb Lignosulfonates Purified bv: Adsorption on ion exchange resins
A p arent
hlethoxyl,
Mop. Wt.,
14.8 11 6 13.9
...
. . ...
8.7 10.1 12.5 11.25 11.4
1500 1460 3200 9350 5600
%
Grams5
Dialysis Precipitation a Calculated from diffusion coefficients ( 8 ) . b Indulin A. Marasperse K.
299 301
Liter/Gram Cm. 9.34 6.92 7.56
Phenolic OH, % 3.87 2 87 3.13
Guaiaoyl Propane, Units per O H 2 1 2.2 2.4
300 300 300 299 299
4.74 5.45 4.91 3.52 3.54
1.96 2.26 2.04 1.46 1.47
2.4 2 .5 3.4 4 2 4 3
Xmsx.,
ma
Aamax..
absorptivity of model compounds with this structure (3,9 ) is very high (20,000 to 30,000), their contribution to the total phenolic hydroxyl content will be small in most cases. The presence of enolic hydroxyl groups in the side chain conjugated with the benzene ring would contribute also to an increased adsorption of the alhline solution in this region of longer wave length ( 1 , 3). While the method should be applicable to hardwood as well as softwood lignins, it has been used so far with lignin preparations from softwoode only. On the assumption that every phenylpropane unit of softwood lignins carries one methoxyl group, an assumption nhich is certainly only an approximation ( I S ) , the number of guaiacyl propane units per phenolic hydroxyl group may be calculated from the phenolic hydroxyl and methoxyl contents of the samples. These values which are less affected by differences in degree of purification of the samples are given in the last column of Table 11. The value for western hemlock native lignin obtained in this manner is of the same order of magnitude as those given by Aulin-Erdtman ( I ) for native lignin from spruce and from western hemlock The values for
the alkali lignins are of a similar magnitude, indicating that all these preparations contain amroximstelv“ one.Dhenohc hydroxyl group for two guaiacyl propane units. The values for the ligno-sulfonate samples roughly indicate a decrease in phenolic hydroxyl content with increasing molecular weight (11) of the preparation.
__
ACKNOWLEDGMENT
The author nishes to thank F. E. Brauns and J. L. McCarthy for the gift of samples, and Elizabeth V. Coffman for careful assistance with the experiments. LITERATURE CITED
(1) Aulin-Erdtman, G.. Suensk Paperstidn., 55, 7 4 5 (1952). ( 2 ) Ihid., 56, 91 (1953). (3) (4)
Ihid., p. 287. Aulin-Erdtman, G.. Tappi, 3 2 , 1 6 0 (1949).
( 5 ) Brauns, F. E., J . A m . Chem. Soc., 6 1 , 2120 (1939). (6) Brauns, F. E., “The Chemistry of Lignin,” pp. 248-54, N e w
York, Academic Press, 1952. ( 7 ) Doub, L., and Vandenbelt, J. M., J . A m . Chem. Soc., 6 9 , 2714 (1947). ( 8 ) Felicetta, V. F., hlarkham, A. E., Peniston, Q. P., and McCarthy, J. L., Ihid., 7 1 , 2879 (1949). (9) Goldschmid, O., Ibid., 75, 3780 (1953). (10) Lemon, H. W., Ibid., 6 9 , 2 9 9 8 (1947). (11) Maranville, L. F., unpublished results. (12) Peniston, Q. P., and Mecarthy, J. L., J . Am. Chem. SOC.,70, 1324 (1948). (13) Schuerch, C., Ibid., 7 3 , 4 9 9 7 (1951). RECEIVEDfor review September 14, 1953. Accepted J u n e 14, 1954. Presented before t h e Divisions of Cellulose and Polymer Chemistry, Symposium on Methods of Cellulose and Lignin Research, a t the 123rd hleeting of t h e AWMERICAN CHEMICAL SOCIETY,Los Angeles. Calif., March, 1953. Contribution No. 10 from the Research Division, Rayonier Incorporated, Shelton. Wash.
Ultraviolet Absorption Spectra as a Measure of Phenolic Hydroxyl Group Content in Polyphenolic Tanninlike Materials L. FRANK MARANVILLE and OTTO GOLDSCHMID Research Division, Rayonier Inc., Shelton, Wash.
A rapid and simple method for determination of phenolic groups in natural polyphenolic materials, such as tannins, has long been needed. An ultraviolet spectrophotometric method employing difference spectra has been developed which is suitable for this determination. Results by the spectrophotometric method are compared with those by the purely chemical 2,4-dinitrophenyl ether method. I’alues obtained by both methods on a series of bark extracts of severai species of softwood and hardwood trees are in qualitative agreement. The ultraviolet procedure is an extremely rapid and convenient method for obtaining a relative measure of the phenolic content of natural polyphenolic materials and protides a convenient means of differentiating different classes of such materials.
LTRAVIOLET spectrophotometry has been successfully applied to the determination of phenolic hydroxyl groups in lignin materials by means of the difference between their
neutral and alkaline absorption spectra (g, 6). This suggested that a modification of the method might be usable on the more complex problem of phenolic group determination in natural polyphenolic polymers such as the tannins. (Polyphenolic, as used here, refers to materials with more than one phenolic group per aromatic ring.) While the structures of the gallotannins (or hydrolyzable tannins) are now fairly well established, those of the phlobatannins or condensed tannins (also known as catechin or catechol tannins) are still in question. No method is available for preparation of pure condensed tannins and all experiments and tests are, of necessity, carried out on tannin extracts. No absolute method is available for determining tannin concentration in tannin extracts (the standard hide powder method is empirical). The amorphous nature of the condensed tannins greatly increases the difficulties of characterizing these materials and of defining their purity. A rapid and accurate determination of the phenolic group content would be extremely helpful in the study of the condensed tannins. Since many of the properties which make these bark materials useful articles of commerce are apparently
ANALYTICAL CHEMISTRY
1424
derived from the polyphenolic groups, the practical value of such a method is also apparent. Chances of successful application of this method to the polyphenols, however, seemed rather slight since polyphenols are known to undergo complex reactions in alkaline solutions. Preliminary experiments with ultraviolet absorption spectra showed the tannins were no exception and that their alkaline ultraviolet absorption spectra change continually with time. The numerous possible different positions for substitutions of two or more phenol groups in an aromatic ring also might well discourage hopes for this method, since each possible combination might be expected to require its own model substance as a standard. In spite of these apparent difficulties, a quantitative ultraviolet spectrophotometric method was developed and applied to a wide variety of bark extracts and tannins. The difference spectra of these materials were compared with those of a group of polyphenolic pure compounds which were used as model substances and standards. Results obtained by this method were compared tvith those by a modification of the purely chemical 2,4-dinitrophenyl ether method of Zahn and Wurz (9).
Partial analyses of these samples appear in Table I. Of interest is the high extraction yield for Sitka spruce. The two hardwood extracts, black gum and alder, have higher methoxyl contents, as expected. Galactose, glucose, arabinose, mannose, and xylose were determined individually by a chromatographic method after an acid hydrolysis and were added to give the total sugar content in the last column. Redwood extract has the highest sugar content (25.1%) and western hemlock extracts the lowest (8.2%).
EXPERIMENTAL
In order to develop, test, and compare the ultraviolet difference spectral method and the 2,4dinitrophenyl ether method, a series of water extracts of bark of various domestic species of trees was prepared and analyzed. In addition, a sample of commercial unsulfited quebracho extract and two specially purified hemlock bark extracts were studied.
Table I.
Analyses of Extracts Sulfated
Yield, Sample %" Water extracts Hemlock Black gum Sitka spruce Southern pine Douglas fir Alder Lodgepole pine White fir W. red cedar Redwood Quebracho ... (unsulfited) Solvent extract of 5.8 hemlock Hide pouTder purified water extract of hemlock Oven-dried basis.
...
Moisture,
%
Ash,
%"
Methoxyl,
%"
Total Sugars,
%"
20
30
40
20
VI
30
40
,IO-3,sm-1
Figure 1. Ultraviolet Absorption Spectra of Neutral Solutions of Various Extracts Ordinate scale indicated for upper curves. Successive curves each displaced o n e u n i t downward
...
4.3
... ...
...
...
8.5
...
1.4
...
12.3
Preparation of Samples. WATEREXTRACTS.Barks of the following species, western hemlock (Tsuga heterophylla), black gum (Nyssa sylvatica), Sitka spruce (Picea sitchensis), southern pine (mixed P i n u s palustris and P. caribaea), Douglas fir ( P s e u dotsuga tazifolia), western red alder ( A l n u s rubra), lodgepole pine ( P i n u s contorta), white fir ( A b i e s amabilis), western red cedar ( T h u j a plieata), and redwood (Sequoia sempervirens) were ground in a hammer mill, and extracted with water in a steamjacketed kettle a t 97' C. under atmospheric pressure. Yields were determined on the extract and first wash which were collected as two fractions, passed through a 325-mesh screen, and after total solids were determined, the more concentrated fraction in each case was spray dried. A sample of commercial grade unsulfited queQUEBRACHO. bracho extract was obtained from the Magnet Cove Barium Corp. SOLVEXT EXTRACT.Bark of western hemlock was also extracted with acetone, the acetone evaporated off, and the residue dissolved in water. The filtered solution was extracted with ethyl acetate and the ethyl acetate solution was vacuum evaporated. Melting point of final product was 132' to 136" C. HIDE POWDERPURIFIED WATEREXTRACT.The water extract of western hemlock bark described above was purified by adsorption on standard hide powder and was removed with acetone-water according to Buchanan, Lewis, and Weber (4).
MODELCOMPOUNDS. The catechol (melting point, 102' C.) and resorcinol (melting point, 108-110" C.) were reagent grade Eastman chemicals. The d-catechin (melting point, 172.5' C.) and dihydroquercetin (melting point, 231" to 233' C.) were obtained from E. F. Kurth of the Oregon Forest Products Laboratories and the 0-conidendrol (melting point, 252'C.) from W. W. Moyer of Crown Zellerbach Corp. All were vacuum dried over phosphorus pentoxide in an Abderhalden dryer a t the temperature of boiling acetone. Method Development. Ultraviolet spectra of the natural materials in nonalkaline solution appear in Figure 1. All of these curves show a maximum near 290 mp with the exception of black gum and possibly redwood which is very flat. Spectra of the same materials in alkaline solution are shifted upward, and the maximum moves toward the red ( 3 ) . At very high pH, the curves straighten and structure disappears. While a high pH for the alkaline solution is desirable to shift the equilibrium nearly completely t o the phenolate form, preliminary experiments on both models and bark materials showed, as might be expected, that the polyphenolic materials reacted rapidly in sodium hydroxide solution or in buffer solutions of very high pH, and that the absorption spectra of these solutions lost their normal shape so rapidly as to be unmeasurable. It was found, however, that solutions prepared with borate buffer a t pH 10 were sufficiently stable to permit measurements and alkaline enough to show pronounced peaks for the shifted maxima. "Neutral" solutions were adjusted to about pH 3 to shift the equilibrium nearly completely to the phenol form. Difference spectra were obtained directly i n the spectrophotometer, alkaline solution us. neutral, as a reference, rather than by
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4 subtracting two separately determined curves. Both alkaline and neutral solutions were made up from the same stock solution in which the sample was originally dissolved in pH 10 borate buffer solution (sometimes preceded by a few milliliters of ethanol). This stock solution was diluted to give a n absorbance of approximately 0.5 for the difference curve maximum. This technique employs the instrument at its highest sensitivity and measures the phenolic content at constant phenolic group concentration. Several measurements of the phenol peak ( Au-.) were taken as a function of elapsed time from the time the stock solution was first made up and the value used for each set of measurements was that extrapolated to zero time. Linear extrapolations were usually possible. Errors from decomposition of the alkaline solutions were thus minimized.
75
350
325
300
275
1
I
I
I
1
DROLAS
fm
1425 catechin has been found to some barks. Douglas fir bark was shown by Kurth (7) to contain substantial quantities of dihydroquercetin. Difference spectra of these two and similar compounds were, therefore, investigated. Difference curves of d-catechin, catechol, resorcinol, and b-conidendrol all showed maxima of nearly the same magnitude and shape near 290 mp. With time, Aumsx. increased for catechin, decreased sharply for 8-conidendrol and catechol, and remained essentially constant for resorcinol. These four compounds each have two phenolic hydroxyl groups per aromatic ring in either ortho or meta orientation, or both. Values of the equivalent absorptivity difference maxima were, therefore, averaged for all four standard substances for the purpose of this method. Difference spectra for the four standard substances are plotted in Figure 4. As shown in Table 11, the average maximum difference in equivalent absorptivity, A h m a xis . 1329 liters per equivalent centimeter. The difference curve for dihydroquercetin, also shown in Figure 4, exhibits a maximum near 330 mp, which did not change appreciably with time, and A b a x . is 5705 liters per mole centimeter.
Table 11. Equivalent Absorptivities of Model Compounds A m a x . , Liter/Equiv. Cm.5
&Catechin 8-Conidendrol Catechol Resorcinol
15
Av. Dihydroquercetin 0 Aemax. extrapolated to zero time. foreach compound.
Amax.,
mfi
1186 291 1593 298 1365 288 1170 287 1329 5705 326 Average of multiple determinations
IC
~
L
k.mp
375
b -
350
325
300
275
a
LOWEPOLE Pub!
C
Figure 2.
Difference Curves for Various Extracts
Difference spectral curves for the bark materials are illustrated in Figures 2 and 3. All show maxima near either 290 mp or 330 mp or at both wave lengths, except redwood which is virtually structureless, and black gum m-hich has maxima at 280 mp and 350 mp. Douglas fir was the only sample with a maximum near 330 mp which did not also have a maximum near 290 mp. With the bark materials Aumsx. either increased with time (at a rate not greater than 20% per hour) or did not change. Difference curves for model compounds were obtained in a like manner. Suitable amounts of those compounds which were insoluble in pH 10 buffer were dissolved in 95’% ethanol and 2-ml. aliquots of this solution were diluted to make the alkaline and neutral solutions. As models, a number of polyphenolic pure compounds were examined and the most suitable were selected as standards for the difference spectral method. I t has been suggested ( 5 ) that catechin is ii building unit of the condensed tannins and free
Figure 3.
Difference Curves for Various Extracts
Several of the bark products also possessed maxima of Au at 330 mp in addition to maxima at 290 mp and determination of phenolic content for these should presumably include both maxima. The t%-o maxima do not overlap seriously, and both were included in the final procedure. Difference spectra for 1,4-dihydroxy, 1,2,3,-trihydroxy, and 1,3,5,-trihydroxy phenols were quite different, as were protocatechuic acid, quercetin, caffeic acid, hematoxylin (tech.), and fustic extract.
1426
ANALYTICAL CHEMISTRY
Values of A b x . for these polyphenol model substances differ from those obtained for the model substances by Goldschmid ( 6 ) . This difference is caused, in part, by the different pH of the alkaline solutions and, in part, by the interaction between the two hydroxyl groups in the same aromatic ring. APPARATUS. A Beckman Model D U ultraviolet spectrophotometer was used throughout this work with matched 1-cm. quartz cells with ground stoppers. A 10,000-megohm load resistor was used, when advantageous, in place of the standard 2000-megohm resistor.
zero time is recorded for each. The difference curve is measured from 260 mp to 360 m p with points a t 2 to 3 mp intervals near the maxima where the times are again recorded. The two maxima are measured a third time and the three maximum absorbances (liter per gram centimeter) are converted to absorp tivities and are plotted against elapsed time (minutes) to obtain the extrapolated value a t zero time for the maxima at both 290 and 330 mp, A b . . CALCULATIONS.Phenolic hydroxyl group content of the at polyphenolic samples is calculated from the values of hmar 290 and 330 mp and from the average values of Aemae for the two types of model compounds, as follows:
+
Moles of phenolic hydroxyl per gram = Aak2/1329 % phenolic hydroxyl = 1.28 AaiEE 0.298 A&:: Aa::,9/5?05
+
0 B-CONIDENDROL 0 CATECHIN 0 CATECHOL
0 RESORCINOL
t
A
2,4,-Dinitrophenyl Ether Method. The 2,4-dinitrophenyl ether method of Zahn and Wiirz (9) was used with the folloa ing modifications: Larger excesses of dinitrofluorobenzene were necessary since phenolic contents were unknown. The ethers of the bark materials were not crystalline materials and were frequently finelv divided and very hard or impossible to filter. Gummy materials present in the reaction mixture added to this difficulty. Samples were, therefore, precipitated in large centrifuge tubes and, after 3 hours, were centrifuged a t 2000 r.p.m. until clear (usually 1 to 2 hours, but occasionally 6 to 8 hours). The supernatant liquid was carefully removed and the precipitates rvere washed twice with 10-ml. portions of 1to 1 methanol and the washings removed by centrifugation as above. The samples were then filterable on fine sintered glass crucibles, as per Zahn and Wurz, without undue difficulty. They were washed thoroughly with water, and were dried, weighed, and analyzed as usual. Some pure polyphenolic compounds were analyzed by the dinitrophenyl ether method a-ith the results shown in Table 111. Catechol, p-conidendrol, d-catechin, and dihydroquercetin react in a normal manner. In quercetin and 3-hydroxyflavanol, however, the 3-hydroxy group is sufficiently activated by conjugation n-ith the aromatic ring that it also reacts quantitatively in addition to the phenol groups.
Tahle 111. Analysis of Model Compounds by the 2,4Dinitrophenyl Ether Method 0
Figure 4. Difference Curves for Ilodel Compounds Scale on left refers to dihydroquercetin and scale on right to other four compounds
SOLUTIONS. Borate buffer solution, pH 10; 43.9 ml. of 0.2M sodium hydroxide and 50 ml. of a solution, 0.2M in boric acid
Compound Catechol d-Catechin Dihydroquercetin
,Moles DNFBa Yield of Ether per Equiv. OH (Theoret.), in Sample %
3-Hydroxyflavone Quercetin 8-Conidendrol
1.05 1.1 1.1 1.4 1.2 LO5 1.36 1.21 2.2
100.1 88 5 85 2 95.0 99.9 88 7 102.4 98.9 103.5
M.P. of EtheriCoirrrected), C.6
138 147 158 I67 188 174 170 167-8
N, 5% Theorct. Found 12.66 11 i 4
11.57 11.57
6.93
12.37 12.37 11.28 8.14
12 5 10.8 11.0 11.2 7 1 11.6 11.7 10 7
Conidendrin 145 8.3 and 0.2M in potassium chloride, are diluted to 200 ml. with a Dinitrofluorobenzene. water: 0.6M sulfuric acid solution. b In agreement with Zahn and Wiirz ( 9 ) . PROCEDURE. F i f t y m i l l i g r a m s of sample are dissolved in 100 ml. of pH 10 borate buffer and time of addition is Table IV. Comparison of Phenolic Group Anal>-ses recorded as zero time. Sometimes it is advantageous to dissolve the sample in Phenolic OH Groups, %” a few milliliters of ethanol before adding Diff. 2,4-DinitroTannins r ‘ ~ of ~, Av. Aamax. spectra phenyl ether in Total Soli$!s4 the p H 10 buffer. One aliquot of this Sample Detns. 290 mp 330 n ;i method method (Std. H.P.), solution ( 1 to 40 ml., depending upon Quebracho (unsulfited) 2 12 0 1.2 18.0 14.4 70.5 phenol content of the sample) is diluted IT. hemlock (solvent to 50 ml. with the pH 10 buffer (Solution extracted) 2 11.2 2.0 15.6 13.8 65.8 W. hemlock A). A second aliquot of the same size (hide powder purified) 2 10.5 2.0 15.3 ... 86.3 as the first is added to 2 ml. of the 0.6 X sulfuric acid (sufficient to reduce the final Water Extracts Black gum 4 5.7 pH to 2.5) and is also diluted to 50 ml. W. hemlock 47.8 with pH 10 buffer (Solution B). Sitka spruce 46.9 Difference curves a r e d e t e r m i n e d Dquglas fir 41.5 Alder directly by measuring the absorbance of 43.2 S. pine A us. that of B as a “blank.” The 83.3 Lodgepole pine aliquots above are adjusted to give 25.8 White fir 19 1 W. red cedar an absorbance reading of approximately 27.4 Redwood 0.5 for the absorbance maximum near 290 mp. The two absorbance maxima a Results on an oven-dry basis. b See Table V. near 290 and 330 mp are measured quickly and the elapsed time from the
1427
V O L U M E 2 6 , N O . 9, S E P T E M B E R 1 9 5 4
fonated quebracho) which are insoluble in dimethylformamide. The sample may be dissolved in a few milliliters of water first, but the resulting 2,4-dinitrophenyl ether is usually not sufficiently insoluble i n solvents containing water. For samples where solubility is not a problem, the 2,4-dinitrophenyl ether appears to be an accurate method. Some of the results shown in Table I11 indicate that certain enolic hydroxyl groups react a i t h the 2,edinitrofluorobenzene. In the spectrophotometric method, nonconjugated aliphatic enols would have no effect while those conjugated with an aromatic ring would exhibit a shift of the maximum in alkaline solutions to much longer wave length than phenolic groups. For example, 3-hydroxyflavone has a maximum in alkaline solution a t 400 mp. The spectrophotometric determination is somewhat mole empirical in nature and is dependent upon the availability of suitable model or standard substances. It should be a useful tool in the examination of widely different polyphenolic materials, and is capable of satisfactorily high precision for routine determinations on materials of a similar nature. The entire spectrophotometric method, including a trial dilution and complete curve, can be run in about one hour. Routine determinations involving only measurement of the peak of the curve could be run in a matter of minutes. In contrast, because of the filtration difficulties in the dinitrophenyl ether method, only a fen samples could be run a t one time and the time from start to finish was two dags, or more.
Table V. Precision of the Spectrophotometric Method Applied to the Water Extract of Western Hemlock Bark Determination NO. 1 2 3 4 5 6 7
8
9
10 11
Aamax. 290 m p 5.5 5.5 5.1 5.6 5.5 5.4 5.4 5.6 5.G 5.5
5.1
330 my 1.5 1.5 1.2 1.3 1.4 1.4 1.4 1.4 1.4 1.2 1.4 1.7
5.3 12 Average ... . Standard deviation = 0.24 Coefficient of variation = 3 . O %
..
Phenolic Hydroxyl, % 8.1 8.1 7.6 8.3 8.1 8.0 8.0 8.3 8.3 8.1 7.6 8.0 8.04
RESULTS
Results by the two methods are compared in columns 5 and 6 of Table IV. Determinations of “tannin in total solids” by the standard hide powder method of the American Leather Chemists’ Association (ALCA) ( 1 ) are listed in column 7 . As a test of the precision of the spectrophotometric method, the phenolic determination of the water extract of western hemlock was repeated 12 times over a period of several months with the results shown in Table V. The coefficient of variation is 3.0%.
Table \-I.
Ether Extraction of Douglas Fir Bark Water Extract % of
Sample Original water extract Residue Extract Sum
Original Extract, byn’eight 100 89.9 10.1
Phenolic OH, Av. h m a x . 330 mw 295 mp 330 mp 295 mp ~
18.7 10.8 45.9
... 0.6
In another experiment, the Douglas fir water extract was extracted with ether to remove dihydroquercetin, and both the ether extract and the extracted material were analyzed by the spectrophotometric method with the results presented in Table VI. DISCIiSSION
As seen in the last three columns of Table IV, per cent phenolic hldroxyl values by the difference spectral method and the 2,4dinitrophenyl ether method are in qualitative agreement with few exceptions. Values of per cent tannin by the standard hide powder method of the ilmerican Leather Chemists’ Sssociation also parallel the phenolic hydroxyl contents. Results on the black gum extract should not be expected to compare too closeljwith the other materials since the maxima of the difference curves appear at different wave lengths than do the other materials or the standard substances. The difference spectral method is capable of a degree of precision satisfactory for many applications, as shown in Table I-. Thus, the method is extremely useful for the determination of even relatively small differences in phenolic hydrox3 1 content between different preparations from the same species. The results of the ether extraction in Table VI point up the necessity of using both maxima in the method. Most polyphenolic materials are a t least slightly soluble in water and solutions of the concentration necessary for spectrophotometric determination can usually be obtained. Solubility occasionally presents a more serious problem in the 2,4-dinitrophenol ether method. If the sample does not completely dissolve, it is questionable whether complete reaction occurs. Difficulties occur with sulfonated samples (such as lignosulfonates or sul-
...
5.6 3.2 13.7
... 0.8 ,
.,
Phenolic OH on Basis of Original Extract, % 5.6 3.6 1.4 5.0
-
Distribution Phenolic OH, %
of
100 64 25 -- 89
The difference spectral method has the additional advantage that the difference spectra themselves are often useful in identifying and classifying bark materials, as may be seen in Figure 2. Since the present work was carried out, Mansfield, Swain, and Nordstrom (8) have published a method for differentiating flavones and their derivatives by means of their alkaline and neutral spectra. ACLVOWLEDGMENT
The authors wish t o thank E. F. Kurth, of the Oregon Forest Products Laboratory, for the samples of d-catechin and dihydroquercetin, and W. W. Xloyer, of Crown Zellerbach Corp., for a sample of P-conidendrol. It is also a pleasure to acknoirledge the careful experimental work of Ruth McCleary. LITERATURE CITED
( I ) American Leather Chemists’ =Issociation, “Methods of Sampling and Analysis, Analyses of Extract, Proposed Methods of 1946,” pp, d7-Al3. ( 2 ) Aulin-Erdtman, G., Saensk Papperstidn., 55, 745-9 (1952). (3) Aulin-Erdtman, G., Tappi,32, 160-6 (1949). Lewis, H. F., and Weber, B. W., J . A n . Leather (4) Buchanan, M. il., Chemists’ Assoc., 45, 513-30 (1950). (5) Freudenberg, K., “Die Chemie der naturliachen Gerbstoffe,” Berlin, Springer, 1920. (6) Goldschmid, O., AXAL.C H n r . , 26, 1421 (1954). (7) Hergert, H. L., and Kurth, E. F., T a p p i , 35, 59-66 (1952). (8) Mansfield, G. H., Swain. T., and Nordstrom, S a t w e , 172, 23-5 (1953). (9) Zahn, H., and Wurz, A . , 2. anal. Chem., 134, 183-7 (1951). RECEIVED f o r review September 14, 1953. Accepted J u n e 14, 1954. Presented before the Division of Cellulose Chemistry a t t h e 123rd Meeting of the AMERICAN CHEMICAL SOCIETY, Los Angeles. Calif., March 1953. Contribution KO. 11 from the Research Division of Rayonier Inc., Shelton. Wash.
