Analytical Method for Thujaplicins HAROLD MACLEANand J. A. F. GARDNER Forest Products Laboratories of Canada, Department of Northern Affairs and National Resources, Vancouver, 13. C., Canada
hexane-chloroform (2 to 3) solvent as a blank. From a previously prepared calibration plot (Figure I), relat,ing absorbance to thujaplicin content, the thujaplicin content on a moisture-free wood basis is calculated. If the thujaplicin material is crystalline or is an oily mixt.ure of isomers, a n-hexane solution of approsinlately 5 my. per 100 nil. is prepared and a 10-ml. aliquot is used as described above
To facilitate investigations of the chemistry and variations in the natural durability of western red cedar, a rapid method for the determination of the substituted tropolones (the fungicidal thujaplicins) was developed. The thujaplicins in a hexane-chloroform solvent are treated with aqueous ferric acetate solution to yield their red ferric chelates, which are then estimated colorimetrically in the organic layer. For analysis of cedar wood, the thujaplicins are extracted from the finely ground sample with hot water and then extracted into hexane, This procedure excludes an interfering phenol which is also extracted from the wood by the hot water.
DEVELOPMEST
Tropolone derivatives exhibit characteristic color reactions with several heavy metal cations due to the format'ion of stable chelates which are soluble in organic solvents. Thus, the thujaplicins react with copper acetate or cupranimoniurn solutions to give a neutral green chelate which is very soluble in chloroform and insoluble in water (3). With the appropriate amount of ferric iron, a red chelate soluble in organic solvents is formed. Both Nozoe (9, I O ) and Cook and others ( 3 ) noted that, v4th an excess of ferric ion or in the presence of acid, the red chelate vihich forms at first is converted to a green watersoluble complex. They also suggested that the red ferric chelate might be useful analytically because of the intense color of very dilute chloroform solut,ions. The formula for the ferric chelat'e of y-thujaplicin is
T""
a-, p-, and 7-thujaplicins (2-hydroxy-3-, 4, and 5isopropyl-2,4,G-cycIoheptatrien-l-one) are potent fungicides occurring in the heartwood of western red cedar (Thuja plicata D. Donn.) ( I , 6 ) , eastern white cedar ( T h u j a occidenfalis L.) ( 7 ) , hiba (Thujopsis dolubrata Sieb. et Zucc.) ( I O ) , and Formosan hinolri (Charnaecuparis taiwanensis hIasmune et Suzuki)
(10).
These extractives are believed to be mainly responsible for the high natural durability of western red cedar ( 1 1 ) . I n investigations of the acetone extractive in the species (Z), and also of the distribution of the durability factors (8),the need for a simple rapid method of determining them substituted tropolones in solutions and in wood substance became evident. A simple accurate method is described which has been used routinely to analyze a large number of small wood samples (8).
I n early work in this laboratory it wa3 found possible to determine the thujaplicin content of chloroform solutions accurately by measuring the green color developed on the addition of excess freshly prepared ethyl ether solutions of ferric chloride ( 4 ) . This method was suitable in the absence of phenols and accurate if water was carefully excluded, because the green complex is very soluble in water and relatively insoluble in organic solvents. I n working with western red cedar a method was required for the routine analysis of stcam distillates and wood extracts known to contain chlorofoi m-soluble phenolic substances which also give green colors with ferric ion. Preliminary experiments had established that other nonphenolic extractives soluble in organic solvents-Le., thujir acid and its methyl estergave no color reaction with ferric iron. Therefore, the utility of the red ferric chelate was investigated. A method of carefully controlling the iron addition so that none of the rcd chelate formed would be converted to the green compound was a primary consideration. Accordingly, a number of ferric complexes of lesser stability, such as the lactate, gluconate, citrate, and acetate, were investigated as the medium for adding the iron. By shaking chloroform solutions of y-thujaplicin with aqueous solutions of these various complexes it was found that mnsimum red color response in the organic layer and absence of any green color was obtained using ferric acetate solutions. Blank tests showed that the reddish brown ferric acetate solution did not impart any color to the organic layer. Both p- and y-thujaplicin had been isolated from western red cedar, and the possibility that relatively small quantities of the a-isomer were present had not bcen disproved; therefore, it was necessary to compare the rolor response of the three isomers and that of the mixture of isomers occurring naturally in western red cedar. Pure samples of the p- and r-isoniers were available from previous work on western red cedar (2). A sample of the or-isomer was isolated from eastern white cedar ( T h u j a occidenfalis L.) by the method of Gripenberg (7).
EQUIP,ME.YT AND R E A G E S T S
The folloning are needed: Fisher Electrophotometer with No. 425 blue filter. Wilev mill. intermediate model. Burfell wrist-action shaker. n-Hexane, boiling point 65-67' C. Chloroform, ACS reagent grade. Ferric acetate solution. Dissolve equimolar quantities of sodium acetate and ferric chloride in sufficient distilled water to make a final solution 1%in iron. This solution has a pH of 4.3. PROCEDURE
Sufficient wood material is ground in the Wiley mill to pass a 40-mesh screen to provide duplicate 2-gram samples for hot r a t e r extraction and duplicate 1-gram samples for moisture content determination. The 2-gram samples are leached for 1 hour with 100 nil. of distilled water in a 250-ml. Erlenmeyer flask, which is placed in a boiling water bath and fitted with an air condenser. The hot liquid extract is carefully deranted through a tared Alundum or fritted-glass crucible. The procedure is repeated, using 50 nil. of distilled water for 1 hour follo\l*edby 25 ml. of distilled miter for 1/2 hour, The sample is then transferred to the crucible and washed with a maximum of 25 ml. of hot distilled water. The crucible may be dried and weighed for a hot v&er solubility determination, if required. mhen the combined water extracts are cool, they are made up to 200 nil. with distilled water. A 5-ml. aliquot of this solution is extracted twice with 5-ml. portions of n-hexane in a small separatory funnel, shaking for l minute in the Burrell shaker for each extraction. T o the 10-ml. hexane extracts are added 10 ml. of chloroform and 5 ml. of ferric acetate solution. The samples are shaken for 5 minutes in a separatory funnel on the shaker, then the organic layer is filtered into a 28-ml. graduate. The aqueous layer is washed with additional chloroform, and the filtrate is made up to a final volume of 25 ml. Absorbance is measured in the Electrophotometer, using a KO. 425 blue filter and a similar 509
ANALYTICAL CHEMISTRY
510 Aliquots of standard solutions (0.5 gram per liter) of the three isomers in hexane-chloroform solvent were treated with ferric acetate solution by the method described, and the results wercb plotted (Figure 1). The values for a- and p-isomers almosl, coincided, with the p-isomrr giving a slightly greater response Both were approximately 20% lower than the values for the -/-isomer. The plot for 7-thujaplicin was a straight line ovei the range tested, but the plots for the a- and p-isomers fell off slightly a t the higher concentrations. The same divergence was obtained by varying the concentration of solutions of the pure ferric chelates of these two isomers, indicating that the divergence was due to deviation from Beer’s lam, and not a characteristic of the chelate formation reaction.
I
-1
ratio of the isomers occurring in the mixture. If it is assumed that the a-isomer is not present, in western red cedar, then the position of the curve for the thujaplicin mixture isolated from mood (W.R.C., Figure 1) relative to those of the p- and r-isomers indicates that the amount of 7-isonier is approximately 60%. I n the event that the a-isomer is found to be present in Canadiangrown red cedar, this rough determination of the content still holds becausr the color response of the a- and p-isomer is almost identical. For the direct analysis of wood samples, an efficient method of obtaining the thujapliein content in solution was required. Steam distillation of cedar wood removes the thujaplicins, mhich me readily analyzed in colorless steam distillates by the colorimetric method. However, to ensure complete removal of all the thujaplicin, a lengthy distillation a t atmospheric pressure is required; therefore, a method of solvent extraction was sought. It was knoirn from the work of Erdtman and Gripenberg (6) that, although diethyl ether is a good solvent for the t,hujaplicins, it is a poor medium for directly extracting them from the wood, because a large portion of the content is apparently retained in the wood by the ether-insoluble “membrane substances.” Hot Trater extraction appeared to be an attractive possibility in that, by determining the weight of the residual wood meal, there could be simultaneous determination of hot water solubility. Furthermore, the aqueous extract would contain the water-soluble polyphenols, which are derivatives of pyrocatechol ( 2 ) that had been found to contribute, along with the thujaplicins, to the natural preservative content of western red cedar ( 1 2 ) .
Table I.
Efficiency of Extraction of Thujaplicin front Western Red Cedar Using Not Water (2-gram sample)
THUJAPLICIN rnq
Figure 1. Calibration curves for red ferric chelate of a-, p-, y-, and western red cedar mixed thujaplicins
Stage 1 2 3 4 5
The plot obtained using the thujaplicin mixture of isomers isolated from western red cedar is also given in Figure 1. Two separate preparations from different samples of local mill-run western red cedar sawdust, using methods described previously ( Z ) , gave identical curves (W.R.C.). Because the three isomers have similar stability under the conditions of isolation, the plot obtained should represent closely the mixture of thujaplicin isomers occurring naturally in British Columbia red cedar, Therefore, it was adopted as the calibration curve for analysis of samples grown in this region. The effect of variation of the pH of the ferric acetate solution on the color formation in the chloroform layer was examined for the B- and -,-isomers. A t pH values of 4.3 (normal for ferric acetata solutions) and 4.9, the response for each isomer appeared to be constant. However, a t p H 4.9, great difficulty was encountered because of the formation of stable emulsions during the shaking stage. At p H values below 4.3 the color response of the ?-isomer increased, a 12% gain being obtained by reducing the pH t o 3.95. However, the value for the p-isomer remained practically constant. Thus, use of a pH below 4.3 increases the divergence between the color response of the two isomers. This would in turn increase any error in the analysis of wood samples caused by the ratio of the thujaplicin isomers being different from that used in obtaining the calibration curve (W.R.C., Figure 1). A solution of a 1 to 1 mixture of the p- and y-isomers was tested by the analytical method and gave a ferric chelate color response exactly midway between those obtained for equivalent solutions of the pure p- and y-isomers, respectively. Therefore, the color developed by a known quantity of a mixture of these two isomers can be used with the aid of the graphs of Figure 1 to calculate the
6
1701. Water, 311. 100 50 25 25 (wash water) 25 25
Time, Hour 1 1 1/2
1 1
Mg. of Thujaplicin Extracted 9.25 1.72 0.42 0.11 0.15 0.09
-
Totals
70 of Total 78.8 14.6 3.64 0.93 1.27 0.76
-
__
11.74
100.00
If an analytical method for the polyphenols could be devised, aqueous extraction would allow simultaneous determination of hot water solubility, thujaplicin content, and water-soluble polyphenol content. The efficiency of a hot water extraction of thujaplicin from :t sawdust sample was examined and the results are given in Table I. The first four extractions accounted for approximately 987, of the total material, while the sixth extraction removed only a negligible amount. Therefore, the four-stage extraction was adopted as satisfactory for the analytical method. Originally it was thought that aqueous extracts of cedar might be analyzed by simply adding ferric acetate solution, extracting the red chelate with chloroform, and measuring its concentration with the colorimeter. However, the mixture of pyrocatecholtype polyphenols contained in the hot water extract of western red cedar ( 2 ) forms an immediate brown precipitate when ferric acetate solutions are added. This results in emulsions if a subsequent chloroform extraction is attempted. Therefore, it was necessary to extract the aqueous extracts first with a solvent which would completely remove the thujaplicins but only small ,mounts of the phenols. Chloroform was used initially, but an iinexpected difficulty developed. Small quantities of an unlcnotvn phenol were found to be extractable with chloroform from i,he aqueous extracts of cedar. This phenol gave a red color
511
V O L U M E 28, N O . 4, A P R I L 1 9 5 6
were made on the color of the chelate from the thujaplicin mixture isolated from different samples of mill-run cedar sawdust. The absorption maximum for these experiments fell at 425 to 420 mp, indicating that the -,-isomer represented approximately 33% of the mixture. This result is in agreement with the figure of SOY0 derived previously from the relative intensities of the colors measured.
I
ACCURACY
The accuracy of the method on cedar heartwood was checked by quantitatively isolating all the thujaplicins as the pure copper salts from a large sample of the wood (50 grams). The yield calculated from the analysis mas 0.542 gram, which was 98% of the yield actually obtained (0.55 gram). This result wan in line with the 98y0 efficiency of the extraction phase of the analytical method.
Table 111. Analysis of Western Red Cedar Heartwood
Figure 2. Absorption Spectra
.,,, -
-----.-. -.
y-Thujaplicin-ferric iron complex Purified western red cedar mixed thujaplicin-ferric iron complex Western red cedar thujaplicin-ferric iron complex b y analytical method Interfering color produced b y western red cedar polyphenol-ferric iron complex
Test NO.
Table 11. Maxima in Visible Absorption Spectra of Thujaplicin-Ferric Chelates Isomer a
P Y
hlaxima, M p 426 41s 43 1
544
540 551
588 584 595
Again assuming that the a-isomer is absent or present in only minor quantities, the position of the maximum in the 425-mp region for the ferric chelates of thujaplicins of cedar relative to those of the pure 6- and -,-isomers is a rough measure of the relative occurrence of these isomers. Several determinations
%
%
0.65
0.616 0.611 4
5 6
with ferric acetate in the chloroform layer similar to that of the ferric thujaplicin chelate, and gave results approximately 20% high. Fortunately, experiments with other solvents for the polyphenols showed that the interfering substance was not ex. tractable from aqueous solutions with n-hexane which did, however, remove all the thujaplicins. Other compounds extracted by hexane-thujic acid and its methyl ester-do not give any color reaction with ferric ion. Although hexane is a good solvent for the thujaplicins, it is a poor solvent for the ferric chelates, with the ?-isomer being insoluble and the a- and p-isomers being only slightly soluble, Therefore, to avoid precipitation of the chelates, it was necesrjary t o dilute the hexane solutions with chloroform prior to the treatment with ferric acetate solution. A 3 to 2 dilution by volume of n-hexane with chloroform was found satisfactory. This mixed solvent was used in the preparation of the calibration curves and in all analytical work. The absorption spectra in the visible region for the ferric chelates of the three thujaplicin isomers as determined with a Beckman DU spectrophotometer are very similar, having three maxima. These maxima, as shown in Table 11, occur approximately 13 mp higher for the ?-isomer than for the p-isomer, with those of the a-isomer being approximately midway between the other two. The corresponding spectra for the mixture of thujaplicin isomers isolated from cedar wood, for the chelate color obtained in the analysis of a wood sample, and for the interfering phenol eliminated by the use of n-hexane are shown with the spectra of the ?-isomer in Figure 2.
Deviation from Av..
Thujaplicin,
7
5 9 10
1.47 1.97
0.608 O.G29 0.628 0.627 0.621 0.621 0.615 0 . 625
1.43
1.27 1.12 0.1F
Std. dev.
Table IV. Test XO.
Analysis of Hot Water Extract of Western Red Cedar Heartwood Deviat.ion from A v . ,
Tliujaplicin.
%
% 0.620
0,610 0.605 0.608 0.620
0.610
i
0.16 0.84 0.80 0.0073
0 , GOO 0.810
Std. dev.
1.61 0.00 0.83 0.33 1.61 0.00 1.61 0.00 0,0068
For routine analyses, Figure 1 shows that, if the naturallj. occurring thujaplicin isomers are in a ratio different fmm that in the material used to derive the calibration plot (W.R.C.), an error would be introduced. Because it is possible that this ratio n-ould vary somewhat with forest growth conditions, high accuracy would require a simultaneous determination of the ratio, which is impractical for routine work. Moreover, in extensive use of the routine method on wood samples originating from different parts of trees of varying ages from several regions of British Columbia (8),no evidence of any wide variation of the ratio was obtained. The same calibration plot was obtained with the thujaplicin mixture from two different samples. Also, the position of the absorption maximum in the 425-mp region in the spectra of the ferric chelates of several other samples underwent no noticeable shift. With practice and the use of machine shaking in the extraction stages the precision was very good. The results of ten separate determinations on one sample of western red cedar heartwood sawdust are given in Table 111, and those of eight detcrminations on one sample of aqueous extract in Table IV. Comparison of these results shows that the precision of the aqueous extraction portion of the procedure is very good. I t was noted during the course of this work that actinic light degrades dilute chloroform solutions of the ferric chelates and the thujaplicins rapidly-up to 40y0 loss in S hours of direct sunlight was observed. The chelates were more stable than the
ANALYTICAL CHEMISTRY
512 free thujaplicins. Therefore, in preparing standards and ralibration plots. all solutions were freshly prepsred and carefully protected from light during storage. LITERATURE CITED
(1) Anderson. A. B.. Gripenberg. J.. Acta Chem. Scand. 2, 644. (1948). (2) Barton, G. M.. Gardner, J. A. F., Pulp & P a p e r .Vag. Can. 55, 10, 132 (1959). (3) Cook. J. VI., Gibb. A. R., Raphael, R. A,. Somerdle. A. R.. 3. Chem. Soc. 1951. 107.
(4) Dutton. G.G. S.. Gardner. J. A. F.. unpublished results. (5) Erdtman. H.,Gripenberg. J . , Acto Chem. Sand. 2, 625 (1948). (6) Erdtman, H.. Gripenberg. J.. Nature 161,719 (1948). (7) Gripenberg, J . , Acta Chem. S c a d . 3,782 (1949). (8) hlaelean, H..Gerdner. J. A. F.. unpublished data. (9) Nozoe. T.. Bull. Chem. SOC.Japan 11,295 (1936). (10) Nosoe, T.. Science Rep&. T6hoku Unio.. FGst SeriCa 34, 199-236 (1950). (11) Rennerfelt, E.,PhusiOl. Plantarum 1, 245 (1948). (12) Roff,J. W., Atkinson. J. AI.. Can. J . Botany 32, 308 (1954). R e c ~ i v ~for o review September 2. 1855.
Accepted January 14, 1956
Semimicrodetermination of Fluorine in Organic Fluoro Compounds CHAlM EGER and ASHER YARDEN Scientific Department, Israel Ministry of Defence, TeI-Aviv,
A s i m p l e s e m i m i e m m e t h o d for the d e t e r m i n a t i o n of fluorine i n organic fluom compounds irr described. T h e sample is ignited i n a modified Parr s o d i u m pemxide bomb and the solution of the m e l t is percolated t h r o u g h an acidic c a t i o n exchanger. The neutralized percolate is t i t r a t e d w i t h t h o r i u m n i t r a t e , using s o d i u m alizarin sulfonateas indicator. In fluom c o m p o u n d s which also c o n t a i n chlorine or b m m i n e , the various halogens can he determined simultaneously.
A
NUMBER of methods have been proposed far the transformatron of organic fluorine into titratable fluoride ions, including: (a)oxidation with gaseous oxygen (7, 14. 65, SS, 85) or sodium peroxide in a nickel bomb (19, 24, 87), with addition of benzoic acid or sucrose as combustion aids and, sometimps, of potassium nitrate or perchlorate as accelerator; ( b ) reduction by the hydrogen flame (11, 86) or alkali metal (16, 17, 20, 28, 80, S4); and (c) fusion with calcium oxide ( 1 , $1, 29) or sodium carbonate (9). Another method involves the reaction of hydrogen fluoride, formed in the destruction of the organic fluoro compound, with silica (10, 12, 18). Occasionally the organically bound fluorine is liberated hydrolytically by aqueous ammonia solution ($6,SO). The present investigation, which has already been briefly noted (15),has shown that the combustion with sodium peroxide is the most convenient and reliable method; however, the 2.5-ml. Parr mierohomb which is ignited by flame did not give reliahle results, and the 22-ml. electric ignition bomb bad too large a capacity. An electrical ignition bomb of 8-ml. capacity was, therefore, constructed and used successfully. The fluoride ion formed in the combustion is contaminated with a relatively large quantity of sodium hydroxide and other sodium salts which interfere with the subsequent fluoride determination. Gravimetric determinations [calcium fluoride or lead chlorofluoride (3711 failed a t the fluoride concentrations studied because precipitation was not quantitative. The usual distillation method (881, even after a number of improvements, tended to give low results, especially when the fluorine content was small. It therefore proved necessary to eliminate the sodium ion from the fluoride solution. This was achieved by means of the acid form of a cation exchanger. In the percolate, which contained, in addition to hydrofluoric acid, small quantities of carbonic and hydrochloric acids (when potassium perchlorate was used as combustion accelerator), the fluoride ion can he determined by titration with thorium nitrate, using sndium alizarin sulfonate 88 indicator. This method (8) has been improved by mme minor modifications.
nzagenrs ana aorunons. ur;tnuaru wmum nuonae solution
(0.1mg. of fluoride ion per ml.) was prepared by dissolving 0.221 gram of s3diurn fluoride (Baker analyzed reagent recrystallized once from distilled water and dried) in distilled water, adding 1 ml. of O.l.V sJdium hydroxide solution, and making up to 1 liter. rnL-~~:..-~ olution. An 84' solution dissolving 5 itrate (tetrad y s e d reaof distilled qe w a s obiim fluoride -:/~~-1~
~i~~~
pemride
1.
bomb, assembled
100 &I,of distilled water (a* proximately 1.W). To this sohtion a ldl acetic acid (AnalnK) sol&ion wa8 added to pH 3.55 on a. Beekman Model G DH motar
Ethyl alcohol, redistilled on&.' Sucrose, AnalaR. Potassium perchlorate, Parr Co. Ion exchanger. I n the present investigation, Amberlite IR112 (Rohm & Ham, Phildelphia) wa8 used. Rohm & Haas have discontinued the production of this specific ion exchange resin, and have substituted for it Amberlite EX-100. Any acidic ion exchange resin can be used in the method described. Anoaratus. An Rml. semimicro electric ienition bomb was constructed (Figures 1, 2, and 3 ) . ~The cups were made of stainless ateel, the ot,her parts of nickelelectroplated brass. A length of 3.5 cm. of fuse wire (Parr Co.) was used, and a resistance of 1 ohm was incorporated into the ignition circuit. The water bath xnd~ignitionunit used were those of the original Parr instrument ~~~~
($8).
~
~
Procedure. PREPARITION OF CALIBRATIOX CURVE. With an automatic microburet of 5-ml. capacity, 1, 2, 3, 4, and 5 ml. of the standard sodium fluoride solution were measured into five Erlenmeyer flasks of 50-ml. captpacity. Water was added to make a total of 10 ml. of solution. The reagents were then added to each flask in the following order: 0.2ml. of indicator solution; hydrochloric acid (approximately 0.1N) drop by drop. until the color turned from red to yellow; 10 ml. of ethyl alcohol; and
