Chemical and Physiological Investigations on Guayule Essential Oil Citation Dubbs, Clyde Andrew (1946) Chemical and Physiological Investigations on Guayule Essential Oil. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8vy8-kz07. https://resolver.caltech.edu/CaltechTHESIS:06012025-093758946 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Haagen-Smit, Arie Jan Thesis Committee: Unknown, Unknown Defense Date: 1 January 1946 Record Number: CaltechTHESIS:06012025-093758946 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:06012025-093758946 DOI: 10.7907/8vy8-kz07 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17337 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 18 Jun 2025 17:58 Last Modified: 18 Jun 2025 18:07 Thesis Files PDF - Updated Version See Usage Policy. 36MB Repository Staff Only: item control page

CHEMICAL ~ND PHYSIOLOGICAL INVESTIGATIONS ON GUAYULE ESSENTIAL OIL Thesis by Clyde A. Dubbs In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy California Institute of Tech:ooloSY Pasadena, California 1946 ACKNOWLEDGEMENT • First appre ci a t ion nu s t be expr'e s sed t o Professo1~ A. J . Haagen- Smit, wit h wh om the present wor k was carr i ed out. Especially must he be than ked for providing unusua l opportunities, providin g for assistance in the more routine portions of the work , and showing patience at times when progress may have been seeming ly lacking. Professor 11a agen-Smit and other staff members, pa rti.cularl y .Professors James Bonner, F.W. Went, and Laszlo Ze chmeister, have given real though less tangible assistance through their lectures or their publications. Dr. Gertrud Oppen heimer and Mr. Glenn Swinehart must be than ked for carryin g out several micro-analyses during the course of the present work. Dr.~otti Steinitz is thanked for a ll of the lengthy fractionations reported in Section III and, in addition, for the great bulk of the ester and physic a l constant data g iven in this paper. Mr. W. Clan cy, with his expert glass blowin g ability and desire to please, has been indispensable to much of the present work and other work not reported upon a t this time. Mr. Robert Dorland and .LJr. J·ustus l:i..irchner have always been very helpful. Dr.Ralph Siu deserves special thanks for providing the rough fractions of guayule oil used in t h e work of Section IV. Mr . .tiarve y 'l'omlin made helpful suggestions in regard to the large scale steam distillation apparatus. Finally, Mr. 'l: omlin, Mrs. Ger- trude Wilson, TVli ss Pricill a rloth and Mr s . Bever l ey Dandli ker are all than ked for t heir part in the work of Section III. SPECIAL ACKNOWLEDGEMENT Special appreciation is extended to my mother, Bertie lJubbs. Mrs. TABLE OF CONTENTS Introduction................................... II Micro-Analytical Methods for the Determination of the Essential 011 Contents of Different Tissues of Intact Plant Organs. . . . . . . . . . . . . . . 12. II. Essential Oil Relations During the Growth of the Guayule Leaf .......................... 89 III.Large Scale Investigations on the Variation of Guayule Oil Composition with the Age and Organ of the Plant ......................• 95 . IV. Appendix: Description of the Precision Fractionation Apparatus; The Detection and Partial Isolation of Many New Compounds from Guayule Essential Oil ................... 152 General Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1eoo Bibliography ........................... . . ..... , J"12 I~ CHEMICAL fJ\ D PHYSIOLOGICAL IN VESTI GA'r IONS ON GUAYULE ESSEN'rIAL OIL Relatively little study has been made of the role which essential oils play in the metabolism of the plant. The prevalent view today is that essential oil compounds are by-products or end products of plant metabolism. 'l'he analysis of the oils according to their locations in different tissues of plant organs is a necessary technique in the thorough study of es·sential oil relations. Appropriate methods have, therefore, been developed for this purpose. 'l' he formation of essential oils during the growth of the gua.;yule leaf has been studied with these methods. A large-scale study has also been made on the compositions of oils from different organs and the changes in composition of these oils with age. Pinally the construction and use of a precision fractionation apparatus has permitted the detection in and partial isolation from guayule essential oil of additional compounds. The biochemical significance of the results will be discussed. J .I,HCRO-ANALYTICAL METHODS FOR THE_ DETER~U NATI ON OF THE ESSENTI AL OIL __CONTENTS OF _DIFFEREN T TI GSUES _OF IN TACT PLAN T ORGN:NS CONTENTS Introduction ......................................... 14 Background.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Obj ec ti ves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, PrelLninary Experi menta l Ob s ervations................ IS Qu.a ntitative Experiment a l Evidence ................... 19 Typical Oil-Bearing Tissue Structures ................ 35 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Sui:nmary: Tentative Scheme of Analysis ................ 42 Volatilized Carbon Dioxide ..... _. . . . . . . . . . . . . . . . . . . . . . 4+ EXPERIMENTAL SECTION Preliminary Treatment and Storage Procedures ......... 57 The Volatilization Method............................ 62 Apparatus and Reage n ts .......................... ~2 General Procedure i :; ............................ Sub-Procedure A; Total Oil Content and Oil Contents of Different Tissues of an Intact ~5 Organ. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ Sub-Procedure B; The Rapid Determination of Total Oil Con tent.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -,3 Procedure for the Determination of Volatilized Carbon Dioxide, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genera l Discussion of the New Method ............ 7'S 7q Surri.: nary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 I)IICRO-ANALY'rICAL I\'. EI1HODS FOR 'EHE DEI'ERMI NA'l 'ION OF '£HE ESSEN'r IAL OIL CO N'rEN'l1 S 01<' DIPFEREN '11 TISSUES OF IN11 ACT PLAN 'I ' ORGANS Procedures will be described which permit the rapid micro-determination of the essential oil contents of small plant samples and which provide for a separation of the oil according to its location in different tissues of intact plant organs. 'l'he present method is based on a simple prin- ciple suggested by Haagen-Smit, namely, the direct volatilization of the small plant sample - a process which is essentially a micro-steam distillation in which the tissue water itself initially serves as the source of st earn. 'l'he volatile oil is subsequently combusted and determined as carbon dioxide. A special feature is the separate and simultaneous absorption and determination of volatilized carbon dioxide (evolved directly from the tissue). It is possible to tak e a small guayule leaf of 20 mg. (or less) dry weight and, within an average ti me of about 30 minutes, when a routine series of determinations is being carried out by one analyst, to obtain the followin g infor•mati on: fresh weight, dry wei ght, a value for "loosely-bound" oil content, a value for 11 total 11 oil content, and the amount of ttvolatilized carbon dioxider' (carbon dioxide which is directly evolved from the tissue by heat). At the end of the run there is available a dry sample, ready for any subsequent analysis that may be desired. The time can be shortened to as little as 10 or 15 minutes if the fresh weight and total oil content are the only informat ion desired. weight, with a 11 }'or a guayule leaf of 20 mg. dr y loosely-bound 11 oil content of about 1.0% of dry weig ht ·(or about 200 of oil), the precision is wi thin 15%. In order to realize the f u ll potentialities of t lIB new technique, more work must proceed on the micro-determination of indi vi dual essential oil co mponents. 'l 'his second phase is now ripe for progress in view of the many color reactions ava ilable for essential oil compounds on one hand, and the curren t rapid development of colori metry , spectro-photometry and related techn iques on the ot h er hand. With a vigoPously sup p orted effort along this chann el, results are assured. the present met h ods ma y also be of interest to the essenti al oil i ndustry since they provide a rapid an d con venien t tool fo1• k eepi n g a runni ng a ccount of t he crop yie lds of essential oil plants. Applications may also be developed for th e r apid evaluation of t he effects on cell structure of various freezin g pro cedures an i mp ortant consideration i n the work of t he food techn olo g ist. By d etermini n r. the amount by whi ch t he rate of oil volatilization i s i n crease d by a prelimina1<y freezi ng pr ocedure, the techn olog ist c an evaluate the degree of cell break down that is c ause d by this particul ar procedure. Baclcground. Various procedures are used to obtain essential oils from plants, including solvent extraction, absor•ption in hot or cold fats, pressin g , and ste am distillation. ~he constituents of t he oil fro m any given plant material will vary a ccord i ng to t h e method used. 'l'herefore, for the present work, Che essential oil s have been defined as those oils ob t a ined by steam distillation. As ste am d istillation is ordinaril y practiced on the ma cro-sc ale for the e x tr a ction and determination of essential oil, the oil is collected in a receiver ~s a sepa r a te lay er us ua ll y flo a tin g on the surfa ce of the wat er. but this oil contai ns more or less wa ter, p ortions of it have b een lost to t he atmosphere, othe r p ortions a re e mulsified. with the water, and still more will be lost durin g the subseque n t transfer and dryinf~ of t h e oil prior to fin a l we i ghing . In the micro- mod ification described in this paper, t hes e difficulties could be avoided. 'l'he pr esen t procedure ma ke s use of a tr a in for carbon microdetermination, as illustrated in 1' i g .15. '1 he sample, cont a i n ed in a s mall tub e (Fi g.16B), is volatilized at a suit ab le temperat ure and the vol a tile oil co mbus ted to c a rbon diox ide which is then absorbed in standard barium hydroxide for volumetric dete rmination. An a ccess o:ry is t he s mall absorption tube (Fi g .16A) which, a ttached to t he sa mp le tube, c at ches all the volatilized carbon dioxide, whi le allowin~ the oil to pass. Subsequently this vol a tilized c a rbo n di oxide may al so be determined by 17 acidification and aeration of the absorption tube in the tr ain of ~ig. 18. ~~r complete details, see the experimental section. Objectives . It is well known that the different or gans of a plant ( as roots, stems, and leaves) produce essential oils of different c hernic s 1 compositions ( Wulff ,1~23; Morrens, 1845 ); furthermore, it is almost certainly true that different tissues of a plant tussue (a s pith , paren chyma, and epidermis) als o produce different oils. ~herefore, in firs t class essential oil work, an adequate means must be made available for separately studyin f•; these different oils. Some oi l separations, as of petiole oil from mesophyl l oil, c an be readily accomplished by prelimary dissection of the organ. But if it is desired to mak e a separation of vein oil from parenchyma oil, the t ask is nearly impossible by simple dissect ion ; and the loss of oil durin g manipulations is a n ever present factor. ~he sectioning of the tissue a ccording to methods of micros copic technique and treatments of the section with specific reagents can yie ld informati on of considerable ~alue fr o m the qualitative standpoint. Moshkina (1940,1 940a) has made such studies on g uayu.le tissue. '.!.'his procedure may well accompany other methods ; but, by itself, t he procedure is inadequate for quantitative work. Let us now consider the basis for the new volatilization method. Preliminary Experimental Observations . .h. turgid guayule leaf', freshly picked from the plant, has a mild odor. As soon as it is crushed or even sli ghtly torn, however, the odo r is g reatly intensified. fhis is a common obser•vation, and it indicates the important fact that the intact tissue structure of a leaf serves as an effective brake on essential oil escape. · ·:-i : bor convenience, let that oil which does escape at the relatively slow rate be c a lled 11 free" oil. hHaagen-Smi t has made quantitative measurements of the 11 free oii of guayule, oil which is libera.tedthroughout the life of the plant). A fresh turgid guayule leaf, boiled in water for about one hour, removed, and the excess water absorbedoff, emits no essential oil odor. VJhen, however, the leaf is crushed, the odor is released. It is noteworthy that, although such a thin t i .s sue as a leaf is subjected to t h is extre me treat ment (in fact, the leaf structure is so degraded that even its petiole is readily ground to a smooth paste between the fingers), it still contains essential oil in its more interior portions. For convenience, let that oil which is tightly retained be called ''tightly-bound II oil; _let the postulated fraction il'.lter11 11 vening between it and "free oil be called "loosely-bound oil. A detached guayule leaf that has dried to brittleness in the a±r gives off no odor, nor does it do so when soaked in water and caut iousl·.v- heated to dryness a g ain. (it has lost its 11 free II 11 oil and its loosely-bound" oil). But let the initial dry leaf or the moist boiled leaf be crushed and the typical g uayule oil odor (of its ''ti ghtly-bound II oil) is a pparent. Similar results have been reported for other organs. "Schi mme l ( Durr ans, 1922) found that uncrushed Aj owan seeds yielded 20 per cent less oil than crushed seeds although the former were distilled twice as long as the latter, 11 and further "found that whereas a certain batch of cut vertivert root yielded 1.09 per cent of essential oil on distillation, a similar batch of uncut root yielded only 0.3 per cent. II Q.uantitati ve ~xperimental Evidence; '. l:issue Lag and the Diffusion Process. 1 Presented in 1' i g . (1) are a set of curves for the steam distill at ion, on an approximate 25 kg. scale (fresh weight), of several lots of similar four year g uayule stems (the important details of procedure being g iven with the fi gure). Cons.ider the di sti lla ti on of the unchopped sterns. 1'he g reat bulk of the oil comes over during the first 30 minutes of distillation; after a bout two hours, the oil distillation has essentially ceased. Yet when we chop these predistilled stems and begin redistillation, there is an immediate and rapid distillation of l a rge quantities of oil. From the initial steepness of the latter curve, it is, furthermoPe, apparent that the oil novr being distilled is at least as volatile as the oil which c ame over first. Due entirely to the structure of the stem, it is feasible to mak e a more or less sharp separation of the oil present in different tissues. 20 ,.._ I:c (!) -LU 5 ::c V) LU LE 0.20 LL a ~ '-' .Q -·w _.J _J ~ o.,o <n -0 _J -0 _J ~ ~ o.oo 0 50 100 DlSTl LLA Tl ON 150 wo iso TIME CM1NUTES) ~ig. (l)') Sharp :Moq2hological Fractionation; Steam Distillation of Four Year Guayule Stems. 0 • Two duplicflte lots of stems, 2_hopved into small pieces with a Wi ley mill {See Fig. 26) and separately distilled. o ■ Two other dup licate lots of stems, distilled without chopping . 4 One lot of predistilled stems ( o ), chopued and distilled again., 21 As a check it is of importance that the total amounts of oil obtained, either by chopping at t h e outset or a fter a predisti l lation, are identic a l (within the variability range of the material). ~he curves for similar larg e scale steam distillations of four year guayule leaves are presented in F'ig. ( 2) . .Lhe gentle slopes of t h e two curves suggest a g radual depletion of the oil content in the tissues by a slow process of diffusion brought into play b y the leaf structure. 'l'he situation is basically the s ame in the stems, except that the oil separation is sharper, apparently due to the pres ence within the stem of a dense - walled structure separating the differen t tissues. Even where such a special separating structure is absent, it is possible that the oil collected in the earlier vol atilization periods is the oil initially present in the more exterior cells, while the oil collected in later distillation periods comes from the more interior cells. 'l1o obtain a quantitative picture of the real si gni ficance of this la g effect, the t wo very steep curves to the left o'f the lea-f curves a re included. 'l 'hese curv es were obt a ined by adding to the still samples of free oil (which had previously been obt a ined by the steam distillation of three year le a ves). 'l 'hese distillations of' free oils illustrate the maximum limitation that can be imposed on oil distillation rate by a limited capacity of t h e steam to carry over oil with it (expressed in the theoretical water:oil ratio). A comparison of these curves with the leaf curves g ives clear evidence t ha t t h e oil:water 0 WID1> ..J _J F en 075 _J .·O _J :c:::r bso Fu. 0 l-z isw <.J 0:: lJ.J Q_ 0 0 50 100 DISTILLATION TIME I I 200 150 (M 1NUTES) I ' Fig.(2 ). 'I 'issue Lag; Steam Distillations of Guayule Leaves and Free Guayule Leaf Oil. o e Two oa Two duplicate lots of four year guayule leaves. duplicate samples of three year guayule leaf oil. , 250 23 ratio, as a limiting factor in the steamdistillation of essential oils fr•orn plant tissues ( even the very perme ab le thi n le af tissues) is entirely , inconsequential ! Fi g . (3) and (4) show t h e effects on oil volatilization of different preliminary treat ments of the plant tissue. We observe tha t the volatilization of 8ample 1, which was g iven no tre a t ment that would effecti v ely bre a k down its structure, lags well behind the other three comparable samples, on all of which preliminary degradin g treatments were em ploy ed (Fi g . 3). Another si g nificant effect is noted in the case of Sample 1. 1''i rst, let it be mentioned that a typica l and na tur a l effect of the volatilization procedure is the cessation of oil vol a tilization when the water is completely depleted fro m the tissue; more water may then be added, about 10 minutes allowed for it to be we ll absorbed, and the volatilization continued. Vihen t his treat ment is applied to Sample 1, i ncre ments of oil are obtained, b ut when, a fter this treatment, the tissue is a lso g round and water added, the oil increment resulting on continued volatilization is si gnificantly g reater - a gain clear evide n ce that the intact tiss u e structure i s a g ood barrier against oil escape. Fi g . (4), less informative in the present co nne ction, shows the added effect of initi a l slow freezin g s at -7°C or a t -l.5°C. The present results are inconclusive, it appe a rin g , however, that -7°c tre a t me nt may help to 11 open up 11 the tissue. It is well 3.0 . ~ -- - 1- :i: l!) w .5 Z.5 - >0 ~ 1A. 0 ~ 20 - .__,. tGRIN~ AooATER _J ,.s - F ~ _J ~ _J 0 __J .... t~ I ·- 1._.. / ~ ~ · 0,0 0 10 • 20 30 40 50 f,Q 7D 60 90 l00 11D 120 130 MO . VOLATILIZATION TI ME (M 1N UTEs } · Fig.:f3 ). Effects on Oil Volatiljz ation of rreatments Affecting ·-,.J.ii'ssue Structure. 1 , o Sample 1. Mild treatment ( a single dry ice freezing and storag ~"""'for less than one day). O sampJ.e 2. Alternate dry ice freezing and thawing every 3 to 5 d ay s f'or a period o'f'"'about 60 days. A. Sample 9 . Dry ice freezing and pulverizing; storage for one d a;y onlY:- 150 160 :z.s ~ :c 3.0 (.!) w 3 r a: 2.5 0 0 w N _J 1.5 r<! _J ~ ;Aoo WATER AooWATER Aoo WATER /. 0 _J 0 _J ~ 0.5 0 ~ 0 10 20 30 40 50 GO 70 80 90 100 110 120 130 140 VOLATILIZATION TIME (MINUTES) Fig. ( 4 ) . Effects on Oil Volatilization of 'l1 reat ments Affe cl:;ing •.i.i ssue ~ruct1.1r€:°-- - - 0 Sampl e 2. AJ.t ernate dry ice f:r-eezing and thawing every 3 to 5 d ays for~p:3riod of - abo u t 60 days. □ Sample 6. Initial slmv freezi ng at-7°c, followed by treat ment of SgmpJ.e 2 . A Sample 7. Initial slow freezing at -J..5°C, followed by treatment of Sample .2 . - 150 160 knovm that slow freezing techniques lead to large ice crystal formation and consequent internal crushing of the tissue. ''If· a peach is cut in half, and one half frozen at -l00°F (-73°C) and the other half at o°F (-1s 0 c), examination after thawing showsthat the former can hardly be distinguished from a fresh peach and that the latter is greatly damaged.'' (Jacobs, 1944). As carried out here, the technique may not be o ptimum, and the result is obscured by the alternate freezings and thawings o ver the period of dry ice stor age. ~he fi gure is most VIS.luable as an indication of the reproducibility that can be achieved by the present method. Finally 1''ig.(5) preseits the effects of different volatiliaation temperatures. An important result to note is that the rate of oil volatilization durin g an initial volatilization to dryness is qu.i te independent of the rate of water volatilization ( again the insignificance of the oil: water ratio factor ! ) . If the water is removed rapidly as at 120° or 110° (see Fig.{6) also ), the diffusion process has a relatively short time in whi ch to proceed and the oil ob tained i .s relatively small in amount. On t .he other hand, if the water is remo v ed more slowly as at 100° or especially 90°, more time is allowed for diffusion and amount of oil obtained is considerab ly g reater. ~ · "1 z.o t- 4-0 0 ~ _J 05 > -0_J 0 0 ,.o - _J <( _J -I- N _ ·I.S w 0 ~ ..__ 0 lL ~ >- ~ 25 l!) 1:r ,-.... 3.0 10 zo 30 40 ~ ro oo + Add water ,Add water f20~ Add wo+e~ oo ~ ~ ~ ~ Q Sample 3. esample 4. ®sample 5. 0 Sample 2. ~ 120°. 110°. 100°. 90°. ISO 160 170 l&O 190 20D tlO Fig . (5). Effects on Uil Volatiljz ation of Various \ Vol atilizati on Temperatures. Grind Add , woter VOLATILIZATIO_N : Tl,M.E (M,NuTe:s) ~ ~------------· ------- I I) .. 120~ Z20 100 " ..,- - - - - -- - - . - - - - ---.--.,..---i..----. ....I- :x: 90 0 J 80 :c (II w LE 70 IL 0 ~ ,o ......., I-: <( 50 _) 0 > 40 0::: w 30 ~ .3 20 _J 4 ~ ~ 10 O· 0 10 20 30 40 50 60 70 60 90 VOLAT. TIME (M1N.) ' Fi g . ( 6). • Effects on Water Volatiljz ation of Various Volatilization iflemperatures. 0 Sample 2. ~ Sample 3 ~ • Sample 4. ® Sample 5. 120°. 110°. 100°. 90°. 100 ,-.. LOO I- ::c (9 90 ~ :c 80 Ill lll tr LL 70 Ii. 0 ~ .._ ,o ~ 50 ~ _.J ~ 40 a:: w 30 ~ 20 ~ _J ~ g 10 0 0 10 20 30 40 50 ,o VOLATTIME (M,N.) Pig. ( 7). Effe cts on Water Volatilization of '.L'reatmen ts Affe cting Tissue structure. Cl Sample 1. Mild treatment (a single dry ice freezing and storager"or less than one day). 0 Sample 2. Alternate dry ice freezin g and thawing every 3 to 5 days for a period of about 60 days. A Sample 9. Dry ice freezin g and pulverizing; storage for one day only. 30 ( It wi 11 be noted that in the studies Just <, presented ~ , no distinctions . • ~wel'le made between the volatilized carbon dioxide arising directly from the tissue and that , resulting from the actual combustion of oil, the procedure for separating the two having not 11 been brought into use in time; and, therefore, the term oil 11 includes both quantities. It is shown later, however, that the curves for volatilized carbon dioxide alone (Fig. 11 to 14") are strikingly similar in form to the present "oil tt curves and quite reproduei b le in magnitude for all the plant material used in the present work. ·i'herefore, if a c o rrection were applied, the 11 oil 11 curves would be merely shifted lower along the ordinate, but would remain essentially unchang ed in typical form and in relation to each other). A. General Data Common to All Samples pl ant hiaterial. Parthenimu argentatum Gray, Variety 593. Date of Sowing. February 12, 1945 - . p1a.ce. Orlando Road Nursery of the California Institute, Pasadena. Conditions. Some time after seed germination in s and flats, these f l ats were placed outside, and growth proceeded under weatlIBr exposure until transfer, at which time the plants, alth ough vigorous in appearance, were much smaller (12 to 15 cm. hei ght) than uncrowded plants of similar age; almost all still had single stems. Date of ·rransfer. - December 14, 1945 Pl ace. One flat was transferred to Room 1 of the controlled conditi on greenhouses of the California Institute (Went, 1944). l his room has a slanting glass roof and aluminum painted walls. 1 1 Conditions. 1s 0 c (65°F) day, 13°c ( 55op )ni g ht. Well li ghted position, although only upper leaves of plants received direct sunli ght. Tissues Analvzed. Large, mature well-developed leaves from the largest plants. B. Harvest No. I II III IV V VI Date Harvesting Data 'l'ime Light Condit ions ~ge ( _ys) 'l1 ime in Room 1 :eer Sam:ele(l {d a ys) 308 3 12/17 /45 2:00 P. M. Sunny 309 4 12/18/45 11:00 A. M. Overcast 96 401 3/20/46 11:00 A . ~.il . Overcast ,:i!'a:U1y 406 101 3/2¾46 11:00 A.M . Sunny 4/1 46 11:00 A. 111 . Alt. sunny & over cas t 413 108 418 113 4/6/46 8:00 A _lVl . Sunny ( 1) ~l signifies slightly less than 1. Ave.h!Q. 1.~a V §§. 1 3 Z'l 2'1 ~l II II II 3°c 51 d., ~ dry ice 14 d. II III 8 9 Freeze i n dry ice, pulv .(2d); p l us 3°C 1 d . -l.5°c & dry :ice 63 d . Fr eez e a t II 7 -7 C & dr y ice 6 2 d. 55 d. 51 d. 4 3 d. 56 d. ( 2b) Free ze a t 11 II 11 11 of.cc} Freeze i~ dry ice II II If II " 6 II II II 4 5 ti ll dry i ce 7 h rs. 11 ..;;nwrw.ta{,2a) f ree ze i. n Pr eliminary Treat ment an d Stor age (2) ·o..,.,. • •· • 11 11 II Har vest No. ( 1) 1 120 120 120 120 90 100 110 120 120 (OC) Vol .T emp . 20.0 23 .o 36.8 41.1 23 .3 32.6 29 .2 34.8 45.5 1.46 1. 63 1. 36 1.56 2.01 1.48 1.28 1.32 0.77 78.9 75.9 74.5 72.9 77.8 72.2 73.8 78.0 75.8 3,7,11 4 4 5, 6 5, 6 5,6 w t-> 3,4,5,6,7 3,7 % Oil(3) ·:1· % Wate r Fi gures mg ._ ( % Dry VV_t~ )_( % F'r~s h_ VVt.) D( y W). 1 rr. C0ND}_iN SED DATA C0NCEF1'1ING ESSE!'n. I AL 0IL(3)DETERMINA'lI0NS AS ILLUS'l 'RA'l'ED I N B'IG. 3 .ro7. 3 2 1 N o. Sample TABLE 33 Notes to Table II (1) See fable I . (2a) S:terilhration'r is a rapld heat treat ment above l00°(see p. 5 7 ) (2b) These lon g storage periods in dry ice a ctu a lly involved alternate freezi ng s and thawin g s about every three days. (2c ) Seep. 58 for the freezing procedure used here. ( 2d) See p. SB f'or t h e pulverizing technique use d here. ( 3) 'l1 h e term 11 oil" a s used he re was determi ned from t h e tot a l am ount of c a rbo n dioxide found after combustion although a portion thereof was carb;i'.on dioxide directly evol \red from the tj_ssue duri ng volatilization. See te xt for discussion. ·l.'he conversion f ac tor for conve rti ng cc. of N/10 barium h y droxide used to mg . oil is 0. 34 , on the ba sis of an oil comp osition of C5 H8 . The values g i ven are for am ounts of oil ob tained during initi al periods of volatil~ ation to dryness, and a re, t herefore , me a SJ. res of t he effects of the va rious treatmen ts. More comprehensive pi ctures of the effects are g ive n in the runnin g accounts of the distillations, a s provided by the fi g ures referred to in the l a st column . • }'reeze in dry ice, & 3° B d. IV 14, 14 Volatilized carbon dioxide ( 11 Co2 r' in the table) is the __carbon dioxide which is released d irectly from the tissue by volatil~ation. It is de termi ned h ere by omitting the combustion process. J;t·or quantitative comparisons with the oil values, the amounts are e xpressed on the C5H8 basis. 78.5 II 11 12 13 13, l1 (2) 0 .72 76.4 80.0 7tl.3 76.8 79.0 77 .7 See Table I. 46.9 0.65 0.66 0.25 0.88 0 171 0.61 14 Fi~ures (% Dry Wt) OZFresh v'lt. % co 2 <2 ) %Water (1) 120 23 .9 16 Fr~~~e in _drg ice, pulv., c< 3 sev .hrs. 120 stertJt-1..:e ti 15 3° ½ hr. 28.9 20.9 III 14 90 33 .0 120 Sfef'i.li~: & dry ice 85 d. II 13 Freeze in dry ice, pulv., & Freeze at ;;4 °c, & dry ice 79 d. If 12 120 32.4 ( mg. ) Dry Wt. 38.8 3°c 51 d., & dry ice 25 d. ti 11 120 [0c) Vol. l'emp • 23 to 120 3°c 51 d., & dry ice 20 d. II Harvest Preliminary No. ( 1) 'l1reatment §ind~ St 0!'a~e CONDENSED DATA CONCERNING VOLATILJZ ED ( 2) CARBON DIOXIDE DE'rERMINA'r IONS AS ILLUS'.L1HA'l1ED IN FIGURES 11 to 14. 10 Sample No. TABLE III. .t,. W 3 5' Typical Oil-Bearing Tissue Structures. It is desirable to get a clee.r conception of the actual physical structures of various oil-bearing plant tissues with si:;e cial regard to the location of the oil therein. 1 1 l he structures and oil di stri buti ons a.re <l!Ui te variable from tissue to tissue and from plant to plant, and it is convenient for practical purposes to make a simple classification of the principle types by means of the much simplified diagrams (in longitudinal section) of F1g.(8). Type A represents the case of an essential oil tissue from which oil is given off to the atmosphere as soon a.s it is formed; in other words, there is no stor~ge. This oil may be considered as one type of the so-called "free" oil. Type B represents a case where the oil is distributed throughout the cytoplasm of the living cells in the form of many tiny globules. Type C is a similar case where the globules are fewer and larger ( Vl.'u l ff, 1933; Moshkina, 1940). A portion of this oil may find its way to the atmosphere as "free II oil, while the rest would be cal led "loosely-bound" oil. 'l1ype D represents the case where specially differentiated epidermal cells or hairs collect and store oil at the very surface of the tissue. 1 'l his type of structure is perhaps the most prolific source of "free" oil. T~pe E represents the case where internal pockets of oil are for.mad. ·rhe oil may be inside special cells, or the gland may be schizogenic, storing oil in a _cavity between cells. This oil can be called "tightly-bound 11 oil. 'I'ype F represents the case where certain colurnns of cells have degenerated and become filled with oil - resin ducts A > ·• . ~ I~ 1-o1 8 · ' ;.,.. f •' It: ""/ i f... ~i ~l ·• - h t · ; l'C:;,: ·: ) ' ~ ,,,, •; •.. :.-1 .• - ~ .... : --. ,:; • ... •' • .• . , . -; ; .: r' ~ .. ~- .:,. ./ .-..~ ...... f'> ' .~ ! .......... . . . . . . . . .. . . . .. .. ..... ..... ... C .. .. .. .. .. .. .. .. ... .. D I E Fig. 8. I ■ ■ ■ I Simplified Diagram Representing Typical Oil-Bearing Tissue Structures. 37 (lysogenic structure)o The oil can also be called a "tightly-bound" oil .. Guayule leaves apparently have a structure and oil distribution corresponding to a combination of Types Band C and Type F (Moshkina, 1940; Artschwager, 1943) 0 3'o Dis cu ssi on. The rate with which the oil is volatilized is determined by the rate at which the oil can diffuse out of the tissues. 'l'his diffusion is greatly :i:nfluenced by par•ti cul ar structural elements between the tissues. 'l 'hese barriers of free diffusion can be broken down by grinding or freezinr: procedures. It is a law of diffusion that the amount of material which will diffuse across a g iven medium in a given period of time is directly proportional - to the concentration g radient across the medium. In the present case, whether the medium be cell membranes and walls or a structureless tissue ma ss, the external concentration is zero, and the amount of oil diffusing out during a g iven volatilization per iod is a dj_rect r11easur·e of t b e oil eoncentrat ion or content in tri.at portion of the tissue f r om vrhich the tissue stru cture pePr:;J. ts d i ffr,si or, to t e!1~e p l 2 ce . .i~ceo:edi:r,r:1y i r Vi· e subl,,it 2, re asona"'.nl y ir1tact guayule le a f to a short period of volatilization, we ma y ssy , fro n: s. kn oyvledg e of the guayule le a f structure, tha t the v a lue is a direct measure of the oil content of rnesophyllic cells, it being postulated that diffusion from the res in ducts of the petiole and veins is negligibly slow. ('I'his mesophyllic oil content is a specific type, thus, of "loosely-bound ,_: II oil). Now if we initially pulverize a tissue and thoroughly homogenize it, all the oil from all parts of the tissue will be thoroughly mixed and distributed uniformly throughout the mass. 'l 'he result of a volatilization in this case will accordinp.: ly g ive a v a lue that is a direct mearu re of total oil content. In a steam distillation of free essential oil, the composition of the oil which first comes over is by no means similar to that initially placed in the still; it predominates in lower boiling constituents. ~his effect is the result of a complex oil-water distilling composition, in which each constituent is represented in proportion to its particular vapor pressure at the temperature of di still at ion. Uf course, the low boiling constituents are poorly represented, and the magnitude of the effect is illus~rated in Fig.(9). Whereasthe water:oil ratio over the terpene range is less than 2, it is of the order of well over 100 for most of the sesqui terpene range. In other words, we may say the amount of terpenes initially coming over will be roughly 50 timesthe amount of sesquiterpenes, regardless of how much sesquiterpene may be present in the initial oil. Now consider the situation for the steam distillation of oils from plant materials, where another distinct physical ' process, diffusion, is in control. 'l'he rate of oil volatili- zation will depend on the rates at which the v a rious oil constituents diffuse from their respective locations within the tissue to the sufface of the tissue. It is another law of diffusion that compounds diffuse at rates inversely proportional to the square Poots of their molecular weights. Therefore, the relative rates at which terpenes (C10H1 5, mol.wt.136) ~o ,-~·- - - - - - - - - - - - - - - - - - - - - - - - - - - - - !! t 0 50 - 0 ~ 30, 0:: _J 0 • • 20 0: w \- 1 ID TERPENE _ ~ G E _ __ 0 0 50 100 150 • 200 zso 300 . B. P. OF COMPOUND CODISTILLING WITH WATER'' Fig.(9). The Compositions ' (Water:Oil Ratios) of Various Cod2stilling Systems of Water and an Immiscible Organic Compound as a Function of the Boiling Points of the Organic Compound. (From data calculated fro m ori ginal data in Youn g, 1922). and sesquiterpenes C15 H24 ) mol.wt.204) di ffus e out will be 204:136 = 1.2:1. In other words, the terpenes d iffuse out but little f as ter than t he sesquit erpenes. 'l'hi s means tha t as the va rious oils, located deeper and deeper within t h e tissue initi ally, be gin to diffuse out, there .will be a mini mum of overlappi ng due tothe more volatile ( s ma ller molecular weight) compon e nts of a more interior oil overtakin g the less volatile (hi ghe r molecula r weight) co mp onents of a more exterior oil; and as soon as e a c h individual oil reac h es the tissue sur•face, it is imme di atel y whisked away i n it s entiretv by t he steam. 'l 'his last stateme nt is true since the am ount of oil li be r ated during a g iven mome n t, even consi d eri nF its content of compound s of rel at i vel y low volatility, is typ ic al l y insuf ficient to saturate t h e c apacity of the water that is a va ilable to c arry it away i mrnediatel y (even in the micro-vol ati liz at ion method , where wa ter is relatively sc arce). Surmnary: 'r entative Sche me of An alysis. ~ A tentat i v e scheme o f analysis is suggested in Figo (lO)oit wi :fl ·b e n ote d th at those most v aluab le p ossibilities, i nv ol ving a s tudy of the oil compositions of t he fractions, are not a t the moment experimentally realized. In order t ha t this t yp~ of ana l ysis may achieve its proper place a s a valuab le tool, analytical met hod s for the dete rmin a tion of specific classes of oil co mp onents or, better yet, specific indi v ictua l oil co mponents must be developed on the micro sc a le. 43 Intact Living Tissue I Freeze ip dry ice, pulverize, mix . tho7hly Volatilize under living conditions (after Haagen - Smit) \ "F'ree II Oil, con ti nuousl y emitted by the plant during life Total Oil (not including "free 11 oil ) I Break up tissue structure I Freezing techniques I Heating techniques I \ Can vary severity of techniques widely >tf i ght ly- Bound U Oil Grind - - : . , . . _ ~ : _ _ - - - - -- - - = - - - - - (in resin ducts,etc.) "LO OS el\oun Ion (Several fractions may be tak en to determine the composition gradient from exterior to interior of the tissue). Fi g .(10) , 'l 'eritative Scheme of lhicro-1\lorphologic al by Volatilization. Fractionation 'I'his is a phase of work that is ripe for ad van cement in view of the great number of highly sensitive color reactions published in the literature, and the present-day rapid strudes ,in such techniques as colorimetry and spectro-photometry. Volatilized Carbon Dioxide. In the course of this work it was observed that considerable amour1ts of acidic material are evolved directly from the volatilized tissue. 11 This material shall be called volatiJ.ized carbon dioxide", to distinguish it from the carbon dj_ oxide fo:rmed by combustion of the oil. .l.'he amounts obtained from different le aves are somewhat variable, but usually lie within a rather limited range of 0.5 to 0.9% of dry weight ( calculated to a. C5H8 basis for quant:i.tati ve comparison with oil values). 'l'he determinations are repre- sented in a series of curves (Fig. ( 11) to ( 14) ) and by the data collected in 'l'able III (in which and appended footnotes all immediately desirable infor'mation and e:x.periment8] qet.ails are condensed). 'Lhe full details of experimental procedure can be found in the experimental section. 'l'he evo1ution of this ma teris1 was originally a source of considerable concern. 1 'he apparent necessity of ma.king separate runs on duplic8te samples in order to deterr1ine the true oil content by difference was a seriousthreat to the method at that t irne. uince the work present eel here, however, t he simple means of quantitatively separ a ting this volatilized carbon dioxide from the oil before con~ust ion was devised. The question is of interest whe re this carbon dioxide finds its origin. h few experiments were carried out to determine Whether this carbon dioxide arises simply by the evolut l on of 45" ~ Z5' 3: - >0::' a"- ~-0 0 ~ .._ 0 uJ N 1$ o.o o 10 20 30 40 so ~o 70 VOLAT. TIME (M1N.) Fig. (11). Comparison of_the Relative Amounts of Volatilized Carbon Dioxide>and Uil Plus Volatilized Carh on Dioxide. 0 Sample 9. Volatilized carbon di oxide plus oil. • Sample 14. Vol ati lized carbon dioxide alone. 0 Sample lS. Volatilized ca~bon dioxide alone 11 11 ( blan ched ). carbon d ioxide he l d by buffers in the tissue sap, or whether it may arise from t he decomposition of or ganic materia l. The possibility existed that, during a g radu al rise of the vol at ilization tempe r ature by reasonably small increments from room temperature, ~h e evolution of carbon diox i d e in some part icular range might suddenly incre a se more t han would, for example, be a ccounted for by disso c iat ion of buffer-b ound carbon dioxide. Howeve~ no such ma r ked evolution could be found as shown in Pig. (12). However, the possibility of sudden decomposition is not at a ll disproved, for it i s very p l ausible to bel i eve that the ol d tissue l ag factor thoroughly discussed elsewhere, and the conseque nt ne cessit y of slow diffusion may effectively ma sk and distri bute any sudden release of c a rbon di oxide within t h e t is sue. ( F i g .(1 3 ) s u ggests such a d iffusion effec t , slower but g reater fi na l evolution at 90° tha n a t 120°). Carb on dioxide within ~he t is sue is comple tely surrounded by water and can onl y escape by solution i n and diffusion t h rough the water . Presumab l y when the tissue beco mes dry, it "closes up" and b eco mes i mp ervious even to c arbon dioxide ga s which c annot le ave until added moi sture reopens a wa ter path to it. ( 'r his view is supp orte d by the acceler a ted evolution a fter fresh a dditions of water to t he tissue ). Our experiments do not permit us to draw any definite conclusj_o ns re gardin,i;; th e specific for•m in wh ich this vol at ilized 47 carbon dioxide j_s present. 'l 'he views of Smith (1940), who attributed the carbon dioxide content of plant tissues to buffer systems, may satisfactorily apply in the present case. ,:r f:2 o.o ~ ..J,S d"' u~ ~-0.5 ~ __J N 0 ~ er ~ 1.0 0 20 ~ 31•1 3G'I , ◄O I 40'1 49•1 ,o I S!tl G7° '7" 80 : 100 '40 l&O 180 Sample 1 3 . . 20Q z20 VOLATILIZATION TIME (M1NUTe:s) 120 Aoo WATER fGRIND Fig. ( 12). Effects on Carb on Dioxide Volatilization of a Gradual Rise of Vol a tili za tion TemEerature. 23•1 0 i • Z40 ~OD WATER loO 280 300 3.20 340 1209 .. 0 '<' 10 20 30 40 E,O 70 80 90 ,oo uo 120 IAoo WATER k~l/'10 130 140 ISO lbO AooWATER T o{o) ONLY 120°-- VOLATILIZATION TIME (M1NUTES) SO ------------------ 10 9, f'z~ no 180 190 AooWATER 200 . • Sample 12. 90° 120°. o Sample 11. Pi g~E_l Effects on Carbon Dioxide Vol a ti l:iz ati on of Two Volatil:iz at ion Temperatures. g o.o ~ _J~ Uvi cfeos ~ _J ~ <( t) ,.o 0 lL. 0 ~ -~' - ,.s 210 ~ ~ 0 la.. ~o:s bF- o.o :<( .... _,~ uf] 0 ::s. 0 <:( ....J ,-..: 0 iO 20 30 40 60 70 80 90 IDO 110 A00WATE"A 120 130 140 Aoo WATER ISO l'-0 . VOLATILIZATION TIME (MINUTES) - 50 DD WATcA __ --... -- .,.. .... 170 o o • 180 t> ample 16. c.tored at 3°c for 3 days. ( Hor-vest DZ") . Samples 11 and 10. Both -sto:r·ed at 3°c for 51 days and in dry ice for about 20 days. (HarvestH). Fig.(14). Reproducibility of Volatiljzed 9arbon ~ioxide Determination. -., -/.0)-• ~ 0 0:: >- ~ i--: ~ 190 200 210 0 U\ 51 A short investigation was made to assure that this acidic material was actually carbon dioxide and did not contain aonreciable quantities of volatile acids native .. to the oil itself ( nor volatile acids arising from the tissue as a consequence of thermal decomposition); and, in addition, to assure that apprecJable quantities of ammonia ( which might arise from ce rtain sensit i ve amides possibly in the tissue) were not produced to depress the actual acidity. A bri ef description of the procedures used and the results are given in Table [ f a nd footnoteso The esBential results are collected in Table IV-B. We can draw these conclusions therefrom: lo The amount of volatil@ basic Lmaterial is negligible. 2~ The amount of volatile acids is probably very smallo (More data are needed, but on the basis of the data ava,ilable, the error involved by neglecting the amount of volatile Rcids is 20% at worst) . More evidence on this point is supplied by numerous acid analyses on a wide variety of guayule oils; the amoun ts of acid are consistently negligible It Therefore, with a maximum errror that is probably well below 20%, we can justifiably call the evolved acidic material 11 Volat i lized Carbon Dio xi de'' o 0.09 18 20 19 0.01 0.01,0.01 0.01 - 17 ,cf Jo NH3 Sample No. 0.40,0.42 0.08 %CO 2 0.01 0. 01 ( 3) 0.01 0.03(2a) 0.28,0.36 32.7 28.3 21.9 21.9 -0.02 %Vol . Acids 11 II 11 20 b . Su mmarized lJata . II II ,, II Basic II Acid receiver first 19 18 17 ~ £ -- 0.01 0.09 o.01(2a) --- --- 0.42 0.36(2b) 0.40 0. 28 ( 2b) -- -- 0.41 0.32 I\\ °' 75 .2 77 .1 77.6 75 .6 Acidic :Material 2f Wate: Ab sorbed Boiled Out Aver. E\r1Dh1'JCE '11 HAT CARBON DIOXIDE IS 'l1HE ONLY ACIDI C (OR BASIC) rviAI1EhI AL DIREC'r LY VOLA'l'JLIZED FROM GUAYULE LEAVES I N APPRE CI ABLE ANIOUNTS (1). A. Detailed Data . 4 ,, , . (zl Basic Ma terial Sa~) h-rr ang e ment of rtecei vers Dry Ab sorbed Boiled Out No. W:C:-(mg.) ·-- TABLE IV. ( 1) All fj_ gures g iven in both parts of this table have been conve_1,ted into terms of % oil (on a dry weight basis). '.!.'his places all figures in the table on a compa rable be.sis among themselves and the fj_ g ures of other t a bles. ( 2) The vol a tilized material was passed throug h two successive receivers, with the acid receiver first in two runs, the basic receiver first in the other two. With the acid receiver first: (a) The ap parent basic material absorbed by the a cid receiver is equal to the actual basic material absorbed ( ammonia ), minus the acidic material absorbed (vol a tile acids soluble in t he dilute H e1). ( When made b a sic, the mat eri a l boiled out is ammoni a alone). ( b) 'l 1he acidic ma teri a l abs .o rbed by the basic receiver must be carbon dioxide alone. ( When made acid, the material boiled out c an likewise be only carb on dioxide). ( The excess of t h e 11boi led out" value over the 11 absorbed II va lue in the case of ~am ple 17 may have b een due to cont amin a tion of the receiver by some carb onate carried over fro m a prior determination). (3) Wit h the basic receiver first, the basic ma terial absorbed by the acid receiver c an onl y be a mmonia. (4) All s amples are from harvest IV. EXPERIMENTAL SECTION EXPEHiillENTAL SEC11 ION The full details of procedure will be g iven in this experimental se c tion. PRELBIINAh'Y TREATMEN'l ' AND ·s'l'ORAGE PHOCEDli"RES The fo l lowinf.-:.: procedures have been used for the preli minary t reat ment and st or age of tis sue s a mples: Procedure I .( Hecommended for Determination of Oil Contents of Different T issues in an Intact Organ. 1. Nu mb er and vreigh an empt y sample tube (Fi g .16B). 2. Harvest the sample and immediately transfer it to the s ample tube. 3. Sterilize the s ample promptly as follows: a. Insert t h e entire len g th of t h e s amp le tube into the close-fitting well of a heat ing block; temper• a ture, 140 - 150°C; 20 seconds. b. Withdraw the samp le tube and immediatel y i mmerse the g reater portion of its len g th in ice water for 10 seconds. nemove and wipe dry. 4. Rewei gh t he samp le tube. 5. Place the samp le tube in a screw cap bottle (c apa cit y of 30 or more tube s ) an d store either in : a. Dry ice, or b. Refrigerator Procedure II. (Pulverizing; Recommended for Determination of :l:otal O:i.l . la. (Replace Step 2, P.I). Wrap the sample in metal foil, freeze in dry ice, remove and pulverize thoroughly with a hammer; 1 b. repeat one or two times. 'l1horoughly mix the pulverized mass and trans fer it to a sample tube promptly. Procedure III . ( Slow Freezing) . 4a.. (Inser•t after Step 4, P.I ). Place the sample tube in a small screw cap vial, which then im.merse in an i ce-sa.ltwater bath of appr•opri ate temperature ( as -2° 1;o -100) and leave overnight in refrigerator. Discussion and Notes . (Note: Figures in parentheses refer to particular steps). In ~rocedures I and III, the sample is initially stored in the same tube which will later be directly introduced into the volatilization apparatus. Thus there is no transfer and attendant losses at any time between harvest and volatiliz 0-'tion a marked advantage. Sierili-ZC#tion is desirable if analyses for physiologically labile substances are to be c a rried out on the sample, subsequent to volatilization., a procedure that will become especially profitable in the future when the present vol a tilization .method is converted to low temperature, low pressure oreration. Other- wise this step may properly be omitted. 5'1 The step is similar to the p r ocess of b l anching used in the food industry. Blan ching, or i mme rsion of the tissue in b oiling wa ter for a few moments, effectively ina ctivates the enzymes which would otherwi'se cause spoilage ( chemical changes) upon storag e; as attested, for example, by the increased stab ility of cert ain sensitive vitamins in tissues so treated. 'r he brreef blan_ching treatment of itself causes only negligible chemic al change. 'l 'he step is inserted here as a simple precaution, since there will often be delays between the ti nie of harvest and the time the s amp le is frozen, or between the time the sample is removed from cold storage and the time volatilization is begun. Data g iven in Table V show that the simple sterili-iotion procedure , as describe d, effectively prevents mold growth which atta c 1rn untreated. leaves repidly, e specially when their cellular structure is first bro ken down b y a prolong ed series of dry ice freezin g s a nd thawing s. The process presumably inactivates enzymes with si milar efficiency. (3b), 'l1 he hot sample tube is promptly immersed in ice-water in order to condense all vaporized wate r (within the tube) on the wa lls of the tube and thereby a void dondensa.ti on on the cork; this condensed water is subsequently reabsorbed by the leaves. Data given in TableYshow tha t loss of water from the s ample tube due to ''blanching " is negligible. Loss of materi a l during per'iods of prolong ed dry ice storag e is also ne g ligible, even after two months. (1 ab). Un til such ti me .as a satisfa ctory means can be found for• pulverizing the fresh sample in the same tube as used for volatilization, the pulverization method will suffer from a considerable dissd.van tage. Otherwise this me thod has particular merit in those cases where it is desired to ob t a in, with maximum speed, va lues representative of t he total oil content. ( 4a ). Work in the frozen food ind.us try has shown that, whereas rapid freezin g (for example, with dry ice) causes the formation of many small ice crystals within the tissue, slow fr.eezi:hg permits the slow g rowth of ice cry st a ls to rel a tively large size. These l a rge cryst als can be effective in l?reaking '4P tissue structure. fl II ti 11 Ster-il ized... : 22 23 24 25 30 ,, II ,, ti none 29 fl II 28 ti dry ice If 27 ,, II 23° II fl 11 26 II dry ice 34 d. then 23° 23° none Storage Treatment 21 Sam;ele No. SO IVIE EFFECTS OJ:i' TABLE V. 90.3 132.0 74.0 147.0 77.7 116.9 - - - 0.2 - 0.2 -4- 0. 3 + 0.3 + 0 .91 - o. 2 ~ ~22.2 o.o !.. -22.7 } - 0.3 - - - -17.5 - - -22 .8} --- Fair g rowth after 19 d. Mold Ct>owth No g rowth a fter four months C) lS' 'rhic k gr owth 6 d.after removal from dry tc e After 51 d.Storage ( mg• ) - (mg.) Chang e of Wt • . After &terili-zatfo n '· - 80.9 63.2 Fresh Wt. (mg.) S "Il:R-JLfZAT10N AN D PROLONGED STORAGE. T HE VOLA'l'ILI ZATI ON METHOD Appar>atus and Reagents The apparatus, as shown in i,·i g .15, is a convention al train for t,h e micro-d etermination of' ca.r>bon. Placed immed- i a t e ly before the coniliustion zone is a s mall heating block, surrounding the vo l a tilization zone in which the sample tube is p l s. ced.. I t is thereby p ossible to volatilize the water and oil from the sam ple at any suita b le te mpe r ature, h eld consta nt within a r ange of two or thre.e de g r•ees; this volatilization step is the essence of the meth od. ~he oil is then combus ted to carbon dioxide which is finally abs orbed and de termined by back titrati on in a receiver· of standard barium hydroxide solution. Appreciable amounts of volatilize d carbon dioxide, evolved directly from the tissue, are quantitatively abs orb ed prioI' to combustion by a special absor·ption tube, attached directly to the sample tube (F'i g .16A), andthroug h which all mater•ial vol a tilized from the sample tube must pass. (The method for quantitatively determinin g the amount of vol a tiJ,ized carbon dioxide is described l a ter). Necessary Reagents are: St anda r•d O. 05 N HCl . Approximately 1 N Ba (OH)2 (carbon a te - f ree if th e a mount of volatilized carb on dioxide is t o be determined subsequent ly). CT T A ,~, p ( Approximate Scale, 1:8). C. Combu stion furnace . R . Re c eiver fo r absorbin g c arb on di oxide. M. Ma riotte bott l e . Complete Train as Used for the Micro-Determination of Essential Oil in Plant Tissues . P. Pressure re g ul a tor . A. Ascarite tube for purifying oxygen of carbon dioxi de . 'E • Thermo me t er. C'l '. Comlmst ion tube . H. Heat in g b lock . Fig.15. C -02 1)1 ~ Fig . 16. Sample and Absor~~ion Tubes. A. Absorption Tube for absorbing vol a tilized carbon di oxide (evolved from tissue). Bo Sample tubes (closed end type preferred). c. Prepared ab~orption tube attached to sample · tube, assembly ready for vol atilizationo General Procedure . Inst·ructi ons for t he g e n eral procedure are a s follo ws: 1. Ad just the combustion furn a ce to the pro!£r temperature. 2. Adjust the heating block flame to g ive the proper vol a tiliz a ti on terJ.pe r a ture ( usua lly 120° or 110°) . 3. AePate the entire train with a current of oxygen. 4. Run the proper kno~m am ount of standard b ar ium • hydroxide s oluti on (8 to 10 ce.) into the receiver and attach it to the apra ratus ; attach the Mariotte bottle to the outlet tube of the receiver. 5. Open up the stopcock to _the lvlapiotte bottle to draw a s mal l current of oxygen through the receiver. Adjust the pressure re gul at or to give a :r:resrure head of about 5 cm. of water. Adjust the arm of the Iviari otte b ottle to secure a proper rate of flow ( 3 to 5 cc./ min.). 6. Prepi re the a ssembly shown in Fig . IGCas follows: Hinse the absorption tube, shaking out excess water, push in a moistened wad of g l a ss wool, to which the n add about 3 drops of 1 N barium hydroxide solution. F'inally attach this tube to the s ample tube. 7. Push this assembly along the inside of the combustion tube until the sample tube is into posit ion within the volatilization zone, thus beginning the volatilization. 8. Continue the volatilization for the proper period. Observe the followin g precautions: a. All water, which may condense ahead of the combustion zone during the oo urse of a run, must be finally driven into the combustion zone with the help of a burner. b. It will also be found necessary periodically to heat the abs orption tube to rr event the condensa- tion of too much water therein. Heat the absorp- tion tube to dryness by the end of the run. c. The tubes followin g the co mbu stion zone will a lso require occ asional heating to clear them of condensing water. 9. Re move the assembly (a glass rod carrying a hooked wire which may pass a long side the small tubes and engage the lip of t h e open end is convenient), det a ch the s ample tube, cork up, and wej_gh when cool. 10. Remove the receiver. Titrate the exce ss barium hydroxide with standard acid while bubbling through a slow current of carbon dioxide - free nitrogen. 11. Sto1"e the sample tube and its contents in a cool dark place to await possible additi onal analyses. 12. Determi ne the weight of oil by multiplying the volume of standard barium hydroxide by an appropriate f a ctor, chara.cteri stic of the oil concerned. In an effi. cient pr ocedure for running a routine series of analyses, each carbon dioxide receiver, upon r emoval, should be immedi ately replaced by a fresh receiver; a new sa mple should be promptly introduced into the apra ratus, a nd the next run begun. Ample time for titration and weighing is a vai lable while t h e volatiljz ation of the next s amp le is under way. 67 Discussion and Notes on the General l'rocedure This discussion will concern itself particularly with experimental details. A more comprehensive discussion, covering this procedure and its sub-procedures from the viewpoint of reproducibility is presented later. ( 2). The tempera.ture of the block, as read on the thermometer inserted into a well in the block, will be higher than the actual internal vol a ti ljz a.ti on temperature by an amount readily determined from a calibration curve. (Such a curve is easily prepired by inserting a second thermometer into the volatilE ation zone and taldng simultaneous readings of the two thermometers over the workin g range). The correction increases with tempe rature a nd depends on the block, it may amount to as much as 12°, for example~ for an internal tempera ture of 120°. The correcticm is negligibly affected by the normal variation in oxygen rate of flow. (5). Rate of flow is not a critic a l factor in the volatilization process and need not be closely controlled. ( 6 ). The absorption tube and g lass wool are necessarily moistened in order that the added barium hydroxide solution will re adily distribute it self uniformly. (8). It is believed that the procedure would be simplified if the absorption tube we re also enclosed b a heating block kept at a temperature somewhat above 1006C. ( 10). It is unnec e ssary to wash out the recei ver after e a ch titration ; it is sufficient simply to pour out the titrated solution and imme diately run in the next portion of standard base. (12). For guayule oil of composition corresfonding very nearl y to C5Hs: Volume of 0.05 N Ba(OH)2(cc.) x 0.34 = weight of oil (mg.). Sub-Procedur·e A: 'l' ot a l Oil Content and Oil Contents of Different Tissues of an Intact Organ . 1. Volatilize the sample at 120° or 110° until drled to brittleness . ( Value for "loosely-bound 11 oil). 2. Li-rind the brittle s ample directly in the sample tube , usinp: a slender r ound- end gl ass rod as pe stle. 3. Add to the g round s ampl e several drops of wat er ( as much or somewhat more t han was in the fresh sample), and allow about 10 minutes for this wa ter to be t h oroughly ab sorb ed. 4. Volatilize 5 minutes. 5. Again add water, let abso rb 10 minutes, an d volatilize 5 more minutes. Hepeat as needed or desir ed. Discussion and Notes on ~ub-Procedure A. The law of diminishin g returns o b viously plays an important role in those volatilizations that have been discussed earlier (F'i g . • 3 to 5). l<'or example, after only 10 minutes of treatment, there may be volatilized from a sample up to 90% or more of the oil t h at can be finally obt a ined durin g that volatilization period, regardless of how long it may be continued.(A volatilization period is defined as t h at period of volatilization durin g which no more water is added to the sample). Such a n extended process involves an unnecessary waste of time and subjects t h e sample to an unnecessarily severe heat treatment. ·i 'herefore, one of the objects of the procedure just outlined is to continue e a ch individual period only as long as it efficie n tly vol a tilizes oil. 1 he second ob~ective is to permit a rather ele mentary morpholo g ical fractionation~ to obt a in values fo r 11 loosely-bound 11 oil and total oil. (1). '.L'he initial period must be continued until the tissue is dry and brittle enoug h to be readily g round. Prr,ctice has shown that this requires a bout 95 % or moI' e of t h e total tissue water to 'be removed. ·i 'his requi r'es ab out 15 minutes at 120°c for' a n a vera ge guayule leaf sample of 100 mg . fresh wei g ht. As ind icated in 1''ig . 7, the various treat ments a ffectin g tissue structure have little effect on the r &te of vn, ter> r emoval ( i n contrast to the e ffect on oil re]Il_oval). The effect of tempera ture is illustrated in Fig.6. 70 It is particularly noteworthy that a temperature of 110°0 is apparently as effective as 120°c, which suggests the use of 110°0 in preference to 120°c in future work. (2) Tbe error due to loss of tissue particles clinging to the rod when it is removed from the s ample tube has been demonstrated to b e con s istent l y ne p; li e; il1le ( by tefore an d a fter wei p hi ng s) . Pj_ f . 17 cm:cl)aJ'e s t r1e res u lts of' this procedure wit h the lonf': er proce dure used earlier. savinr: s i- - "'•~· <-c, It is cle ar• what D g reat cco·rr:n 7 ~L s·11ed • .i:<'0111.., short p eriods co mp rising ..1 r . l .:. -- - • • .... ~ only 27 minutes total time ( and vol at ilizi ng 2. 97 % oil) are essentially equivalent to four lo n r; per·iods co mpr isinf; 120 r:1inutes total time (and volatilizing 2.63% oil). It is noted that Sample 8, submitted to the more repid procedure has a g reater oil content t han the o ther sample; this is annoyinf for purposes of the :rr esent nature. It is interesting to note in passing, however, that Table II lists for Har>vest II two samples with especially lower weights than all the rest (therefore, s maller le a ves), Samples 5 and 8; and, furthermore, that they are the two _, sample s which show the highest oil contents of the harvest. ·J .'his observation fits in nicely with other facts to be presented on another occasion. .i. ' he point in the present connection is that the ' 1bad behavior" of ~ampl e 8 is probably inherent in .:iample 8, not a fault of the vol a t ilizati on procedure l 3.5 71 tAOD WATER ,,,.... 3.0 ~ t1: "jjj ~ >- 2;5 c!sLI. tAODWATER 0 tAOD WATER 0 w --N _j 15 r-<( _J ~ ,.o _J 0 _J ~ 0.5 0 ~ o.o o 10 20 30 40 so 60 10 so 90 100 110 i:w 1so VOLATILIZATION TIME (MINUTES) Fig. 17. Reduction of the 'l'otal 'l 'ime Heqi ired for Exhaustive Volatilization. 0 Sample 2. iSuccessi ve vol a ti liz ation periods of 30 minutes each. • Sample e. Successive volatilization periods of 15, 6, 6, 10, 10, and 10 minutes. Sub-Procedure B. Oil Content. 'l'he Rapid Determination of Tot al Take the pulverized tissue mass which has been prepared according to the instructions of Procedure II, and simply volatilize it under standard conditions for a single suitable period: 5, 10, 15 or 20 minutes. In any case, as was clearly demons t rated on p.39, the value obtained will be a direct measure of the total oil content. 'l1his method is valuable si nee it obviously gives a very rapid technique for comparin g a series of more or less similar samples (as a series of leaves). If all determinations are carried out under standard conditions and for a specified time interval, the relative values obtained for the differer-t samples will directly measure their relative total oil contents. JJ.1 he principal disadvantage of t he Jr esent procedure, referred to previously (p. sq ), is the increased handling and exposure of the samples, occasioning losses and some annoyance to the analyst. It is believed that in the future this disadva ntage can be overcome by improved technique, specifically ·b y usin g sample tubes made of a material that is flexible and tough enough at very low temperatures to permit freezing and pulverizing of the s ample directl y t h erein, and a materi al t h at at t h e same ti me is capable of wi thstending temperatures above 100°c 74 without softening or deteriorating, and whi ch is preferably chemic a lly inert; and actually a tailor-made material would n ow seem to be in the offing - a silicone plastic ! ( Colling s, 1945). Procedure for the Determination of Volatilized Carbon Dioxide. As an integral pa rt of the general procedure for the determination of essential oil, the volatilized carbon dioxide is directly absorbed, prior to combustion, by a si:e ci al absorption tube. If the precaution has been taken of usin g ciarbonate-free barium hydroxide, it should seem possible to determine the volatilized carbon dioxide simply by acidification and aeration of the contents of the absorption tube after the run, absorbing the released carbon dioxide in st andard barium hydroxide solution in the usual manner. Actually this is done in the apparatus shovm in Fig.18. 'l1he procedure is as follows: +J Attach a barium hydroxide receiver to the outlet tube of the apparatus. 2. Add 1 to 2 cc. of 6 N hydrocloric acid to the U-tube (U). 3. Insert the absorption tube (A) as shown. 4. Pass a gentle current of carbon-dioxide free nitro gen through the apparatus for 5 or 10 minutes. (The acid will be force d up into the absorption tube as a liquid column, alternately rising, collapsing in the expanded tube (E), and percolating back into the absorption tube). 5. Turn off the nitro gen flow, disconnect and titrate the excess standard base; ready the absorption tube for a fresh charge. The completeness of the aeration may be judged by the data of 'l'able VI ( and al so that of '11 able VII). The procedure is quite satisfactory for the present purposes. 77 E 6 A T F:ig . 18. Apparatus for Aeration of Carbon Dioxide from the Absorption Tube. A. Absorption Tube . B. Bubbler tube (to collect any acid thrown over from E). E. ·Expanded tube (to as s ure collapse of rising liquid column). '1 '. Trap ( cont aining glass wool (to catch any acid spray). U. U-tube containin g 6 N H2S04 initially. ( Ap pro x . Scale, 1:5) Table VI . Evidence on the Rel:ia bi lit y of the Ae ration Procedure in the Determination of Volatilized Carbon Dioxide. Sample No. Amount of CO2 Ae rated (ml. N/20 Ba(OH)2) During first 5 Min. During Second 5 Min. % Aerated during F'irst 5 min. 31 0.14 0.02 88% 32 0.32 0.02 94% 33 0.24 0.03 89% 34 0.52 0.11 83% General Discussion of the New .iviethod . The reproducibility or precision attainable by the new method has been rather clearly evidenced by the studies previously presented in graph form. Despite initial differences in appa rent oil contents, reflecting different initial treatments that modify cell structure, the curves in general show a strong tendency to converge toward a common ultimate value. In fact, as shown in TableYII, after a process of relatively exhaustive volatilization, the precision · seems to be well within 15%. In addi tion, analyses have been made on s a mples that are completely comparable from the outset, these samples being aliquots of a common well-mixed, and minced leaf mass. 'l'he results, g ive in Table ]ZllI, show a standard deviation of 5% among 5 samples, a maxi mum difference or ran ge of 10%. Other evidences of reproducibility are given by the curves for vol atilized carbon dioxide. 3 2 4 4 4 17 0.65 2 3 1 14 Max. 0.03 0.01 0.66 0.03 0.34 0.17 0.49 0.26 (rta n g e) Ui f feren c e 1.14 1.14 2.97 2.75 2.96 + 0.20 2.67 Max. Value 11 6 15 1 3 3 10 % Llifference 0Be sample 11 ( 2) A volatilization period is defined as that period of volatilization during V'hi ch nomore water is added to the s am ple. ~ blanched 11 No difference Vol. 1'ernp o Leng th of I ndividual Vol.Periods 'l 'i s sue Structure Vol. 1'emp. Tissue Structure Initial Bifference(l in 1i ssues or -Conditions (1) It is well to note that the various sets of results given in this table included samples for which there existed initi a l differences in some factor, affectin g initial rate of volatilization, it being evident, however, that the initial rate effect had been overcome after several volatilization periods. It is a lso well to note that these samples con sisted of ~arate le a ves, subject to some normal v a riability. 1.11 3 3 1.11 2.63 2.58 2.67 2.41 Min. Value 13 2 B. Volatilized Carbon Dioxide . 4 5 5 3 4 Essenti a l Oil. , No.of(2) No. of Vol.Per. Sam.£ l.es 'I'a.ken 3 1-1. . Fi g . REP RODUCI'.BILITY (PRECISIO N) (l) Vol at ili zed l\l aterial (% Drz Wt.) . TABLE VII. Table VIII . Reprodu cibilit y (Precision) . Sample No. 'I 'reat ment 35 Un tr e ated 36 II 37 II J oil % wat e r (% fresh wt)( ~ dry wt.) 11 38 Bl an ched" " 39 Mean Storage + 25° 2 hrs. 71.l 1.00 3 hrs. 7O.O 0.96 70.6 0.90 71.2 0.91 71.O 1.01 Dr y ice 1 d. none Dr y ice 1 d. st a ndard deviation % Difference between extreme values t 70.8 0.5 d 1.770 :t: 0.96 0.05 10% Description Top leaves were minced into very small pieces with a razor blade, the mass mixed, then divided into simil ar s amples. Vol atilization temperature: 120°; vol a ti liz a ti on time, 20 minutes. It will be noted that the previous results were obt a ined before there wa s introduced tl:Je technique of simultaneously determining oil and volatilized carbon dioxide, by the use of a special absorption tube. It is, therefore, now necessary to present some specific evidence a.s to the reproduci tili ty of the final p!Voee.dure o 'l'he results of this work are presented in table IX. It is clear that the determination of oil is quite precise (10% difference between samples). The precision of the volatilized carbon dioxide determinations is considerably less on a percentage basis (msximum difference of 33%), but this is attributable to the smaller amounts of this material. '1'he results a re very pleasing. Finally let us give some clean-cut answers to certain questions of si gnifi ca nee. t'he se answers are found in Table X . 1. Does the rubber tubing which is used to connect the sample and absorption tubes evolve any appreciable am ount of carbon dioxide on heating? No, not even when the rubber is heated to slight tackiness, is measurable carbon dioxide obtained. 2. Does the carbon dioxide retsi ned by the absorption tube fillin g evolve again when the tube is heated? even wh en the tube has absorbed a full qi ota No, of carbon dioxide, and is then heated so hot that the glass woQl therein turns somewhat brown, the carbon dioxide evolved is negligible. 3. Dees the essential oil pass quan ti tati vely through the absorption tube? for Ye s, the values obt a ined "% oil & % CO 2 11 are essentially the same whether the absorption tube is used or not. 30.0 35.1 18.8 - - -t + - - 43 44 45 46 28 .5 10% 33~t 0.34 :t. O. 05 0.89 0.30 0.40 0 .36 0.40 0.33 - 0.30 0.27 ± 0.06 - - - e 0.93 0.84 9% 1.17 ± 0.06 - - 1.19 1.22 1.11 1~ Oil ~ CO2 % Oil + Jb C02 (% Or y Wt ) ( o Or y Wt ) ( % Ory V/t . ) % Water 6% :± 1 .3 73.0 73.6 75.7 73.1 71.8 73.5 71.7 72.2 72.1 (% fresh W'c.) All samples were of Harvest )ZI" ; aliquots of a common pulverized leaf mass. Volatilization te mperature : 120°; volatilization time: 20 minutes. ( 1) 'l'he plus and minus si g ns indicate whether or not the absorption tube was used, or whether or not combustion was employed. 'rhus all combinations were tried, oome in duplicate. % Difference between extreme v alues Mean -± standard deviation------------- 47 25.5 + - 42 29 .o ,30 .9 + + 41 - 28.3 + (mg. ) DPy Wt. + Condit ions ( 1) Abs.I'ube Comb. 40 Samp. No. TABLE IX . EVIDENCE SUPPORTIN G THE REPRODUCIBILITY CF 'l' HE SI M.ULTANEOUS DE'rER.MI NA'l'IONS OF ESSENTI1\ L OIL AND VOL A'l'ILI Z ED CARBON DI OXIDE. ~ Table X. Evidence Supporting the Re liability of the Simultaneous Determinations of Essential Oil and Vol at iljz ed Carbon Dioxide. Run Volatili?: at ion Material & Condit ions Vol . 'l 'ime Volume of N/20 Ba se Used (min. ) ( m l. ) 1 8 mm.section of No.2 bl a c k rubber tubing 10 (material used to connect sample and -t 15 absorption tubes); heat same . o.oo o.oo 2 a.Guayule leaf (l)volatilized; absorp10 tion tube (containing glass wool saturated with barium hydroxi de solution) attached to sample tube 0.00 b. Heat abs orption t ube to dryness + 10 c. Heat absorption tube more strongly + 10 o.oo o. oo d. Heat absorption tube so stro ngly that + 15 <0.01 a. Same as 2·, heat absorption tube strongly. 10 c .02 b . Continue heat on ab sorption tube + 15 c. Add water to the drie d. le a f and repeat -t lO o.oo o.oo the glass wool browns and the rubber connection becomes slightly tacky 3 (1) '1 .'ms gus.yule le a f would ordinarily have given -off enough volatil:i.zed carbon dioxide to consur:1e about 1.00 ml. of the standard base. Several remaining potential sources of error in the new method require but brief' mention. '.11hese are the following: 1. Leakage of carbon dioxide into the train. 2. Completeness of carbon dioxide absorption. 3. l'he accuracy of the carbon dioxide determina. tion by ti tre.tion. Adequate determinations have been made to ascertain the errors possibly involved in the above eases, leading to the conclusion that they are . entirely negligible. SU1vIMARY 1. Procedures have been developed which permit the rapid and precise mi era-determination of the essential oil content of plant organs. 2. Procedures have been developed which permit the semi- quantitative determination of the essential oil content in different tissues of an intact o~gan. 3. It has been demonstrated that the major process in oil volatilization from plant tissues is diffusion. Especially sharp oil separations are possible when a dense- walled structure separates different tissues. 4. It is possible to determine for a 20 mg. (dry wei g ht) guayule leaf in an average ti me of ab out 30 minutes ( in routine anal y ses) the following info1~mati on: a . Pre sh weight. b. Dry wei g ht 11 c. A ~alue ·for loosely-bound" oil 0precision of 15% or be tter). d. A v a lue for total oil ( precision of 15% or better ). e. The amount of volatilized carbon dioxide (evolve d directly from the tissue by heat). 5. 'l 'he procedure finally yie lds a dry sample suitable for furt h er analyses. 6. It is possible to determine a v alue for total oil content al o ne in 10 or 15 minutes from the time volatilization is begun. 7. l'he potenti al v a lue of the new methods is suggested to g rowers of essential oil materials and to food technolo g ists, as well as to those who are interested in the problems connected with essential oil met ab olism. 8. He commendations for furt h er improvements of technique have been g iven. 9. It is recommended tha t re search be a ctivel y carried out on me thodsfor tl.1e quantitative microchemical analysis of specific esse n ti a l oil components. - TI. ESSENTIAL OIL RELATIONS DURINGTHE GROWTH - OF THE GUAYULE LEAF bk· ESSENTIAL OIL RELATIONS WRING THE GROW'r:fl OF THE GUAYULE LEAF The course of essential oil formation during the growth of the guayule leaf has been studj_ed. It has been shown possible to segrega.te this process into three rather distinct phases, coincident with three phases in the morphol0gical development of the leaf. These results have been confirmed for a series of leaves on each of four different guayule plants, each plant growing, for a period of four months, under an individual temperature combination. Combinations employed were: 27°c (80°F) day and night; 27°c day and 16°C ( 60°F) ni g ht; 27°c day and '7°C (45°P) night; and 18oc (65°F) day and 13°c (55°F) n~ght. Procedure. The plant mater•ial used was taken from the lot of guayule plants described in Table I ( p.~ l ). were sown from seed on February 12, 1945. These plants On December 15, 1945, small groups of about 10 plants each were carefully transplanted, the roots being disturbed as little as possi9le, into 4-inch pots of sand. Thereafter each pot of plants grew under its own t emperature combination in the controlled-temperature g reenhouse 'of the California Institute (Went, 1944). They were waterea daily with nutrient solution. (A very complete record of the growth of these plants was kept over a period of more than three months_; these data shall be presented on another occasion). Over the three day period, April 9, 1946 to April 11, 1946, a representative series of leaves, beginning with young leaves less than 2 cm. long, were harvested frGm a typical plant of each group. Values for "loosely-bound 11 , mesophyllic oil content (see page3'f) were determined for each leaf by means of the new micro-method that has been describEd in full detail in the previous section. Resultso The principal results of these ' investigations are concisely presented in Figo I~ a The full line curves show the values for mesophyllic oil content for leaves of increasing maturity (dry weight). The dotted line . curves give the corresponding-average elongation rates of the respective leaves over a period of 12 to 15 days prior to harvest$ Dur ing the period immediately prior to rapid elongation., there is an extra large production of essential oil (first ohase). This large initial oil production is apparent in the case of every temperature combination. These values are ~eal since even the smallest initial oil value of 0.04 mg~ corresponds to Ool2 cc~ of standard 0~05 N base in the o i l determination procedure, whereas blanks are consistently less than O.02 cc o ( Po 65 ) o During the subsequent elongation period, howe~er, oil formation is suppressed (second uhase) . Then as elongation subsides, the rate of oil formation gradually increases again (third phase). It is noted that this oil increase¢ of the third phase occurs earlier in those cases where the or ~or-~ ';Jn,.dW1-l1 elongation subsides earlier (27'°C a_ay); : m :d occurs later>-when the elongation subsides later (i8° day). Th ese observati ons suggest a connection b etween the elongation processes and the suppression of oil productiona It is well to note that the growth rate curves given a r e for a period prior to harvest of about 15 days. The curves for the two or three day prier period, which :;nay be more pertinent in the present connection, would have their peaks shifted somewhat to the left o These results agree with the findings of Frey-Wyssling and Blank (1940) with Salvia seedlings. They also found that a seedling accumulates the maximum amount of oil just prior to the maximum elongation per lode: (Fig. /'IA ); thereafter the oil content actually decreases ( pres:umably by loss to the atmosnhere). Whereas in the guayule leaf, essential oil formation is only suppressed during elongation, it apparently ceases altogether in the Salvia seedling)o Another observation is that thermoperiodicy apparently causes no marked differences in mesophyllic oil contents among the various aampleso Thia contrasts with the case of rubber in guayule; Bonner ( 1943) has shown that rubber P_ccumulates rapidly in guayule stems and roots under a temperature combination of 27°c day and 7°C night, whi le failing to accumulate at all, fo r example ., at constant 27°C temperature. Ho wever, this is not surprising in view of th e fact that mesophyllic oil is apparently not a storage oil: it gradually disappears from older leaves in which oil production apparently diminishes or ceases~ the oil originally contained in the mesophyll therein being lost to the atmosphere ( or possibly solubilized as glucosides or the like and transported to more interior portions of the plant for storagej., Perhaps the process of essential oil formation in the leaf may be simply explained as follows: There are in the leaf at least three semi-independent biochemical systems affecting oil produ.ction: The primary system is perhaps tied in with the laying down of dry weight material and is in operation throughout the course of leaf developmento The second system ,..,t~~rve is concerned with active c~ll division and is in Aoperation for only a relatively short period prior to cell elongation. The third system is concerned with cell elongation and somehow draws upon or depletes the oil•pr-oducing systems. Tha t we may be dealing with se~eral different biochemical sources of essential oil in a single tissue is in agreement v:lth the view that different sets o f metabolic reactions give rise to different sets of essential oils (p~ 3 )# :) 0.15 0.2.D _J LU 0.00 [I ' 0 I - ~ - /tI 'r' A I I/' ,; \ \ \ \\ \ \ \ \ ~\ \ I\ \ \ - \ \ I I I I \ \ \- A f. ~ j -; ' 0 ., I • ,. and ' \ ' \ \ \. \ 10 100 SALVIA SEEDLINGS I QI I I I I I ~ r--=------0 I \ ' F~q-W1Jsshrr9 8/onl< (ICJlfO) '' ~ o•.oo , d tol l -30- - '4'6 ----------,,-------""' 1,to ,:to ,10 so "° ?b 8" ja 0 ::! , m a.. 0 1.as rn w ~ p.10 ::; -----~---- \ 20 2S 30 85" q.o ..,5 so 55 DRY WEIGHT OF LEAF (Mg.) 15 \ A in the Guayule Leaf~ Fig. l'l. Three Phase Essential Oil Product;i_on 10 . - - -- ·- • &O (mm.) fd5 ·• 70 I . . ._ -. . . . . . _ LENGTH ' 'I' - (): . . .f,fYPOCOTYL __ ~~ \ ~ '~ \ . I '■ I \ CJ 'CA \ - /A, - -....,.~_,.,..,._...,.___ I f I I ,1, , 'U;.i rv,,:. - I / I / I - 0 • 27 °dJ?J an4 l6 °nigbt . • .r,7 °dq and i7<? n1-t., 0 l8 °de,y ~6.'.n ~ l:J- n19nt...1 0 • a7°de.,ttnd--~, n.fini:l , I / /.g, ,.,.J,. , o / / I AMOUNT OF 01 L pe R LEAF - - - - L~AF lrl.ONGA,-IOH RA"T"l!E (AV£"AGli RATlii oveR PRav,ous •"2.-15 DAY P~f\1O0) _ I 000! ---~ . <( 0.05 :E ~ o.osO 0.10· I 9 r--' w z o.,o .z o (9 0.25 Q_ 0.30• · _j tr LL:0, - 0.15 w Q:: 035 (t z 0 _J ~ O.lfO 0.45 0,50 0.5S O.f.O <( l-: u.. w ~ Cf\ ~ ., ~ 0.20 ( "--' ~ ~ a c::t ~ I .~ .. ·I' .; .- ...t· ; ! !• t .0 III'. LARGE S C~LE INVEST I GAllQNg__QN__'rliE.__YAfUAT ION OF GUAYULE OIL COMPOSITION• WI TH THE AGE AND ORGAN OF THE PLANT _QONTENTS Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Treatment of the Pl ant ~aterial ................ ... 100 Physical Constants and Quantitative Chemical Analyses of the Unfra ctiona ted Oils............. /J :, Fractionation and Investigation s of the Fractions. 1~4 DiscussionO' .... ................................... '° l'tl Not es on Future Procedure ......................... /42 Qua.ntita.tive Distribution, PhysilZal Constant, and Ester Content Curves •....................... 143 S wnmary . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . , so ]I.LARGE SCALE INVES'l'I GA'l 'I ONS ON THE VARIA'l 'I ON OF GUAYULE OIL COMPOSITION WI 'I1li 'l 'HE AGE AN D ORGAN OF I' iiE PLANT The followin g investigations were initiated in order to determine the chemical compositions of guayule essential oils from different organs of the plant, and also to determine how these compositions change with the age of the plant. The present in vestigations have concerned the oils of leaves, stems, and roots (and some sub-fractions thereof) from plants of approximate ages of one, two, three and four years. These investigations have been carried out on a relatively large scale. .:,omewhat over 1500 kg. ( or a bout 3000 1 bs. ) by fresh we:i.ght of plant material have been sectioned and steam distilled, the oils being collected, dried, and stored for subsequent analyses. Over 70 individual steam distillations in 50 gall on stills were involved in this phase of the work. 1 '1 he physical constants (density, refractive index, dis- persion, and optical rotation) as well as ester contents have been measured for al 1 oils. l 'he primary method of at tac k has been the technique of fractional distillation. Fractional distillation permitsthe separation of the individua l consti- tuents of an oil to a greater or less de gree, thus providing a picture of oil compositions, and also yields fractions that may be the subjects of further study. After the plant material an d the treat ments to which it has been subjected (sectioning and steam distj_llation) have 'f"I been descri b ed, the work on the various resultin g oils shall be t ak en up in two pha ses : .B·:ir st, the work on the vn.fractionated oils; and second, the fractionations and the study of the resu lting fractions. A subst ant i B.l improvement in pr ocedure has been the construction and application of a precision fractionating column , wh ic h has been of value, not only in the present work, but which has contri buted significantly to our chemical lrnowledge of guayule essential oil (p. 158 ). 100 TREATMENT OF' THE PLANT MATERIAL The history of the plant material, which was furnished through the courtes y of Dr. Hamil ton 'I'raub of the Unit ed States Depart ment of Agriculture, is g iven in 'l'able -xr . When received at t h e California Institute, the material was promptly sectioned and steam distilled. Sectioning Procedure In view of the g reat quantity of material, a very careful separation of the plants into their parts was impractical. Sectioning was accomplished simply by use of a cleaver, eac h plant being chopped into the three portions, leaves, stems, and roots, in the manner shown in Fi g . JclJ. ~L Fig. 1:0. Typical Manner of Sectioning . It is to b e noted that the portion c a lled ''leaves u actu ally incl udes a proportion of twi g s and small stems. On the other hand, the ste m portions were pic ked essentially free of the rel a tively few lea ves they possessed initially. ~ypical e xamples of the material ¢lealt with here are pictured in Fi g . :ZJ. to -i..5 . Ab out one-half of the stems and roots were g round ( more correctl y , chop ped) in a s mal l Viiile y mill before distillation. The typical nature of the g round ma teri a l is shown in Fig . ;it:, • (bJ .,q9e: 3 y ~ Ftj-22.'.. 'sfem~ Aqe .3'.fea,.s (Full 5 i:rel ng. i ,.;~~~~1,,- Roo~ A9e 3 ~ears (~~/I .Size) ' ,, J ,r=-;9. ~5.-. l?cJats (FullS/ze) A 9e~ ~ I/ears ' - - -: .- ----- --- ~------- ~--~""'~7 P'.t.:J, 2 G;,, ~ice,/ !root dnd Si'elM. F,qj11te;t· After Gr/nt7hn~. A 3 v.J ¥,:J&gro 593 593 III IV 4/20/41 3-4/-/42 11/-/42 11/1/44 10/9/44 10/7/44 12/18/44 11/1/44 12/26-27 /44 11/24-25/44 Dis tille d ~ 11/22-30/44 10/12/44 10/19-25/44 10/ 13}44 10/21-25/44 - 11/9/44 Ba led J2fil& ,11/9/44 Dat e Harve sted(3) &ge 42 30 23 9 8 ( 111 0.) 21-29 10-16 . 14-15 8-9 23-24 (day s) period of dorman cy for guayule . ( 3 ). All harvests were wi thin the peri od, October to December, whi ch is during the warm, a nd overg rovm with mold. IJ) 0 - Period f;t;:QW Ha rv. tQ Dist. (2). 'rhese plan ts were subjected to rain during transit and arrived a t the Institute (1). All plants are fro m non-irrigated plots at Sa linas, Cal ifornia. Spence Block I Bardin 406F Spence II 3 /9/44 Spen ce 593 I Date Planted Hans on & Lee 3/9/44 Bloc k 7 Pield 593 Varie t y I M( 2 ) Plants Hi story of the Plant Mate rial ( 1 ) . Guayule, Parthenium Ar g entatum Gr ay Table :xI . steam Distillation ProceQure. Some of the important aspects of steam distillation have been discussed in another portion of this thesis, where typical. steam distillation results, obtained at this time, were pre.sented. Fig. r and i2. give typical dis- tillation curves for leaves and stems. Apparatus. Each 50 gallon still is provided with a wire mesh false bottom in order to keep the plant material from direct contact vdth the hot outside bottom of the still which is heated directly by air blast burners. Each still is so supported that it can be readily tipped for convenient removal of spent plant material. The flanged still head engages the top of the still with a tight friction fit which, however, is readily disengaged when desired. the still head connects with a large water-cooled metal condenser. The condensed material is received in a special flask which separates out and collects the oil in a graduated neck, permitting a continuous record of the amount of oil distilled; meanwhile the distillation water pi. sses ' directly back into the still (cohobation). Procedure. Initially several gallons of water are added to each still and, while the water comes to a boil, plant material is weighed into the still by small box loads, the still head swung into position, and soon the distillation 107 may begin. Additional steam may al so be supplied from outside ste am jets connecting with inlet tubes passing into and al most reaching the bottom of the still. Between each run it is necessary merely to turn down the flame, remove the still head, tip the still, r ak e out the spent contents, an d prepare for the next run. Since the coho bation process, which recirculates the distill ation water, is used, additional water need be added only infrequently. 'l 'i me Analysis. The followi ng rough estimates of the ti mes required for the various ore rations can be given: Sectioning : 60 kg. (120 lbs.) of plant per hour per person. Grinding : 20 kg . (40 l b s.) of plant per hour per mill. (Sma ll Wiley mill used). Steam Distillation: 25 kg. (50 lbs.) of plant per hour r:e r still. 'l'hi s fi gure ass umes use of the still at maximum capa cit y , ab out 50 kg . (100 lbs.), and with a two hour distillation period. Fresh Weight Dis tri butiori; Oil Yields. The ori ginal dat a collecte d in the presen t work have been : (1) The fresh wei ghts of the three fractions ; le a ves, stems, and roots. (2). The fres h weights of single plants o (3). The amounts of oil obtained from each fraction. These and derived data are collected in Tables XII to xv. Table JZll . Fresh vVeight Distribution of Leaves, Stems, and Roots For All Plants Massed ~ Portion 'I1otal Fresh Weight (kg•) 1 yr. Moldy 1 yr. 2 yr. 3 yr. 4 yr. Leaves 125 70 248 398 103 Stems 27 22 70 276 106 Roots 23 16 28 95 53 Total 175 . 108 346 769 262 Grand 'l 1otal 1660 Percent of Fresh Weight Portion 1 yr. Moldy 1 yr. 2 yr. 3 yr. 4 yr. Leaves 71 65 72 52 39 stems 15 20 20 36 40 Hoots 14 15 8 12 21 'l1otal 100 100 100 100 100 110 'I'able JlIIT . Fresh Weight Di stri buti on of Leaves, Stems, and Roots For Individual r 1ants . Portion Fresh Weight per Plant (gr.) 1 yr. Moldy 1 yr. 2 yr. 3 yr. 4 yr. Leaves ( 1) 43 39 108 130 390 stems (1) 9 12 30 90 400 Roots (1) 8 9 12 30 210 Tot al ( 2) ftgpr~xipat~ .o p an s used (3 ) 60 60 150 250 1000 2920 1800 2310 3076 262 (1). These values were derived b y takin g the proper percentage (as found in 'l able 1 ) of the wei ght of a corresponding tot a l plan t. (2). In lj_eu of a count of the total number of pl ants used (which woul d have been preferable, although many pl ants were muti lated and inco mplete), a few plants deemed bypical by rough observation were we:n.ghad and the above fi gures derived as the best possible values; a llow a rough deviation of 30%. (3). No. of plants - '.11 otal fresh weight Fresh we i g ht per plant l II TABLE XIJZ., ESSEN'l'IAL OIL OBT AINED FROM All, PLANTS . Portion Wei g ht of Oil (gr.) 2 yr. 3 yr. 4 yr. 236 291 895 286 46 33 53 247 }74 Roots 30 34 26 110 J. 27 Total 249 303 37 0 1252 527 1 yr.Moldy 1 Leaves 173 Stems yr. Gr an d 'l'otal 2Y/Ol Fer<:ent Y:i P ] d of C:t l r;f ( ('-' o f Fre:::ch y,;t . ). Portion 1 yr. Moldy 1 yr. 2 yr. 3 yr. 4 yr. Leaves 0.14 0.34 0.12 0 .23 0.22 Stems 0.17 0.15 0.08 0.09 0.16 Roots 0.13 0.21 0.09 0.12 0.24 Average 0.14 0.28 0.11 0.16 0.20 TABLE ::1aZ: Essential Oil Per Plant (Derived Data) . Portions Wei ght of oil 12e r J2lar t (gr.) 1 'Yr. Moldy 1 Yr. 2 Yr. 3 Yr . • 4 Yr. Leaves 0.04 0.13 0.13 0.29 0.86 Stems 0.01 0.02 0.02 0.08 0. 66 Hoots 0.01 0.02 0.01 0.04 0.49 Total 0.06 0.1'7 0.16 0.41 2.01 (1) The values for this table were derived from values found in previ ous tables. Physical Constants and Q{uantitative Che mi cal Analyses of the Unfractionated Ojls • The physical constants, including densities, refractive indices, dispersions, and optical rotations, and ester contents have been measured for 27 unfractionated oils. fhe system of designating these oils is explained in 'l'able XVI. Results. Table XVII rresents the complete data. To facilitate the comparison of these data and aid the possible detection of correlations, the data are repeated in different manners in Tables XIX and XX. iJiable XIX-A lists the oils in decreas- ing order for e a ch type of measurement. Table XIX-B repeats Table XIX-A, but includes only those oils of primary interest. Table XX lists the measurements separately for each of the gr oups : leaves, stems, roots; one, two, three, and four year old plants. No marked correlations are ap:r:;arent in the cases of dispersions, refractive indices and densities. In the case of optical rotations, ro me tendency is noted for the rotations to become less negati ve going from one to four year old plants. ihere is a decided tendency for the rotations to become more negative going from leaves to stems and roots. All root oils (vdth one exception) exhibit far stronger n e gative rotations t han any other oils. 11 4 Correlations . are evident with the ester contents. Ester content decreases steadily going from one to two to three to four year old plants in all cases. Moreover, ester content also decreases (with one exception) going from leaves to stems to roots. Table XVIII shows the effect of mold. 'i 'he oil from moldy one year plants (plants ~~ich arrived warm and overgrown .wi tli mold) is comp ared with oil from fresher one year plants. In all cases the mold apparently caused decreased ester content. Some favorable information on the reliability of the steam distillation procedure is given by the comparison of oils III L, III Lb and III Lr (III Lb was obtained from a particular run of three year leaves, during which the still was accidentally allowed to go dry, al lowin g cha rring of the bottom portions of the charge. 'l 'he oil is very dark and has a strong burnt odor. III Lr is some of oil III L that was poured back into the still and re-steam distilled). 'l 'he measurements for these oils are quite similar ( 'l'able xrx:), s uggesting that a treatment more .severe than normal steam distillation has little effect on the oil compositions. 115 Further favorable evidence is given in regard to the reproducibility of the steam. distillation procedure , that is, the similarit y of oils from different runs on the same plant material. I Oils IIIS and IIIS al 38, which are dupli- cates, show quite similar measurements. Procedures. IJ.' he d ensities were determined with a one cc. py:onometer. The refractive indices and dispersions (drum reading s or 11 z n values) were measured on a Zeiss Abbe refractometer. 'l 'he ester contents were determined by. hydrolyzing approximately one gram samples in standard alcoholic sodium hydr oxide under reflu x for about one hour, then back titra tin g the excess base. The satisfactory pre cision obtained in the ester deter- minati ons ia shown by the duplicate values reported in 'I1able :1Cl1.:C. Conclusions. 1. Ester content decreases steadily with increased age of plant; decreases steadily going from leaves to stems to roots. 2. h oot oils have far stronger negative optical rotati0ns than any other oils. 3. l\Iold growth on guayule plant sharply decreases the ester content of the oil obtained therefrom. 4. Some favorable evidence is g iven that the steam distillation procedure gives fairly reproducible results and causes only small changes in the oil composition. TABLE~. The System of Designating Oils I, II, III, IV -------------Approx. ( l) a g e of plants ( years). L --------------------------Leaf oil . T --------------------------Twi g oil ( 2 ). S ---------------- ---------Stem oil- H -------------------------- Root oil, ti (3)-----------------------"Loosely-bound" oil (bark oil), p (3 )---------------------""'.-"Ti ghtly-bound" oil (wood oil) . M --------------------------011 from moldy plants (I only) . Example: IRMp ----------------------- Wood root oil of mol dy one year pl a nts. (1) For the exact a g es, see Table-:xr.. . (2) 'l'he "twig $tt consisted chiefly of very s mall stems and t wi g s plus some other material (IV on ly). (3) The letter· "~ 11 desi g nates oil from ung round material. In accorda nce with nomenclature introduced elsewhere ( p. J'if ) , this oil may, therefore, be c a lled bound 'f oil. 11 loosely- .l.'he letter "12." desi g nates oil obtained after grinding and redistilling material that had already been predistilled un g round, and ma y be cal led bound" oil. 11 ti f; htly- 'rhe letter rrg_n designates oil from material 117 g round at the outset, or total oil, u + p. Since after a distillation, the bark of stems a nd roots is apparently well-steamed and can be readily stripped from the wood, it is probabl y t h at the "loosely-bound II oil represents chiefly barl~ oil and the chiefly wood oil. 11 tightly-bound 11 oil represents ( See Fi g . I ). /1'3 Table :lYI[o Physical Constartts and Ester Contents of Oils f~om Different Ages and Portions of Plants. d~.?.5 -25 Oil n25 7) z25 ~5 Per Cent Es t er (l) Ave~ of 2 Detnse IL 19.9 20.8 20.5 19.8 4.9 4.g 2.9 2.3 (2.5, 3.2) 20.0 20.0 20.2 3.9 5.9 2 .9 (3.7, 4.1) (5~4, 6.4) (2.7, 3.0) 19 .8 19.5 19.7 2 .6 1.6 2.4 (2a5. 2 .7) (1.4, 1,7) (2.3, 2.4) 20.0 20 . 2 4.5 ( 4. 2, 4.5. 4o7) -0.,!4 3. 9 (3.8 , 3.9} 1.4822 -5.00 20. 2 3.4 ( 3 •.2 . 3.6) 0,883 1.4840 -10.15 20.0 2,4 (2.3, 2.4) 0.873 o.879 -3.04 e.s12 1.4800 lo479C 1 .4805 2 0.3 2@.0 20.2 4.5 4.1 4.1 (4.5. 4.5) (4.1, 4.1) (3.9, 4,3} IIIS a:11s• o.874 o.876 1.4742 1.4782 -5.,61 -5025 20.2 19.9 2.7 3.0 (2,17, 3o2) IIIR o.884 1.4821 -10.50 20.0 2.2 (1.9, 2.0, ::2.2,2.: IVL 0.881 1.4829 -4.67 21.0 3.7 (3.6, 3.8) J:tT o.887 1,4829 - 9.46 19.9 2.8 (2.'7, 2.8) IVSg 0.812. o.897 0,877 1.4795 1;4840 1,4827 -3. 07 -5 .64 -3.04 19.5 2,1 4.1 (2.0, 2.2) (3.9, 4.2} IL3b ILM 0.880 0.885 0.905 o.894 1. 4809 1. 4840 1.4922 1.4869 !Su ISp O~a353 0 .. 906 1.4805 1.4840 0.879 1.4792 o.885 -.il.30 0.953 1.4827 1.4893 1. 4913 IILf o.891 0.874 1. 4868 it: ,4808 -4.71 IIS 0.890 IIR IIIL IIILb IIILr IL3a ISMg IRg IRMu IRMp 0.900 IIL IVSu !VSp IVRu - 0.9 -0.40 -0.77 -7.62 -5.72 -7.06 -7.65 - 21.05 -14.18 -2.45 -1.34 20.0 20.0 2.2 0.880 1.4805 -9.00 IVRp 19.9 1.3 e. 883 1.4830 -4.59 20.0 2 .0 (2) 0 .865 1.4744 -2.70 (4o5, 5 .3 ) (3.9, 4.1) ( 2. 3, 2.3) (2.5, 2.9) I ( 2. 0, 2.3) ( 1. 3, 1.3) (1.8, 2.2) 3.5 ( 1) . Ester content is calculated on the basis <if acetate (molecular weight= 198). 0 bornyl { 2). Total 2 Year Oil of Haagen-Smit and Siu {1944). TABLEXlZIIT. EFFECT OF MOLD ON ESTER CONTENTo - O IL N: on-1'loldy Moldy PER CENT ESTER s ISp ISu 5 .. 9 ISMg 2w9 IRg 2.6 IRMp IRMu 1.6 3.9 2.;4 ISp 5.9 IL . 4.9 IIL 4.5 IIIL 4.5 IIILb 4.1 IIILr 4.1 IVSu 4.1 IL3a 4.0 ISu 3.9 IILf 3.9 IVL 3.7 IIS 3.4 IIIS' 3.0 IL3b 2.9 ISMg 2.9 IVT 2.8 IIIS 2.7 IRg 2.6 IHlv'lp 2.4 IIR 2.4 IIJv1 2.3 IIIR 2.2 IVSp 2.2 IVSg 2.1 IVRp 2.0 IRMu 1.6 IVRu 1.3 ~ Ester 0 0 IVL 21.0 20.8 IL3a IL3b 20.5 IIIL 20o3 ISMg 20.2 IILf 20.2 IIS 20.2 IIILr 20.2 20.2 IIIS ISu 20.0 ISp 20.0 20 0 IIL IIR 20.0 20 0 IIILb IIIR 20.0 IVSu 20.0 IVSp 20.0 IVRp 20 00 IL I 19.9 IIIS~ 19.9 IVT 19o9 IVRu 19o9 IIJv'l 19.8 IRg 1908 IR.Mp 19.7 IR.lvlu 19 .. 5 IVSg 19.5 z2S IL3b IR.Mp IRMu ILM IIL IL3a. ISp IIR IVSu IVRp IVL IVT IRg IVSp IIS IIIR IL IILf ISu IIILr IVRu IIIL IVSg ISMg IIILb IIIS IIIS ·- \ -A 1.4922 104913 1.4893 1.4869 1.4868 1.4840 1 •.4840 1.4840 1.4840 104830 1.,4-829 1.4829 1.4827 1.4827 1.4822 1.4821 1.4809 1.4808 104805 1.4805 1.4805 1.4800 1.4795 1.4792 1.4790 1.4782 1.4742 r?.' · IRg IR.Mu IRMp IIIR IIR IVT IVRu ISMg ILI,J! ISp ISu IVSu IIIS IIIS IIS IIL IVL IVRp IVSg IIIL IVSp IIILb IIILr IL3b IL3a IILf IL 1' L«lo -21.30 -21.05 -14.18 -10.50 -10.15 - 9.46 - 9.00 - 7.65 - 7.62 - 7.06 - 5.72 ~ 5.64 - 5.61 .;. . 5;;25 - 5.00 - 4o71 - 4.67 - 4o59 - 3.07 - 3.04 - 3.04 - 2.45 - 1.34 - 0~77 - 0.40 - 0.14 - o.o TABLE.:x:IX.PHYSICAL CONSTANTS .Al.1D ESTER CONTENTS OF OILS LISTED IN DECREASING ORDER FOR EACH TYPE OF MEASUREMENT. ISp IL3b IRMp IRMu IVSu ILM IIL IIS IVT IL3a IRg IIIR IIR ISu IVRp IVL IL IVRu ISivlg IIILb IVSp IIIS IILf IIIS IIIL IIILr IVSg ~ O.9O6 O.9O5 O.9O3 O.9OO o.897 o.894 o.891 o.89O o.887 o.885 o.885 o.884 o.883 o.883 o.883 0~881 0.880 0.880 o.879 o.879 o.877 o.876 o.874 o.874 o.873 o.872 o.872 ,, 1.S 0 r-) .IYRu.. IIIR IVSg IVRp IIR IIS IVT IIIS IRg rv1 IIL Flfui IIIL ISU IL I.SP IRg 2.,0 ,,a IL IVT IVRu IVRp IIIR IIR IVL IIIL IIS IVSg IIIS ISu ISp IIL 5.9 4.9 4.5 l .=:3 4.5 3.9 3.7 3.4 2.8 2.7 2.6 2.4 2.2 2ol 19 .,8 0 21.0 20.3 20.2 19.5 20.2 20.0 20.0 20 0 20.0 20.0 20.0 19.9 19.9 19.9 ISu IVRu IIIL IVSg IIIS IL IIL ISp IIR IVRp IVL IVT IRg IIS IIIR 1.4-868 1.4840 1.4840 1.4830 1.4829 1.4829 1.4827 1.4822 1.4821 1.4809 1.4805 1.4805 1.4800 1.4795 1.4742 8 12 rvRp IVSg IIIL IL - o .o - 5.04 - 9o46 - 9.00 - 7.06 - 5.72 - 5.,61 - 5.00 - 4.71 - 4.67 - 4.59 - 3.,07 IVT IVRu ISp ISu IIIS IIS IIL IVL -21.130 -10.50 -10.15 IRg IIIR IIR lV,?e:t IVRu IIIS IIIL IL IVT IRg IIIR IIR ISu IVRp IVL ISP IIL IIS o .tcr72 o.887 o.885 o. 884 o.88.3 o.883 o.883 o.881 o. sso 0.880 o.874 o.873 0.906 o.891 o.890 tJ -- L 4.9 4o5 4.5 3.7 L_ 2 1. 0 IL 1.4809 IIL 1.4868 IIIL 1.4800 IVL 1.4829 .IYl... IL 19.9 2O.O (, IIL IIIL 2ma . IL IIL IIIL IVL 1,__ ISp ISu IIS IIIS IVS IVS ~s ISp ISp ISu IIS IIIS IVS 19.5 t&; 20.0 20.~ 5.9 3.9 3.4 2.7 2.1 lo/4.84O 1.4805 1.4822 1.47Li2 lo .4795 s s s IRg IIR IIIR IVRp IVRu & IVRu IRg IVRu IVRf IRg IIR IIIR 2.6 2.4 2.2 2.0 1.3 PERCENT ESTER IL ISp ISu IRa I il IL IL ISp ISu IRg REFRACTIVE I NDICES 19.9 z6'~ 19.8 20.§ 1.4827 1.4840 1.4821 1.4830 1.4805 R R fij~i IIL IIS IIR 1.4809 I IIS u~ IIL IIL I IS IIR 19.9 4.9 5.9 3. 9 2.6 104840 104805 1.4827 I DISPERSION (DRUM READINGS) R :to,o III m~ III IIIL IIIS IIIR 20.0 IIIL 20.2 IIIS 4.5 3o4 2.4 III 21.0 2.8 2.2 2.0 1.3 ,.i N - 1.4829 1.4829 1.4795 1.4830 1.4805 IV ½~~6 iii IVRu 19.9 IV IVT IVSg IVRp IVRu IV IVL - 3.7 20.3 IVL 20 02 ~0 , 0 4.5 2.7 2.2 1 o L1-868 IIIL L48OO IVL 1~~822rIIIS l.47/.i2 IVT 1.4840 IIIR 1 ..4,821 IVSg IVRp IVRu II II II TA.BLE:m.. PHYSICAL CONSTANTS AND ESTER CONTENTS OF OILS LISTING THE MEASff.REMENTS SEPARATELY FOR EACH OF THE GROUP: ~LEAVES, STEMS, ROOTS; I,II,III,& IV YEAR PLANTS. IL IIL IIIL IVL IL IIL IIIL IVL 0.880 o.891 o.873 Oo881 L -o.o -4.71 -3.04 -4.67 L ISp ISu IIS IIIS IVS ISp ISu IIS IIIS IVS - 0.906 o.883 o.890 o.874 o.872 s -7.06 -5.72 -5.00 -5.61 -3.07 s IRg IIR IIIR IVR IVRu IRg IIR IIIR IVRp IVRu R R IL ISp ISu IRg o.885 o.883 o.884 o.883 0.880 IL ISp ISu IRg DENSITIES -21.30 -10.15 -10.50 - 4.59 - 9.00 I SPECIFIC ROTATIONS 0.880 0.906 o.88J o.885 -0.0 -7.06 -5.72 -21.30 I IIL IIS IIR IIL IIS IIR III o.891 IIIL o.890 IIIS o.88J IIIR II o.873 IVL o.874 IVT o.884 IVSg IVRp IVRu III -4.71 IIIL -3.04 IVL -5.00 IIIS -5.61 IVT -10.15 IIIR _-10.50 IVSg IVRp IVRu II ol ~ o.881 o.887 o.872 o.883 Oo880 IV -4.67 -9.46 -3.07 -4.59 -9000 IV IZ4 FRACTIONATION AND INVESTIGATIONS OF THE FRACTIONS In line with the general purpose of deter~ining the quantitative chemical compositions of each oil, the followingoils have been fractionally distilled: one, two, three, and four y ear leaf oils; one year moldy leaf oil; and three year stem oil. The refractive indices a nd di sp ersions o f all the resulting fractio ns have been ~easured. The optical rotations of the fraction s of three year leaf oil have been measuredo In addition, ester determinations have been carried 0 11t on those fractions comprising rich ester re;ions. For each oil the data have been plotted on a graph (Fig. 28 to 33). The data for the previous distillation of Haagen-Smit and Siu (1944) have been replotted on a similar graph (Fig. 27) to facilitate comparisons .. 'I'he Proble m of Interpreting the Distillation Data. 1°he fractional distillation of a mixture yields · a series of fractions, boiling and distilling over at a successively higher temperatures. It is then possible to obtain a quantitative distribution curve by plotting, as ordinate, the weight of each fraction, or a more convenient equivalent as~ the weight per 1000 grams per degree Centigrade, against, as absc issa, the temperature range over which the fraction distills. With a perfect fractionation instrument, each fraction would be a pure compound (or a constant-boiling mixture .of quantitative composition fixed by the co mponen ts, or (whi ch is very unlikely) a mixture of several compounds of ide ntical b oiling points), the entirety of which would distill over at its one characteristic boiling point. The ~orresponding dis tribution curve would consist of a ser•ies of completely separated vertical lines located at the respective boiling points of the various constituents. ~uch an ideal situation of complete resolution of components woul d obviously present relatively little difficulty of interpretation from the sta.ndpoint of quantitative analysis. Actual l y with a finite apparatus, the degree of separation cann ot be this sharp. t'he compound of one fraction wil l be contaminated by compounds fro m adjacent frs ct ions to a greater or less tiegree>L .: ba,iefly, there will be more or less overlapping. The actual extent of overlapping will be conditioned by many factors; On one hand, an incfeased efficiency or resolving power of the apparatus will minimize overlap p ing; on the other hand, many fact ors, but especially the closeness of boiling points of adjacent compounds, will increase overlapping. ·.i.·he corresponding distribution curve will con- sist of a series of peaks of varyin g hei ghts, widths, and shapes; some peaks will be well separated and distinct, so me less distinct, others almost merged. It is such curves as these that are presented by the present distil19:tions of guayu l e essential oil. Let these latter distribution curves now be considei->ed more closely. The original work of Haagen-Smit and SuQ (1944) on two y ear total oil gave a distribution curve (Pi g . ?-7) comp osed of four well-defined groups, identified respectively as terpenes, oxygenated terpenes, sesquiterpenes, and sesquiterpene alcbhols. The four g roups contained ten peaks, in each of which the presence of a different compound was established, with the probability t hat this compound was the major component of that peak. This initi ally suggested the possibility that any guayule essential oil would yielq, upon fractionation, a distribution curve of similar qualitative character: groups, ten definite peaks. four major In such a case, the method of interpreting the curve would be fairly simple an d direct; the very nature of the distribution curve would serve as a guide f "2.7 for dividin p up the fractions into ten divisions, representin g the amoun ts of the ten major compounds present;. An additional guid e, if nece ssar y, wo uld be the temperature of distillation. One year moldy , two, three, and four year leaf oils, and three year stem oil VJere distilled with the apparatus used in the ori ginal work, a hel:ic es fractionation coiliumn with the total condensation, variable take-off still head of Whitmore and Lux (1932). ( Howe ver, on ac count of the time factor as well as to minimi..ze losses, each oil was fractionated only once, and not twice as in the original work). The distribution curves obtained, however, are not in accordance with t he si mple situation t h at has been antici pated above. Indeed, the curves are all qµite erratic and show little apparent correlation with one another, either as to g eneral form or as to disti llation temperature. There is no separation into four major g roups, much less into ten definite p ea ks. In short, any immediate interpretation of the curves in terms of quantitative analysis is quite impossible. Much better results were obt ained when there was finally brought into use an improved apparatus, featuring a silvered vacuum-jacketed meter long fractionating column, six mm. inside diameter, with a nichrome spiral as packing (p. JS3). Une year leaf oil was fractionate d in duplicate with this apparatus, giving the di stri but ion curves dravm on Fi g . 2.<3 • Of particular si gnifica nce are the very g ood results obtained in fractionating the terpenes, the for ms of the distribution curve s alone permitting immediate quan titative division of t he terpenes into four groups. However, there again is no evide n ce of four ma5 or g roups or a specific numb er of peaks for t h e whole distillation range. It was such results as those which have just been recounted , which suggested that the increased fractionation power available in the new appar a tus be app lied to about 100 cc. of one of the hi g her boiling fractions previously obt a ined in the ori ginal investiga tion; and, indeed, the results were such as to warrant the exten sion of the refractionation procedure to a number of additional such fractions --- the o v era ll result, a much fuller, more comprehensive picture of the chemical composition of guayule essenti a l oil, and clear evidence t ha t guay ule oil is really very complex. This complexity can account fo r much of the difficulty exrerienced in the fractional distillation of the v ari ous oils. Despite the difficulties which have been pointed out, it has been possible to obt i:d n so me fairly reasonable results from the distillation data. 11 he method which was chose n to accomplish this will now be considered. The l\Iethod of Interpreting the Distillation Data. First this question, in regard to the li mitations of the method, shoul d be considered ; Shall it be possible to determine the amount of an ind ividua l compound, or shall it only be p ossi b le to determine the ag g regate amount of several compounds ma ssed toge the.r in a group(?). An examina tion of the present g raphs ind icates that it should be reasonably possible to de termi ne the am ounts for the follo win g divisions, but no subdivision, without, at least, supplemm t ary chemic a l analyses, is possible. Division No. Probable Chief Constituents I -------------« - pinene lI ------ ------- -fl - pinene, un known terpe n e .Hf- ------------ limonene, neasily ox idizable ter pene' 1 N ------------- I------------- carbonyl compounds, ester m------ - ------ sesquiterpenes JZII------------- sesquiterpene alcohols W11( r'esidue) --- (re sin s) IJOC(trap) -----'i'his summar izes the limitations of the present method. It is r a ther fortunate and important that eac h of t r.te vari ous d i visions is probably comprised chiefly of . compounds of si milar chemical ch a racter ; and, therefore, a knowledge of t he amount of material within each division has real c h e mical and biolog ic a l si g nificance ! )oO The actual me t h od of dividing up the fractions into the aforementioned divisions may now be outlined as follows: 1. 'I'he ori g inal distribution curve of Haagen-Smit and Sui (1944) has been chosen as the workin g pattern. It has been divide d up into the appropriate divisions on the basis of its characteristic shape in conjunction with the knowledg e of where the various comp ounds are located. Vertical lines at t he base of the g raph have bee n drawn (as in all subsequent g raph s) to mark off the divisions. 2. The refractive index ranges for the various divi- sions have then been taken and ·recorded in Table ::z:xr.:, _ 3. These refractive index values ba.ve been chosen as the primary criteria by which all the other curves have been divided. The exact places of division have been modified in those cases where the forms of the distribution curves indicated a sli ght shift. Also two secondary criteria for· certain dividing li n es are mentioned in notes to 'r able J2QCL.~ -: . Re fractive index values have bee n chosen as criteria because they are readily and accurately obtained. 4. Several check s on the correct placing of any di vision line may be had by reference to the re gion in which each of the characteristic physical constant and ester curves is intersected by this line. Note: The curves for the terpene rang es of IL and It ; obtained with the improved apparatus, can be divided directly on the basis of their very characteristic and reproducible form alone. TABLE "5lXI' . hefracti ve Index Ranges for the Various Divisions. Division No. Refractive Inde~ Range I 1.466 - - 1.470 B 1.470 - - 1.475 m: 1.475 - - 1.480 ]l 1.480 - - 1.479(~) r m 1.479(~)- 1.496 w mr 1.502(2) ( residue ) ~ (trap) 1.496, - - 1.s62( 2 ) (1). A more reliable criterion for the division between groups ]f' and 'His the ester value; the ester content rises sharply going into Division Y. (2). A more reliable criterion for the division between g r-oups E' and Wis a point about one-half way .up the side of the first hi gh peak of the refractive index cur-ve. 132 Results. The consideration of the results will be in two phases: First, the for::ns of the physical constant and ester curves will be examined. Second, the actual quantitative chemical compositions of the o il s, accar~lng t o th e divisions, will be tabul~ted and discussed. ~he Forms of the Physical Constant and Est. er ,J:.1r ·;res. Two purposes can be achieved by the examination of the forms of the physic8.l con st!J,n t i.'.nd ester cur.ves: First, any unusual curve, presumably sug3esting an unu a :.1al oil 1 can be singled out for special atte ntion. Second, the reli ability of the methoo. of di v ision, as 2,lr eady sta t ed, can b·• judged by noting if all the curves a.re intersected by the division lines in th e :)roper r egio ns. The following points may be stated in regard to the interpretation of the physical constant curves: In the case of a. 2,er:[ect fractiona t:Lon , the form of a curve would depend solely upon the ldeg~_lli of t h e cdmpounds upresent a nd would be e ntirely independent of a~ounts of these co m,o ~ndso I t1 ~he case of an actual fractionation, however, the form of a\ curve will d epe n d on both the 'ident ity and the amounts of the compounds present and will be further modified by such factors HS where the cuts between f ractions happen to be ~ade . For example, let us say that in the case of one oil we obtain a curve with a certain peak, while in the curve for another oil, this peak is absent. 'I 'his difference may be explained in either of several ways: 1. The compound responsible for the peak may be missing from the second oil. 2. 'l'he amount of a neighboring compound in the second oil may be so large that an amount of it overlaps and distills with the compound responsible for the peak, an amount sufficiently large to completely mask the peak. 3. The arbitrary cut between fractions in the second case may have been so made as to divide and distribute the responsible compound to its neighbors on either side, thus eliminating the peak. It should also be mentioned that the temperature measurements are relatively inaccurate, so that displacements of port ions of the curves from rip-.,ht to left have little if any si gnificance. To compare corresponding re gions of curves, use the divisipn lines as the proper primary guides. It would seem to follow from these :i:e- st considerations, that the primary value of the physical constant curves, from . . the viewpoint of analysis, lies in their ability to detect the broader, more fundamental changes in oil composition. Consider now the curves o f f;'.'.1~ •~ 121 to '$3 •' ., All the refractive index curves have the same general form, suggesting that these oils are roughly similar in composition. 1'he curves for III S and ILl\'l may show enough modifica- tions to suggest somewhat different compositions. 1;he re g ions where the curves are intersected by the dividing lines (marked .- tt · ') a re essentially the same in all the curves, thus checking , for these oils, the method of division used. However, in the cases of ILM and, especially, III s, the chang es in forms of the curves are sufficient to arouse some doubts, for example, as /34 to the correct placing of the dividing lines that bound Division min the III S curve. All the dispersion curves have the same general form. 'l'he intersection regions check quite well again, al though there is again some doubt in the cases of III S and IL Ni . VJ'i th III S, a number of peaks are missing or merged (?); with IU1, the latter port ion of the curve is strang ely depressed. IL 1 Minor variations in form are noted for IL and (in which· cases the two peaks of Division Eare seem- ing ly missing). '.I'he optical rotations have been determined for the fractions of only one of the oils, III L. It is interesting that in this case the distribution of optical activity is precisely the same as was observed in the original work of Haagen-Smit and Siu on two year total plant oil: excesses of t-oc-pinene, d-1imonene, 1-phellandral, 1:_-sesqui terpene s, and d-sesquiterpene alcohols. It does seem desirable to determine the optical rotations for the other oils also. All the ester content cur v es exhibit the same form, a h i g h pe ak loc ated in Di vision Z i_n all c a ses (a furt her favorable chec k on the division method). In addition, some curves show a tiny peak i mmedi a tely a fter the ma jor pe ak and located in Di vision W: Vlhere investi gated, s mal l er percentages of esters have .be en found in other re gions a l so. 'i'he major peaks of ILl\i a nd IIIS are noticeabl y lower t han t hose of the other oils. r35 In the fore g oi n g discussion, it has been s h own quite clearly, a mon g other th in g s, that the me t hod of d ivision is qui t e satisfactory for oils IL, IIL, and IIIL, perhaps somewha t less s a tisf a ctory for ILM and I IIS. .L'he se evalua- tions may be held in mind whi le considering the resulting data which wi ll now be presented. The C {uantitative Chemic a l Compositions of t he Oils. 1'a.ble:xx:zr gives the results for one year leaves for which the improved apparatus was used. These results are better t han those for any other oil and illustrate the reproducibility possible for the terpene ran ge when using this apparatus~: Table:x:xDf collects the quantit a tive chemjcal composition s of .a ll the oils. Finally, Table XxW is a presentation, unfortunately imcomplete, of the quantitative distributions of esters in the various oils. After the data were tabula~ed, an effort was made to evaluate the reliability of each figure, reference being made to t he ori ginal data and to the various graphs, and consideration being given to the points of the previous discussion. An asterick (,i- ) indicates that a figure is re l iable. A question mark(?) indicates that a figure is doubtful. In the case of esters, parentheses indicate that the fi gure is a minimum value only, saponification values for all the concerned fractions not being available. 11 ABLE XKU • Quantitative Chemical Composition of One Year Leaf Oil Expressed as Weight Percentages of 'r ot al Oil Illustrating Reproducibility of herults with Improved Appa ratus. Di vision Im12ortant Components Wt. Percentage A12rox. Wt.Percentag IL IL 1 IL IL 1 Ave. a-pinene 20.2 19.7 20 20 20 S-pinene (unknown terpene) 27 .o 15.3 11.7 23 .2 14.0 9.2 27 • 15 12 23 14 9 25 15 11 limonene m m,u,m. terpenes 15.4 15.6 15 16 16 62.6 58.5 63 59 61 'N 1,.·3 1.0 1 1 1 :t I[ i Ira ll'b 'IC carbonyls,ester 5.8 '7. 6 6 8 7 w sesquiterpenes 8.6 8.0 9 8 9 EI sesquit.alcohols 14.1 12.1 14 12 13 iJB(residue) (resins) 5.5 :tr(trap) 1,: 3 i 99.2 6 0.5 1 1 1 13'6 T ABLE K.&D1 • ~uantitative Chemical Compositions of Oils Expressed as Weight Pe rcentages of Total OiL Division IL II L III L IV L I LM III S :r 52(?) lI m 5 (? ) 13~23 {?) 57•r. 12{?) 16-r.- es ter non-est er 4>,!- 10{?) 14{l- 16 {? ) 21 ( ? ) 4 (? ) 6(?) 9(?) 3(?) 6 4 3 4 3 9 98 98 99 96 93 93 T ABLE x.x; 02' . Quantitative Distribution of Esters Expressed as Weight Percentages of Total Oil. II L III L IV L I LM III· S Division I L 1 0 .7~~ (0.3) g 1.3~~ (0.1) BT 0.9~· E T, ]I, 1Sl 2 .9..;~ g 0.3-J:· O.Hr 0.2* 0.1 -r.· I 2.7{:• 2 .6-r.- 3 .6~- 2.1* 1. 7 ➔~ 1.9 ➔~ Et 0. 9-r.- ( 0 .7) (0 . 2 ) (0 .2) (0 .9) 0.H:• (0.4) -0~· (0.1) 2.7(?) ( 0 .5) 3Z[[ 0 ?E- 0. l·K· ( -0) ( 0 .1) :sen. iX' - 0~- 0.3 (7.1) (3.4) (4.6) ( 2 .8) (2.6) 4.8( ? ) r4o, The following fairly reliable points of interest can be extracted from the tables: 1. The terpene contents of all the oils are substantially the same, with the except ion of I LM, which has a much lower terpene content. Ostensibly, the ter_pene contents (exceptin g I LM) range from 55% to 69%. However, in different distillations, varying arn01.mts of material pass over into the trap (Div.'Ei), thus introducing another complication (which, fortunately, is large l y eliminated vv'ith the improved apparatus). From experience, the author would judge offhand that this material in the trap consists largely of terpenes (pinene and limonene), plus further material that belongs in .ui v.N. Further, I suspect that tb.e material in Div.lli"consj_sts largely of another ter-:p3ne. If the amounts of_ these divisions (I,11,J:B,Wan dJI.) are combined, there are obtained the following values for "terpene content", with a ran g e (exclusive of I LM) of 61 - 70%: I L II L III L IV L I LIVI III S 63 64 70 64 48 61 2. The relative proportions of the individual terpenes are substantially the same, on the basis of the reliable results available for IL, III L, and IV L. If the amounts of the various divisions are converted into the percentages of total terpenes, these figures arise: I L III L IV L I 33 33 36 II. 41 48 JiI 26 19 :i H,m 67 67 64 £I,n,m. 100 100 100 Division 14-0A 3. 'l'he non-ester 11 carbon-yl" content of Div. :E' is the same for all leaf oils (3-4%), is mucti higher for III S (14%), and is probably hi gher for I LM (lo%?). 4. 'I'he ester block of Div. IVto Div. W (but contained primarily in Div. Ji) is substantially the same for oils I L (3.9%), II L (3.4%), and III L (4.0%), but si gnificantly less for oil IV L (2.4%). It is also significantly less for I LM (2.6%) and III S (2.0%). ( '.I'hese values, it is reminded, do not represent the total ester contents for these oils). 1 5. 'l he s.esquiterpane content (Div.E) is the same for oils I L, II L, III L (9-10%). It is probably higher for IV L and I LM. It is definitely lower for III S. 6. 'l 'he sesquiterpene alcohol content ( Div.El) is also the same fol" oils I L, II L, a nd III L (10-13%). It is definitely lower for III s. 1 7. r1 he combined sesqui terpene and sesqui terpene alcohol content is sub stantially the same for all leaf oils (19-23%), g reater for I LM (30%), and much less for III S (7%). 141 Disclilssion. It has sometimes been proposed that the sesquiterpenes, diterpenes, trit erpenes, tetraterpenes, and polyterpenes (rubber) are perhaps formed by simple polymer ization of the terpenes~ If this viewpo int were correct, i t would be exp ected th2,t, as a plant grew older and the po lymerization proceeded, its oil would contain an increasing percentage of the hi ghe r ~ole cular wei ght compounds . A second viewpoint is that terpenes are not intermediates in the formation of the higher compounds (Prokofieff, 19 40), but that thes~ higher compoun6.s · may arise directly from more or less specific prec~rsors. Accordingly, Haagen-Smit and Siu (1944) sugg ested that rubber may have "its origin i n polyoses like star ch and inulin, whi l e the terpenes are derived in a similar way from s impler sugars as postulated by Emde" (1931). Haagen-Smit and Siu reasoned that, since the terpenes of guayule essential oil are at le ast 90% cyclic, it seems unlikely that the stable six-carbon rings wou l d open up, a necessary s t ep if they were to serve as precursors of the aliphatic rubber molecule. Our present work gives additional support to the iatter viewpoint. Th e content of sesquiterpene~compoun~s, as wel l ,'3. S thR t of terpenes, is ess en ti ally constant for the one, two; thiee , and f our year leaf oi ls . Since after four years no noticeable accumulation of sesquiterpene compounds occurs, the polymeri zation of terpenes to sesquiterp enes is unlikely . 142 Notes on Future Procedure. If work of the present nature is to be prosecuted most effectively, better analytical methods must be applied. The use of a precision fractionating c9lumn, as in the later stages of the present work, is a great aid, but is far from enough. Specifically we need: 1. Methods that are capable of handling smaller amounts of oil satisfactorily. a. Less plant material is thereby required. b. More sele ct plant material can be choseno c . The present high expe~diture ot time and effort can be iteducede 2. Methods that are capable of directly analyzing oil mixtures without preliminary sep~ration. Spectrophotometry is perhaps the most valuable method of this kind and should be applied .. The author has carried out considerable work and study on quantitative ozonization and predicts that this method may also be a future tool in this field. Q98 20 . ;I Fig. Z7 ~ Quantitative Distribution, Bhysi gal Constant, ·and Ester Oontent ·Curves fo r the Fractionation . of. Two !ear Total. Gua ule Plant Ha . n- Smit and Siu l 44 . 144 :IL, IL ' .! I I ' '' J r \ ' \ \ •0 60 Cl: w t- 20 _ 21 N 80 J.50 20 I' /' ~· 1~ I - -.--n -.----..--- r - - '\ \,. .\ ' Fi g 2~~ Dunlic a t e Quantitative Distributioni Physical Constant , and Ester Content Curves for the Fractionation of ONE YEAR GUAYULE LEAVESo /45 .Ir L - - 19 18 • -- ---- - - - - - + - -- - I _; Fig. ~ - Quantitative Distribution. Physical Constant, and Ester Content Curves for the Fractionation of TWO YEAR GUAYULE LEAVES. 14(<1 ID.L a> 0 21 18 1.50 +30 · .a, l49 100 80 •IO ' 0 0 't:1 -10 -a, -,a : -40 - ,, r Fig. 3Q. Quantitative Distribution. Physical Constant. and Ester Content Curves for the Fractionation of THREE YEAR GUAYULE LEAVES. 14"1 ·00 &) ei t- "'w 4i) I-{ al ·o I 21 • 20 19 18 L49r---r- -- - t -t -- - + - - - t - - - - -/1 - j - - - , - - - + - - --+- -~ 'Fig, 31-. Quantitative DistributionJ.- Physical Constant. and Ester Content Curves for the Fractionation of FOUR YEAR GUAYULE LEAVES. ILM ··: , - -(I) ~ - •• , .,~:i: ... - · - - - · · - · - - · - -·-·· -·-. --- :r_t_; .. : • - ·--· ___·,;...+-;- ··' w 1 1---401P: "':!..........--l------+-- --+-- - - + - - - + - - - - - - ' - - --+---------:----+--t--t - 20 . • I~ L "I\ A • LJ_-...---'f ... ... · - L __ ----- IOI · .,,. .. le . 80 u -·.- ·- ~ 6( ?a: 1---------·- • C) 40 ~L-0 vi" :::E . 2C ~ I I 1 "' i T .. , IIJl..:....111• TEMPERATJJRE 'C ,. . -i-- i .. T D u-0 •- Quantitative Distribution, Physical Constant , and Ester Content Curves for the Fractionation of MOLDY ONE YEAR GUAYULE Fig .:32. LEAVES. ms I I ,,r ;. . . I 'II '' j ... ), t. I· •' I ' I I. I; ....,_;;jil,,....-~~~·. Q,uanti tatlve Distribution, Physi'cal ' Constant. and Ester Content Curves for the Fractionation of THREE YEAR GUAYULE STEMS. . Fi_fu __;58. J. Summary 2 lo Investigation·s on the chemical compositions of the essential oils of leaves, stems, and roots from guayule plants of approximate ages of one, two, three, and four years, have been carried out. Over 1500 kg. of fresh plant ;naterial have been handled by steam distillation. 2. Physical constants and ester contents have been determined for all the oils: a. Eater content decreases steadily with increased age of the plant. b. Ester content decreas·e s steadily going from leaves to stems to roots. Root oils have far stronger negative optical rotations than any other oils. Co 3. One , two, three, and four year leaf oils, one year moldy leaf oil, and three year stem oil have been fractionally distilled. The results thus derived on the quantitative chemical compositions of the oils have been carefully evaluated~ a. One, two , and three year leaf oils are substantially similar in the following respects: 1. Terpene content (65%) . 2. Dj_vision V "car bonyl" content (4%), 3. Division IV, v, and VI ester block content (3q8%). 4o Sesquiterp ene conten t (10%). 5. Sesquiterpene alcohol content (12% ). bo Four year leaf oil is similar· to the one, two, and three year leaf oils in some respects: 1. Terpene content. 2. Division V '' carbonyl" content. 3. Total sesqu iterp ene and se squiterpene a lcohol content. 1st B. (cont.) Four year leaf oil is different from one, two, and three year leaf oils in other respects : 1. Division IV, V, and VI ester block content less (2.4~ ). 2. Sesquiterpene content perhaps grea te r. c 0 Three year stem oil is similar to the one, two, and three year leaf oils in only one respect, terpene content. It is .9:if:f erent fro m tbe:m in th ene respects : 1. Division V 11 carbonyl 11 content much greater (14%). 2. Division IV, V, and VI ester block content less ( 2 • 0% ) . 3. Sesquiterpene content much less. 4. Sesquiter p ene alcohol content JilUCh, less. (Total sesquit erpene and sesqaiterpene alcohol content is 7%). d. One y ear moldy leaf oil is different f rom the one, two, and three ye a r -leaf oils in all observed respects: l . Ter-pene con tent much lower ( 50%). 2. Di vision V II ca.rbonyl " content probably gL~il~J:. 3. Division IV, V, and VI ester blockcontent less (2.6%). - 4. Total sesquiterpene and sesquiterpene alcohol content greater (30%). . 4-.. The significance of some of the results in connection with essential oil ra l ations in the guayule pl ant has been discu ssed. ., ]Y.APPENDIX IY.APPENDIX DESCRIP'rioN OF 'r HE PRECISION FRAC'I'IO NA'J:IO N AP PARATUS The present appa ratus is similar to that described b y Podbielniak (1933). It is shown i n Fi g . 33 . A performance curve is g iv~n in F1 g . 3 4 . Column. The column consists of a meter- lon g fractionatin g tube, 6 mm. inside diameter, packed with a single nichro me helix, 2 turns per cm. Heat insulation is secured by a jacket; the inside of the jacket and the out s j_de of the fractionating tube are silvered, several small windows being provided at appropriate places to allow observation of the packing; the jacket is kept eva cuated by permanent attachment to a mercury diffusion pump during the course of a fractionation. 'i 'o avoid bre ak a g e due to differential temperature elong ation (of the hot fractionatin g tube over the cold jack et ~ the fa c ket is sepa rated at one place and recoupled with a rub b er sle e ve. Still Head. 'I1he still he a d is ma de small md compact to mi n i mize hold-up. A tiny thermo-couple well repl a ces the usua l thermometer. Mea n s for chan g i ng the receivers without i n terruptin g the fr a ctionation a re p rovided by a 3 way t a k e-off stop-cock. Pressure Control a nd Me a suring System. The vacuum source for all fraction a tions is ·the mercury di f fusion pump, back ed by a Hy vae oil pump. In t h e c a se of 20 mm. pressure, a si mple mercury pressttre re gulator is interposed in the system; while, in the c a se of very low pressures, the same regulator, but with 154 butyl sebocate substituted for mercury, is used. Instead of b~ the troubleso me bubbling of nitrogen through the boiler contents, bumping is a voided by sealing a platinum wire into the bottom of the distillation flask. .1.'he pressure is measured wj_th a suitab le manome ter or McLeod gaug e attached as near as p r a ctical to the top of the fr a ctionating column. Temperature Control and Measurin g System. Instead of a troubleso u e oil bath, a Glas - Col heating man tle, regulated by a Varitran, is e mployed. The temperature of the boiling liquid (inste ad of the less i mportant bath te mperature) is meas ured b y a thermo meter inserted into an appropriate we ll. 'I'he heat insulation of the column has already been mentioned. The temperature of distill at ion is me a sured by me a ns of a thermo-couple system. One junction is inserted in the well at the top of the fractionatin g column, the other j unction is a ttached to a t h ermometer bulb, which is also wrapJX:ld with glass cord and a heating wire. Vi.hen the galvanometer included in the circuit registers zero current, the tempe rature of the thermometer, as readily regulated by the heating wire and an a ir jet (for cooling ), records directly the distillation temperature at the top of the column (a sugges tion of Dr. V'~.B. Dandliker, communicated through Dr.J-.O. Kirchner). Miscellaneous Apparatus. A dry ice trap is provided to catch any uncondensed va pors pa ssing o v er. Of part icular note 155 are the vacuum-jac k eted standard taper joints (Podbielniak, 194 1 )> located at points exposed to hi g h temperature, which, lubricated with silicone g rease, a re hi ghly lea~ -proof and otherwise connnendable. '1;he oil fractions are directly rece:i. ved in small screw cap vials in which they are stored. PRESSURE RECORDING SYSTEM TEMPERATURE RE:CORDIMG SySTE:M ~ ~ PRESSURE R.E C.VLATOR DRY ICE TRAP SILVERED VACUUM JAtKliT WITt+ WINDOWS Ff)A VISl811.ITY' 6 MM. lNS IPS O IA MerTE:lt NICH"OMI! .5PlltAL PACl<ING TO t)1FFUSION PUMP JAC.Ke• se PJ\ R;.11·e: o,- - ·- - - - . Jo1~1eo sy Ru~SE>{ COIVNS'C.TI ON (TO AVOIO •l'l ■Al(l'I, • • ) Fifu-2)• High Precision Fractionatio~paratus. ;. +• :t t:ct:i TT+[. ·H -on -r 1+' THE DETECTION AND PARTIAL ISOLATION OF MANY NEW COMPOUNDS FROM GUAYULE ESSENTIAL OIL There is presented in Plate Ga composite curve bringing together the results of one primary fractionation and five refractionations of crude guayule oil fractionc. The cheuiical complexity of e:uayule essential oil is clearly inc:Ucated. The detection and partial isolation of many new compou.nds from guayule essential oil is demonsti-'ated. The f ull presentation of this work ( including Plates A to F for the individual fractionations) wi ll be given elsewhere, '' 1• ! - t· 1.50 ~ l."19 X ~ ~ UJ > ;:: );J 1.48 ...a: \· UJ a: 1.47 1.-46 • (•c). ~~ c.o ,. Ot-- -+---+---+--+- - t----+--+-- -+- ·-+- ----j- - ' -+---+-- -+-- -+--+---1 '2 Ot-- -t-- -t-- --t+--t--t----+-- +-- -+- -+- ----j- -f--- +---t-- - + --+---1 <r I I Ot-- -+---+----t+-1- I rP -fl- - -- II G t--- +-·- - - - ----- I -,--- 1' ' I --t-1 ----r- i I - - _J___ O t--- 1-------<- - - to +- ' 4~--.,t i "' i-- 2 1-- - , - -- ~ I I :::i- , I I -- . " . ~ ." ~ --~--- I )< 1A · - - f--------- 4= ~ , x_ ~.;, >"i ~ ~ ,t Ji GENERAL SUMMARY GENERAL SUMMARY Several more detailed summaries will be found appended to t~e more lengthy individual sections ( p . 8i)JSO). 'r he principal results of the thesis as a who l e are collected here. 1. Procedures have been developed which permi t the rapid and pr·ecise micr o-determination of the essential oil contents • of plant organs and the oil contents of diffe rent tissues therein. 2. <1t has been recommended t ha t work be carried out on the next phase of t h e program, namely, the micro-chemical determination of specific oil components, so that the compositions of the oils from d ifferent tissues can be studied. 3. It has b een shown possible to segregate the cours e of essentia l oil formation in the young g rowin g guayule le a f into three pha ses, correspondin g to three phases in the morphological development of the le a f. 4. c h emic a l Lar g e sc a le inve s tigations have been c ar ried out on the co mp ositions of t he esse n tial oils of le aves, ste ms, and roots from guayule plants of one, two, three, and four ye a rs. a. Ester contents decrease stead ily wit h increased a ge of the plant; decrease steadil y g oing from leaves to stern s to roots. b . Root oi ls have far strong er negative optical rotations t han any othe~ oils. c. One , two, three, and four y ear le a f oils a re of r a t l1e r si mil a r composition, the four y ear oil showin g perhaps some differences. d. Th1° ee y ear ste m oil has a much g reater flcarbonyl con tent and a much s rna ller,cont ent of sesquiterp e n es and derivatives than t he leaf oils. 11 e. rhe biochemical sig nific ance of some of these results has been discussed. 5. A graphical communic a tion of t he detection an d partial isol a tion of many new comp ounds fro m guayul e essential oil i s appended BIBLIOGRAPHY BIBLIOGRAPHY Art schwager, E.. , U.S. Dept._ Ag:£. __ Tech. __Bull. 812- , l (1943); Biol. Abstr. 17: 22323 (1943). Bonner, J., Bot, Gaz. 105:233 (1943). Collings, W.R~, Chem. Eng. News 23: 1616 (1945)J Durrens, T., The Distillation of Essential Oils; in Young, So, Distillation Principles and Processes, MacMillan, London (1922)& Emde, H., Helv. Chim .. Acta, 14: 881 (1931)@ Frey-Wyssling, A. and F. Blank, Ver hand lungen der Schweizer. Na-turfo_rschenden _Gesellschaf-t, Locarno +940 : 163 Haagen-Smi t , A. .T., and R. Siu; Jour o Amer. Chem. _Soc. 66: 2086 (1944)e Jacobs, M. Bo, The Chem~stry aa d __Technologz___9_f FQ.od__ .:1. :_:1.d fo_o_d_J~:r-9.d~gt,s II, Interscience, New York Tl944), p.73b Morr ens, C. , .9:e__Gand ~g.l}g,_!_~~ - --Q..§ ___ l~_ §_Q .9i.~.:t~___FZ9y9J._~ _d ' A g r i c u 1 t _ ~ r ~. i, 1845); in Nicol , H.. , f .~:t::'J'. .!. E~_e L _QJ_l _E.~.co.X.'. d. 1936: 328. Moshki(na, M.S.,, Bull. __acad. sci. _URRS..i.___Ser. __ biola 4:6 14 1940); Chem. Abstr. 35:2931. ,Khim. Referat. Zhur. 1940E2): 41 ; Atst_r_ . 36: --1982-.. ·- ----- --- ---- Ch em, ·- ---·--·- Podbielniak, W,., J_pc._.L En ,q· . Ch em, , AnCi.l~ Ed. 5:119 (19 33);; Proko fieff, J_ zvest. ___Akad. Nauk. S.S ._~_!_;RG 589 (1940)., Smith, Jo, Plant Phv s .. 15: 183 (1940). 17!ent , F. W.. , ~mer. Jour. Bot. 30:15'7 (19 43 ) "' Whitmore, F. and Lux, Jour. Amer~ Chem. Soc" 54:3448 (1932) ~ Wulff, E.Vo, Essential Qll Pla nts, The:tr Cultu:re_and Es~epti a l Oils, Lenin~rad (1933). ieneral References ,, " Gildemeister, E. and Fr. Hoffmann, Die Atherischen Ole, Schimmel and Co. (1910). Allen' a Commercial Org_ani.c AnaJ.isis IV; Blal:ci ston, Phi lac el:_",l':i ;_~ ( 1925) ~