University of the Pacific Scholarly Commons
University of the Pacific Theses and Dissertations Graduate School
1967
Studies on the essential oil of Anemopsis Californica
Ramesh Narmadashanker Acharya University of the Pacific
Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds
Part of the Pharmacy and Pharmaceutical Sciences Commons
Recommended Citation Acharya, Ramesh Narmadashanker. (1967). Studies on the essential oil of Anemopsis Californica. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/400
This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. STUDIES ON THE ESSENTIAL OIL OF ANEMOPSIS CALIFORNICA
A Thesis Presented to the Graduate School University of the Pacific
------·
In Partial Fulfillment of the Require~ents f or the Degree Mast er of Science in Pharmacognosy
by
Rarnesh Na r~a i as hank e r Acharya
May 1967 This thesis, written and submitted by
is approved for recommendation to the Graduate Council, University of the Pacific.
Department Chairman or Dean: ./ J ~~ (_./' -~ ~ / /}----o~--C'-~-'<- \
Thesis Committee:
Dated ACK N O\~LEDGm1ENTS
The author wishes to express his sincere gratitude to Dr . ChalJbal for his encouragement a nd guidance throughout this wor k . Without Dr . Cha ubal ' s assistance , thi s work could not have been done . Sincere thanks a re also exnressed to Dr . Roscoe f or his helo in this wo r k .
A gift of some authentic sarn ! ~ l es from M/S Fri t zsche Br ot her s Inc ., New Yor k is gr a tefully ac kno t•rl edged . Fi nancial assistance from the Pfeiffer Founda tion and from the University of the Pacific i s 1eepl y ao n rec i a t e~ . TABLE OF CONTENTS Page Acknowledgement ii List of Tables iv List of Figures v I. Introduction 1 II. Extraction and Physical-Chemical Constants of Oil of Anemopsis 4 III. Functional Group Separation of Oil of Anemopsis 8 IV. Fractional Distillation of Oil of Anemopsis 21 V. Chromatographic Methods Introduction 24 Thin-Layer Chromatography 26 Thin-Layer Chromatography of Oil of Anemopsls 29 Gas Chromatography 38 Gas-Liquid Chromatography of Oil of Anemopsis 47 VI. Summary 67 VII. Recommendations for Further Research 68 Bibliography 69 Appendix I 74 LIST OF TABLES TABLE Page 1. Phys ical a nd Ch em ica l Constants of Oil of An emoosis 7 2. Ii.entity of Unknown l? henolic Fr a ction with Thymol 11 3. Fractional Distilla tion of Oi l of An emousis 23 4. Spray Reagents of Thin-La yer Chrornato grauhy 32 Gas-Liqui1 Chromatogr aphic ?roce1ure 50 6. Re tention Time of Ma jor ?eaks of Oil of Anemoo s ts l.J·9
Concentra tion of Major Co~~ onents of Oil of Anernops is 66 LIS1' OF F'IGUHES
FIGURE Page 1. Functiona l Group Separa tion of Oil of Anemons is 9 2. Infra - re1 Soectra of Thymol and Unknown Phenolic comoounl 1 2
3. Thi n-layer chr0matogram of oil of £1-n emo p~_l.§. 33
4. Thin-la yer chro~ata g ra~ of Phenoli c Fracti on 35 5. Thin-laye r chroma togram of Ca rbonyl Fraction 36 6. Thin-la yer chromatogram of Fraction from Fractional Distill ation of Oil of 37
7. Schematic Diagram of Ga s -Liquid Chro~a t og ranh 39
8 . Gas -Liquid Chr omatogram of oil o f An e~oos is us:i.ng FFAP as a sta tionary ohase ------51
Gas-liquid Chroma togram of oil of Ane~onsis us ing Hy :"'~ erose s?..:: so as a s t a ti o na :r.·y.~--- · ohase 52
10 . Gas-Li qui1 Chro~ato g ram of Fr action # 1 from Fr acti onal Di s til lation of oil of Anemousi s 54
11. Gas-Liq1.tii Chr ~ >mato g ra :n of Fr a c t i on # 2 from Fracti onal Di s tilla tion of oil of Anemousis 55
.12. Gas-Liquid Chroma t ogr a:n of Fr a ction !f 3 fro~ ?r act i ona J Oi stilJation of o il of --Anemon----s i s 13. Ga s-Li qu i d Chr omatog ra~ of Fraction J 4 from ?ractional Di s tj_lla t i on of o i l of -An---emonsi s- 57 14. Infra-red S~ect ra of Methyleugenol and Peak !f 2 59 vi
FIGURE Page 15. I nfra-red SDe ctra of ?. i oeritone and Peak !J 3 62 16A. Infra -red S oectra of Peak # 12 6Lt- 16B. Inf r a - red Spectra of Peak ll 12 65 CHAP1'ER I
INTHODUCTION
The roots and rhizomes of Anemonsis Californica
(Nutt. ) Hook and Arn . (family Saurura c eae )have been used by the early Spanish America ns a nd I ndians to treat a variety of ailments . 1 ' 2 ' 3 ' 4' 5 ' 6 The plant is commonly known as Yerba del Mansa, Yerba Man sa , or Manza. The family Saururaceae is renorted t o consist of
s-recies r enorted , belong lng to the genus ~l}_~.i~P-~-1..~?.• 1'he plant i s na t ive t o the South Western Un ited States and t o Northern Mexico . I t i s c ommonJ.y f ound in wet alkaline soils at e l evations b elow 6500 f eet . 5 Its range extends from California , Utah , Arizona and Texas to New Mexico . The :.Jlant is vJell d i stri but ed from Lo we r Sacr amento Val ley, San J oaquin Va lle y , South Coast Ranges , Inyo County t o Southern Cal i fornia . 4 An e11 opsi. s Ca l i_f ornica_ a n:l o ther members of the f a mily Saururaceae are consi.der ei t o have little econom ic3.1 i :noort !":'. nce , anart fro~ the "nati ve" meilcina l uses of Ane~ons i s .
A~oPs i.~ i s an herbac eous , DPrenni a l d ico t y l e - doneo~s ~ l an t . The nla nt has bgsaJ. l eaves which are 2 large a nd fleshy. The plant blossoms from May t o Augu s t and has an inflorescence c onsisting of ma ny small per fect a~etalou s flowers on a s oike. The bra cts a t the base o f the spike a re white. The pla nt has pr o~ ine n t r oots and rhizome s wh ich exud e a strong a romatic spicy od o r. The r oots a nd r h i zomes a r e c on s i d ered t o b e a very usef u l remedy f or c u t s a nd br u i ses , s or es and spr a i ns . Anemo os i s has a lso been r eported t o b e usef u l as a d iure tic , in rhe uma tism , a ni as a blood ~ urlf ie r in asthma , dys ent e r y a nd gonorr hoea.2
There a r e only a f ew r e uorts pub l 1sh ed on the chemical investigation of t he p l ant Anemoosis . Hort on a nd Paul? i n 1957 first report ed the pr esence of Methy l eugenol as a ma in c onstituent of the essential oil 1erived from this p l a nt. Pickering8 in 1958 , c onf i r med the presence of methyl eugen ol (4-allylveratrol). Ch i lds9 in 1 963 , also c on fir ~e1 the or esence o f methyleugenol and re ~o r ted the oresPnce o f a l e uc oant hocyaniii n in t he pe tr o l eQ~ ether ext ract o f the ul a nt. Th e aut hor obser ved t hat the tot a l an ti spa s~oi ic activity of the plant c o uld n o t be due to ~ethyleugeno l a l on e .
Childs and Col e l O have a l so r e ~o r ted on t h e phar- . macol ogi c a l a nd a n t i t umo r pr operties o f t he extract o f the roots and r h i zomes of Ane~oos i s . The paucity o f r eoor ts 3 on chem ical investigations and the r eported antitumor activities of the olant drew our attention to the drug .
We ~ecided to investigate the essentia l oil of An e~ oosis
Ca lifornica in greater detail . The r esults of our investi gation to date are included and described in this thesis . CHA PTEH II
EX.TriACT ION AND PHYSICAL-CHEfiliCAL CONS'TANTS OF OIL OF ANEMOPSIS
Crude Drug Material The crude drug Yerba Mansa i s commercially avail able . It i s used at pr ~ s e nt in «native « Mexican medicine .
We obtainAd the dried whole roots and rhiz o~es of Anernoo sis Ca lifornica from M/S Ha thaway Al lied Proiucts (2024 West gate Av enue , Los Angel es 25 , Ca lifornia ) in t ~ r ee batches . These batches wer e supolied as (i) Lot- RM - 65- 167 , (11)
Lot-RM- 65-1029 a nd (iii) L ~ t - RM - 6 7 - 9 1 .
Extraction of Oi l Guenther11 has 1es cri ~ed various "methois of extr a c tion of essenti al oil fro ~ ~ l a nt s . The f our ~a j ar ~e th ods used are (1) Hydrodis tillation , ( i i) Solvent extraction ,
( iii)Enfl eurage and (iv ) Cold exnression . The ~e th o1s of hyirodistillati on a n1 solvent extra ction are most often used . The othe r two ~ethoi s a r e of limited a ~n lic a tion in suecial circ ums t a~ c es . Horton? ani Pickering8 employed hy i roi istillati on of Mansa f or their s tud ies . Childs9 on the other hand , 5 used sol vent eY. traction Hi th pntrolc:u!n ether a nd m e th ~-· noJ. .
PreJiminary experiments showed that t he yield of the essenti~1. l oil by solvent extr Hycl rodj_s tillTtion g and excessive fo~m ing a l s o oc cu r~ ed . set u~) j.rE.:er3 :i. a tely ~.;o 8.void eny losses of volatile !::c:tteri::1. J. s due to e t ~ o sph e ric ex9os ure . A mortified C leven~er ' s appa ratus 6 The oil collected was only slightly lighter than water. Some oil globules, espP.cially those collected towa rd the end of the distillation, were heavier and were observe1 t o sink in water. Also at the beginning of the distillation , the color of the o il was light yel low b ecoming deep g reen at the end of distillation. The distillate was allowed to stand for 10 hours; the oil which separated was withirawn and ke~ t ov er anhydrous sodium s ulfate (l-2% W/V of oil) for 30 hours to remove the l ast traces of water from the oil. The dry oil was ke?t in a well stopoered a~ber colored bottle and stored in a refrige r ator. As a large still was unavailable , the d istillation was c arried out i n batc hes of about 300 g of the crude drug. The yields of the essential oil ra ~ge1 from 5.5 to 6.5 V/W of the crui e drug. All batches of the distilled oil were mi Yed together for our studies. Physical and Che~ic ~ l Constants of Oil Some commonly employej physical ani c hemica l measure~ents on essential o ils are useful in q uality control, once t ~ e se values a r e well established f or a particular oil.11 In the c ase of a n ew oil these cons tants are of a limited use . However, t hey ~ay give a r ough iiea of the types of c onstituent s present in the oil. Table I 7 (page 7) gives the physical and chemical constants of the Mansa oil. TABLE I - PHYSICAL AND CHEI1ICAL CONSTANTS OF OIL OF ANEMOPSIS Constants Values Specific Gravity d25 0.9980 25 C(25 Specific Rotation D = less than - 3.5° Refractive Index n~5 1.5195 Acid value less than 1 Saponification value 6 to 7 Addition of alkali solution changed the color of the essential oil to a deep red-brown. This color inter fered with end point determinations for the saponification value. This value was determined on the phenol-free frac tion of the oil (see page 9). The end point for the sapon ification value was determined potentiometrically12 as refluxing ,.,i th alcoholic potassium hydroxide caused a color change to brown, even after the complete removal of phenols prior to the saponification of the oil. C H AP 'J' j~~l I I J OI L OF ------A~SVOPSJS---·- T~e component s of cssent~al oils can be separit ed into fractj_ons accor dine to f unctional groups . l l , 12 Figure l (page g) is a flow di aGr8n of the procedure empl oyed f or such a fur"c tional group sepe r-FJ.t:Lo11 of the oiJ o C fi.~l1Sa . of tbe o:U .. The phenoJj.c f r ~ ction ~ns obtai n e~ after con s i~e~~bJc r.:; '..-~ ro xi:-1 e ( ? ~~ ) vr8.s requj_:;.r~d to !'·~ ::-: ove the !>henoJ.fj compl etely. ~' he "':ld i +i on of 2 . 1:<:~ 1 i c:'18 n .:~ e d the color· of the 9 Oil ( 20 g) (i) Ether 50 m1 ( II) Aqueous Na 2co3 (5%) ( 6 ml x 3) \ f Ag,u e ou~ Phase Ether Phase Ti) Acidify ·Ni th 10% H2so4 ( i )-Aqueous KOH ( 5%) (ii) Ether (10 ml x 3) 20 ml each several times Ether Phase --=---ous Phase Ag,ueous Phase (i) Remove (i) Acidify with (i) Remove · E'ther 10% HCl Ether (ii) Ether Residue //1 Disc!a rd (15 X 3) Free Acids 0.0287 g Ether Phase Aque us Pha:?e Oil (17. 73 g ) DiLard Treat \'Ji th Girard 'T' (page 19 ) JR-;sidue /1 3 ~r~ony l Fraction. (i) Divtd.e lnt o three port ions Portion #2 Portion #3 1(i) Saponify !( i) Acetylate Control Saponified Acetylated ou:- - Oil on I _l___ i fa____ Thin--Layer and Gas - Liqulcl Chromatography F'ig . 1 Functional Group Separation of Anemopsis Oil 10 aromatic odor reminiscent of oil of thyme . Thin-layer chroma tog r a ?hY showed the or esence of one major compound which gave a positive t est f or phenols.. The brown oily liquid was distillei a nd clear c o l o rless distilla t e b o iling a t 230- 238° was colle cted . This d i stillate had a strong odor of thymol . On keeping i n a refrigerator for 4 hour s the liqui1 solii ified . The adher ing oily film was washed with c oli hexane an'i the solid ourlfie·d 0 a nd recrystallized f r o~ boili~g h exane ( b . p . 68. ? ). Thi s recr ystallize1 c om nou.n-i 1·m.s observ ei to t:e thym0l . Ta ble II ( Pag e 11) s h o •:1s the v a rious t es t s carried out t o show the i ientity of the subs t ance with thymo l. Str~ctur e o f Thymol Tests f or Thy~ o l ( Tabl e II) Snot Test (1): Di azot i zed Sulfa nilic a cidl3 Di ssolve 25 g o f S'J. lfanilic 9.c i d in 1 25 '211 o f a lOt sol uti on o f so1 iu~ hy1roxi ie . After c ool i ng , add 100 ml of 10{ so J ~ ti on o f soii um nitrite . The soJ.ut i nn 11 Table II - Identity of Unl{nm·m Phenolic Fraction 1-11 th Thymol 'rest Unknmm Thymol (f1erck ) Reagent Appearance crystalline crystalline Color white l'Thi te Odor Harm thymol thymol odor like Tests 1WDiazotized sulfanilic yellOl'T yellow· aci d phenol phenol ( 2) USP test for thymo l + + (J) USP test for thymol + + (4) ZnCl& + Phthalic + + Anhy ride , fuse + NaOH (5) H2so4 + FeClJ ( sol ) + + ---- - ____ .... Helting Point 48° -49° 49° -50° ---- Mel ting Point of Deriv atives (1) N- Phenyl urethane ( 2 ) oC - Naphthyl urethane r------~------~------~ Thin- Layer Chroma tography Both Have Same Rr Values t---·------· ~· ·------Gas-Liquid Chroma to grapl~ Identica l Retention Time ------1 Infra -red analys is Identica l Inf r a-red Spectra ~------+------· CM- .· ------· ·- --· ·- · ------.. ,. ----- 2000 1500 1 1000 900 800 700 1.. 1 l I I r ': 1 I I j r 1 r I I • I I I I I l I I I . I I 1 I I I j I I I I' I I I T I t____!____l__t-t ~ i f I ' I i i 1 li I I :I I 1 I 0.0 LI A ; I ' I I ·-rr % 1~ · ' ~~- .I I I j I I I f I I ! 1 ~ 1 I I II 1 1 I I I I w ' -,;;;;;;:;;: . ''~- ..,...... __ t 71V Li t .,w I. I I I , j ..... I I ' f I I l'li 'I o 'I j . •. .~~ -. j 'I . . I I l' I • I 'I .10 111111 I I ; i I I I I 1 I • t li T I I I I I 1 I I o I I'U1 ~ I 1 I i r-j ; • • H .20 . . ~ IO Of l I I I I' ol i__t_il j f.30 II I I I II r II ; I I II I I I t t_f. I 1 I I I _j_j. t T'";' ' . I I I I I 1 ' I I I • I I ' • I I .40 ., f I I t==FFI ~ . .50 .60 .70 111 11111 U II [lJ II JJI IJlJ I 111 11111 11 111 1111111 111 11111 11 l 11 11111 I III I I I II i l l II I I I III I I I I i I iI fi 11111 11 if I U TU 1.0 1 1 1Tii1: T r1 111! 1 T r t1 rl i n t 1 1 1t 1 : I I I T l i I 1 I 1 1 I I 1 1.0 oo I I r 1 1 1 1 1 1 1 1 1 1 1 1 . u j · . · . I 1 mg. in 300 mg KBr 1 ~ •1 - 1 II ..'__. ' I I I I I I T I T 1 I - - ~~ ~ ClC) 3 . 4 . . 5 6 ···~ · . . 7 . . ... 8 9 . 10 11 12 13 14 15' ····': .--:·.. .- . -- .. ,;.- ·,: -·.,, . ...J... . _:,· .- . . . . ) .. ~ - - ~ ·::.··· · ·... ~=: ,·. .: ·:::· _:·:""-:.-.:·._, ·_-:- ~: ·(.. _ ·.:~ .. : _WAVELENGTH {MICRONS . - .,. ·- .. .. . --~-: ...~ . ":'• - :~~ :.. -~ ~ ...... ·~ .,-· · - ...... A = Thymol B = Unkno~m Phenol ic Compound Fig . 2 I nfr a -red Spe ctra of Thymol and Unknown Phenolic Compound 1-' N 13 is added gradually, drop by drop into lee-cold hydro chloric acid (40 ml HCl in 20 ml water) while stirring. The reaction temcerature must not exceed 8°. The diazotize1 salt formed is filtered by suction and washed successively with ice-water, ethanol and ether and air dried. The spray reagent is prepared by dissolving 0.1 g of diazotize1 salt in 20 ml 10% aqueous NaOH. Thymol gives a yellow color. Spot Test (2): USP XIII test f or Thymo114 When 0.1 g of thymol i s heated in a test t ube in a water bath with 0.5 ml of a 10% solution of NaOH, a cle ~ r, colorless , or nal e r ed soluti on i s f or~ei , without the separation of oily dro os . Upon the aidition of a few dro~s of chlor oform t o t hi s solution and agit ating the mixture, a violet color is pro du c ed ~ Soot Test (3): US P XIII t est f or Thyrno114 Dissolve a very small crystal of thymol in 1 ml glacial ace tic a cid, add 6 dr oos of concentr a ted s ulfuric acid and a drop of conce ~ trat e 1 nitric acid; the liquid shows a deeo bluish-green color when viewed by reflected light. 14 Soot Test (4): Thymolphthalein test15 Place 200 mg of anhydrous zinc chloride in a dry test tube and hea t the tube in a small flame to be sure it is anhydrous. Add JOO mg phthalic anhydride and 50 mg thymol; heat enough to fuse and then cool. Add l ml 2% sodium hydroxide solution a ni stir the mixture to break the fused :nass. Add more sodium hydroxi:1.e solution to make the solution alkaline. A blue , ourple-red or green color develoos in the alkaline solution. The color dlsaooears on making the solution acidic and rea ~pea r s on ma~in g it alkaline. S oot Test (5): Thymol sulfonic a cid testll Dissolve 40 mg thymol in concentrated sulfuric acii, thymol forms a thymol sulfonic acid; the l a tter produces a violet color with a f ew crystals of f e rric· chloride. Preoara tion of Derivatives N-Phenylurethanel6 Take a small amount of the s ubs tance (50 mg ) in a dry test t ube , and aid a few drops of phenylisocyanate. Catalyze the reaction by aiding 2-3 drops of d ry pyridine. 15 Warm the solution on stea~ ba th for 5 minutes. Cool the contents of the test tube by imm ersing the tube in an ice beaker and scratch the sides of the tube with a glass rod to induce crystallization. Purify the urethane by dissolving it in a minimum a r.ount of ~etroleum ether and filt ~r when hot. Cool the filtrate, filter the cry~ta ls of urethane and dry on a clay plate. Recrystallize the urethane from hot ~ etroleu~ ether till it g ives cons tant melting point. 0( ··Na nhthyl uretha ne1 5 Dry a test tube in a small flame. While the tube i s c ooling , fit a cork with a c al ciu~ chlori1e t~ be . PJ ace rapidly 100 mg of thymol a nd 0.15 ml <.( -NaPhthyl isocya nate and stop oer it with the cork hol ding t he ca lcium chloride tube . Heat the t~be by means of a small iirect fla~e so tha t the mixture boils gently for 2 minut es . Allow the ~ i x tur e t o cool for J minutes a n1 then induce crystalli zat i~n by rubbing with a glass roJ . Add 5 ml of hentane and filt er . Extract the r esidue with hot hept ane ani c oo ]_ the h e ot~ne solution to crystallize the thymo l 0( -rl'3.-:)hthyl ure tha ne. Fi 1 t er an:l lry the crys t a ls on a cla y ol a t e . Recrystal lize from hot heptane till it gives a c onsta nt T.elttng ooint. 16 Carbonyl Fra ction: Residu e# J (p . 9) The aro~a or odor of an ess ential o il i s d ue ~ainly to the p r esence o f oxygenat ed com~ounds - alde- hy·ies ani k e tones, esters , ethers and ohenols . Even ::ninute a•nounts of these oxygena t P.d. c omuounds may c ontri bute a major portion of the a r oma of the t o t a l o il.11 The frac tion conta ining the carbonyl co~uounds in the essential oil can be separated from total oil. Addition of r eage nts such as sat ur a t ed aqueous or a lcoholic sol ution of 2 ,4-dinitro oh enylhyd r az i ne ( 2 ,4-DNPH ) to the essential oil r esul ts in t he precipitati on of crystal l ine or o ily c o m ~ l e xes from the oil.l?,lB After the sena ration of these nreci n itates , the carbonyl c ompoun'1s c a n b e non-iestructive ly r e generated from the comnl exes , extrac t ed with ethe r ani usei for f urther analy sis. These me thods enabl e one t o concentrate aljehy1es ani ket ·.Jnes fro·:-1 a c o:npl ex: mixture of c ompounds such as c onstit ~tes an essent ial o il. 2 ,4-Dinitro9henylhy1razine (2,4- DNPH ) is c onsidered to b e a more soecific rea gent for c ar ~ on yls . Initial spot tests and thin-layer chr omato- f.ra9hy 1sin ~ 2 ,4-0N ?H a s a s ~ray r eagent (o. 32) gave n egative t e sts . Howev er , as uo lnted out by Stahl 11 k e t on es such as pineritone , f enchon e and menthane would g ive a negative 2 , 4-DN ~ H test if pre sent i n J.ow concen- 17 trations in the oil. Hence the n egative test could not be t aken as indicative of absence o f ca r bonyls in the oil of Mansa. Pool and Klosel9 claimed a quantitative reaction of c a rbonyls in benzene with 2,4-dintrophenylhydrazine that had been adsorbed on to a n alumina c olumn. Subse quently, Schwa rtz and Parks20 sugg ested that a sol uti on of 2,4-d introphenylhydrazine in 2 N, hydrochloric aci d c ould b e replaced _advant ageously by 2,4-DNPH in 66% phosphoric acid. An elegant microanalytical metho1 reviewed b y Parsons21 incorporates both of the above ry rocesses. It is possible t~ c oncentrate, det ect and isol ate c a r bonyl c omuounQs as their 2,4-dinitronhenylhydra zon es from micro quantities of essential oil by this method . A modified methoi was em ~ l oyed for the Mans a oil as follows: Analytical grade Celite 545 (10 g) was impregnated with a solution of 500 mg of 2,4-dinitroph e nylhyira zine in 6 ml of ~ hospho ri c ac i d ( Density 1.75) and 4 ml of deionized water. The i~u regnate - t adsorbent was then transferre(l to a glass c olumn (2. 1 em inner diamete r) fill ed with cyclohexa ne. The stationary ~ has e was trans f e rr e~ in s~all ~ortions to achi ev e uniform oacking . The c ycloh exRne wa s r un off the column a ni the a~ sorbent 18 washed with 50 ml of benzene (Reagent , A.C.S.) to r emove the excess of unabsorbed 2,4-DN ?H . A solution of 0.5 ml of Mansa oil in 0.5 ml of cyclohexane was a pplied on top of the column ani the column eluted with cyclohexane until the eluant gave a negative spot test with vanillin sulfuric a cid reagent. The col umn was then eluted with benzene. The benzene (eluant) was initially deep orange red; the c haracteristic color of 2,4-dinitrophenylhydrazones of carbonyl compounds . This indica ted. that S1lall amounts of carbonyl coml)ounds w·ere oresent in the Mansa oil. The 2,4-iinitrophenylhydrazlne, finely disper sed on celite , c a~ e in intimate contact with the carbonyl comryounds in the essential oil. The derivatuves thus formed were adsorbed on the colu~n while the non-carbonyl compounds were eluted by cyclohexane. Successive elution by benzene re~ove d the derivatives of the carbonyl comDoun1s of the oil . Girari 22 r e norted a ~ethod f or quantitative isol a tion of carbonyl compounds using Girard 'T' (carboxymethyl-trimethyl a~~on ium chloride hydrazide) or Girard 'P' {carboxymethyl pyridinium am~o nium chloride hydrazi de) reagent. This methol has the aivantage of yielding wa ter soluble 1er\va tives of ca r bonyl comQounds with the r eacents which can be easily seoarated from the water 1n solubl~ non carbonyl fraction of the oil. 19 Addition of exc ess f o rmaldehyde or concentrated hydro chloric aci d t o the aqueous s o l u tion o f the c ompl ex quantita tive ly libe r a t es the c arbonyl compounds from the c o~p l e x mixt11res . The liber a t ed carbonyl c omooun1s ca n then b e extra cted by e the r or a ny other or ganic s o lvent. Teitelbaum23 used a modified Girard method t o i solate successfully carbonyl c om ~ o un is from the c omnl ex ~ ixture under n eutral c ond itions. Th e f ol l owing me tho1 was emp loyed to isolate the c arb onyl. fra ction of the Mansa o il. A mixtur e of 5 g Girari 'T' reagent ( Eastman Organic Ch e~ ic a l s ), 5 g o il of Mansa and 0.1 g of Dowex 50 W-XB wer e aijed t o a 50-ml r oun i bott on fl as~ c ont a in ing 10 ~1 of absol u t e a lcohol. The mi xture wa s rcfluxei f or l t o l ~ hours during which period t he Girard 'T' reagent d i ssolvei . Th e hot sol ut i on was l ecant ed into 40 ml of ice -water with vigorous s tirring . The solution wa s all owei to stan i f or JO min ut es a nd then extra cted with e thyl e ther (15 ml Pach x) }. The c omb ined ether extract was drie1 ov e r anhyir o us soldium sulfa t e f or 10 hour s . Ether wqs removPi unde r vacuum t o obtain the non-carbonyl fra ction of the o il. Th e aqueous sol ution c ontaining the Gtra r·i c om~lex was then treat ed with 10 ml · of hyi r ochloric a cil and kep t for 1 2 hour s . The libe ratei , o ily , carbonyl f r a ction was e xtract~1 with 20 ether (20 ml each x J }. The combined e thereal. extracts were driei over a nhydrous sodiu~ s ulfate for 10 hours an::l then the e ther was removei under va cuum . 'rhi s ca rbonyJ frAc tion (Res t::l ue If) , uage 9) gave a Do sitive spot test with 2 ,4-dinitrophenylhydrazine (yellow brown color). This fr3ction was f urther investiga t ed by thin layer ( nage Jl} a nd gas-liqui·"i chroma tographic methods ("aaee 60 ) • CHAPTEH IV FRAC'rrONAL DISTILLATION OF OIL OF ANENuPSIS Ess enti al oils c onta in t er pen e hyd r oca rbons and oxygena ted c omoounds with wiiel y 1 i ver gent chem ica l cha r a cteris tic a nd b o iling r a nges.11 ' 24 Due t o this d iver sity o f c omnon e nts p resent in a n e s seatial o i l , n o single ~ e thod of i solation g ives c omple t e ly satis - f actory s e p a r ~ ti on . Iso l a tion of i n1 ivi1ual c on s tituents i s bes t achiev ed a fte r · pr e li ~ inary separ ation s of gr oups of homo gen eous c ompounds by chemica l a n j nhysica ]_ ~e3 n s f ollowe1 by chroma t ogr a phic me thods suc h as thin-layer ani gas-liqui i chroma t ogr aohy . 25 The t er oen e hyiroc a rbon s pr e sent i n an essential oil ca n b e r emov e i , mor e or l ess c o~ D l e t ~ ly , f r~~ t h e e sse ntial o il by r e ctifica tion. The pr ocess of r ecti- fi c a tion--fract t nnaJ. rlisti l l a t i on o f essent ial oil under at~ o soh e ric or r eiuc ei 1r essure --yie l ds ter~e n e a n1 ter- nen~le ss fra ctions . Th e t e ro- enel ess o ils ( e . g- . Le ~ on Oil) a r e c o m me rci ~ ll y a va ilabl e f or p e rf u~ ery uses . 11 The 0rocess o f rec tifi c a tion o f an essent ial oil yiPlds fra ction s c o n t~ lning a f ew c omooun 1s in c onc en - tra tion s h ig h ~ r tha n th~ t i n the t ot a l oil . The in1 ivl- d 'J.a l c 0 m ~0 n en t s in such fra cti0ns may be better ·ietected 22 and i solated by subseq~ent ch ro ~a to gra~hic proc edur es as the ir c oncentr a tion s are higher in such rectified fra ctions . 25 The Nester-Faust Annul ~r Teflon Sptnning Band Di stillation Anpar atus (Nester-Faust Co ., 2401 O~l et own Road , P.O. Box 565, N ~ wa r k , Jelaware ) is an ~xcc llent fra ctionating equi pment . Thi s column has a l a r ge n umber of theor etica l el a t es a n i CQn senarate fra ctions wi thi n 2°-3° boiling uoint r ange if oresent in reJ.ative l y simple mixtures . Accor 1 i ng t o our nreli~ in ~ ry gas-liqui J chromatographic studies ( uage 53), the essential o il of An e~ oos i s conta ined at J.east 20 c ornoo~n ds . Consequent ly, v ery share seoaration c oul d no t be exnect ed . However , collection o f fr ~ ction s with c ertain amo unt o f oartia l c oncentration of c onstituents was ex9ec t ei . The follow ing methoi was usei t o fra ctiona t e the essen€ ial o i l of An emoosls . 40 g of the oi J. of Mansa were d istillei using the 2 feet l on g Sp i nning s~ nd d istiJ.lation c olumn un1er r e i uced nr essure o f 11-12 m~ Hg . ThP c o l : J ~n was a ll ~ wed t o equl l ibr ~ te f or 3 h o~r s nrior t o a ny t ake- o f f . After the equilibration o f the c o l 1 1~n , a r e fl ux r a tio o f 20 : 1 was ~a int a ln ed iurln s a ll t a~e - offs. f he f our c uts maie a r e s ho wn i n Ta ble J. These fra ct i ons were stu·iieJ by 23 thin-layer and gas-liquid chroma tography {pages 34,53). Table 3 - Fractional Dis till a tion of the Oil of AnemC2J?E.iS Tempera- Fraction ture Pressure Height GLC TLC If oo mm Hg e % H/ H Fig.// Fig.// -·"- --- ·-~ --·- 1 75- 100 11- 1 2 3.687 9 . 33 10 6 2 100- 125 11-12 3. 861 9 . 65 11 6 3 125-150 11-12 12.101 30.25 12 6 4 150-160 11-12 15. 148 37. 88 1 3 6 Residue - - 4.280 10 . 54 - - -·--·-···- ~~--~ Recovery - - 39 . 077 97.70 ,...... ,. __ ·-'------CHAPTER V CHROMATOGRAPHIC METHODS Introduction The word tr chromatography" is at present used as a collective term for a group of methods that at first sight may appear somewhat diverse. Chromatographic methods are based on the distribution of components of a sample be- tl'leen t1im phases and subsequent separation of the compon ents from these two phases. Keulemans26 has defined chromatography as · chromatogr aphy is a physical me thod of separation, in which the components to be separated are distributed between two phases constituting a stationary bed of large surface area, the other being fluid hed that percolates through or along the stationary bed, Chromatography can be classified into three methods based on the type of stationary bed employed. (i) column chromatography (ii) paper chromatography and (iii) thinu layer chromatography. A technique developed by Martin27 in 1952, is called gas chromatography indicating that the mobile phase in this case is gaseous. Tswett i s given the credit of first employing chromatography (column ) in 1906. Sin6e that time various techniques of chromatography have been developed so that at present 11 chroma tography'' has become an indispensable 25 tool for separation of components of mixtures. The t ech nique of paper chromatography has been so fruitful in biologica l research that the developers of the technique, Martin and Synge, were awarded the Noble Prize in 1952. 1'hin-layer chromatography Has first described in 1938 by Izmailov and Shraiber. 28 However the credit of developing the technique to its present status i s given to E. Stahl. 29 In the last ten years this technique has re ceived wide recognition.3° Essential oils are quite complex mixtures of chemi cally closely r el ated compounds. These compounds have a l so fairly close boiling points which makes the task of sepa ra tion of the individual components quite difficult. Until the introductlon of chromatogr a phic techniques only a f e vT major constituents of a small number of essential oils were i sol ated. Guenther et. al.3l.3Z in the review papers on essential oils have reported that during the l ast seven years there has been a continuing lncrease in the use of chromatographic methods, particularly thin-layer chromato graphy as \-Tell as gas chromatography. These methods are being adopted by va rious l abora tories d.ealing l'li th perfumery and essential oils to the solution of difficult analytical problems. The t1Ho methods , thin-lay er and gas-liquid chroma - 26 tography, pr oved to be the mo s t successful tools for the investi gation of the oil of Anemopsis a nd are described in the follm·dng sections. Thin-Layer Chromatography Essentially, the thin-layer chromatography involve s the following procedure: A thin layer of adsorbent is applied to carrier pla tes ( usually glass ; recently glass fibre pa per33 v.nd plastic sheet [Kensington Scientific Corp., 1165-67th Street, Oakland, Calif J have been developed). A small volume (10-100)A2) of sample solution is then applied to the pla te nea r one edge and the spot is dried. The spotted pla te i s then placed in a n a ir tight j ar containing a suit able mixture of devel oping solvents. On closing the j ar, deve lopment of the chromatogram t akes place in the ascend ing fashion. After a short period of development, the plate is t aken out and air-dried. Various r eagents may then be sprayed on the plates to visualize the components separated as color spots. In this technique it is poss ible to apply rela tively large volumes of sampl e solutions as compar ed to paper chromatography. The devel opment time of chromatogram is also short (paper chromatography up to 78 hours ; thin-layer chr oma tography up to 3 hours ). In mo st cases the separa tions are comparable to those obtain ed by paper chroma tography. 27 In vie~>r of these advantages, thin-layer chromatography has become an indispensible tool for analysis of complex mixtures. Stahl29 is responsible for developing thin-layer chromatography and its techniques so that they could be used easily and with reproducible results. He devised a spreader to achieve uniform coating of adsorbent on uni- formly flat glass plates. He also developed the use of adsorbent mixtures with gypsum such as silica gel and alumina gel {Silica Gel G and Alumina Gel G; E. Merck, Darmstadt) as adsorbents. Addition of gypsum gives good adhesive properties to adsorbents. r1oreover Stahl devised the apparatus for thin-layer chromatography as a complete kit enabl1.ng uniform applications of the technique. Kirchner and CO\'lorkers34, 35 reported the applica- bility of the chromatostrip method inresolving the compon ents of essential oils as early as 1951. Stahl1 3 and Randernath36 have \'lri tten comprehensive manuals on the applications of thin-layer chromatography. These books extensively discuss the different parameters concerning the separation of different classes of compounds. Stahl has devised a useful system to examine the factors concerned in good separation of various types of compounds, involving the relationship between the polarity of the adsorbent, solvent and the components to be separated. 28 Attaway and his associates37,38 have separated terpene hydrocarbons and. oxygenat ed compounds by t hin- layer chroma tography and have published the ir Rf values . These authors have used non-polar fluorocarbon solvents for separation of chemically related sesquiterpenes. l1onoterpene hydrocarbons are separated by thin layer chromatography with difficulty. The techniques of using thin l ayer of Silica Gel G containing silver nitrate hav e been used by Barret t . et. a l. 39 ~1orri sLw and Gupta and sukdev41 to achieve separation of terpene hydrocarbons and unsaturated hydrocarbons . Klou1-mn and Heide L~ 2 have report ed.· on · t he separa tion of pbonols and phenol ethers by thin-layer chroma to- graphy . The authors have observed t hat mlxtures of polar and non-polar sol vents such as benzene , chloroform and ethyl acetate are gener a lly suitabl e for separation of phenols and phenol ethers. Christensen43 has reported on the characteristic colors produced by 25 different alcohols with 25 different spra y r eagent s . Stahl13 has r eviewed extens ively the publications on the application of thin- layer chromatography to the ana l~rsi s of essential oils. 29 Thin-Layer Cnro~otography of Oil of Anemopsis The 'Desaga ' (Brikmann Instruments Inc., Westbury, N.Y.), 1evise1 by Stahl was used in the exryeriments . The genera l conii tions of chro.natograohy usEd throughout our work are summerizei below. Carrier plates: Glass plates grt X a ~ 2" X 8" Adsorbent: Silica Gel G (E. Merck) Thin-Layer: 250 y.. thiclmess Solvent: Benzene + Ethyl acetate (95.5) Activation: Dried at 110° for 2 hours and kept in desicca tor over drierite before spotting. Sample size: 10 y.t Development: Room tempera ture (22° - 27°) The glass plates were thoroughly clea ned with chrom ic a cid solution followe1 by soap solution a ni thor- ough rinsing in 1eioni zA1 water . The plates were d riei in an oven a n1 were mounted on a template. 3efore ap9lying the aisorbent, the pl ~ tes were again cleanei with ac i dic methanol followei by nure me thanol. Twenty-five grams of Silica Gel G were weighed out in a glass morta r a nd 35 ml of ieionlzej water were gradua lly added with trituration to f or m a untfor~ slurry. This 30 slurry was further diluted with constant stirring with 15 ml of deionized water and immediately poured into the spreader placed on the first plate in the template. With a uniform motion the spreader was drawn across the plates to spread the slurry on the plates. The plates were allOl'led to air dry for *hour and then activated at 110° for 2 hours. The activated plates 't'Tere stored in a desi,;. ccatorover drierite until they were used. The samples for analysis were prepared by dissolv ing in an equal amount of hexane and applied to the pre pared pla tes 1 em distance from one edge, using disposable capillary micropipettes. The developing tanks were filled >vi th the solvent mixture to a depth of 1 em. The t anks l'Tere alloTJred to equilibrate with the solvent for 4 hours. Two spotted plates were placed in each tank and developed until the solvent front had travelled to the 10 or .15 em mark from the starting line. This usually required about 40 minutes . The plates were then removed from the tank and air dried. The plates were then sprayed l•Ti th various spray reagents to develop the color. Spray Reagents The diversity of the types of compounds present in the essential oil of Mansa required the use of different spray reagents to visualize the spots of compounds with different functional groups. The reagents and their 31 compos 1· t 1o· n are g1ven· 1· n T abl~~ 4 (pag_ -.e ~ 2 ) . Results of Th in-L~y er C hr o ma to~raphy Fig . 3 (page 33 ) shows t he thin-layer chromatogram of the total oil of Hansa sprayed with the above f our r eagents . Anisal dehyde , a general pur pose s pr ay r eagent for t erpanes , s teroids and sugar~ showed abo~t ten cl ear spot s . The s epa r ation was the best t hat coul d be a chieved with a vari ety of s olvent systems examined . Vanillin- sul fur i c acid spr ay r eagent 2l so showed about eight spot s . Va nillin is al so a general purpose spray reagent . Dia zotized sulfanilic a cid , which i s a specj.fic reagent for phenols gave onl y one s pot ind ice.ting the possitJ.e· presence of one phenoli c compound . 2 , 4- Di nitrophenyl hydraz i ne ~~ve nesat ive t est wi th t he t otal oi l . Fi gures 4 and 5 (pages 35 , 36 ) sho\·T t he chroma.t o gr ams of t he phenolic f r acti on (residtie #2 ) and the carbonyl fraction (r esidue #3 ) besi de t hose of t he t ot al oil of Anerr.ousis . Although t he oil of f:Ie.nsa gave negative resul ts vlit h 2, 4- dinitrophenyl hydrazi ne , the carbonyl fraction gave one colored s pot ~ i th t hi s reegent . As descr ibed previously (page 13 ) the increased concent ration of car bonyl compound i n t he carbonyl frac t i on made i t possible to obtain a pos itive t est with t he r eagent . The phenoli c fracti on gave a yel l ow spot ~ i th d i azoti zed sul fani l ic aci d . The compl ete r emoval of t he phenolic fraction f rom t he oil was d i ~f i cul t 32 TABLE 4 - Spray Reagents for Thin--Layer Chromatography --f--·------1 No. Spray Reagent Composition C1asse of compounds detected --·-+------+------t------i l. AnisaJ.dehyde Anisal dehyde ( 3 drops ) + 'rerpA11es1 3 mi xture of crr 3cooH (gl )(l ml) Steroids and. CH-aOR ( 10 ml), ad.d. H2s o4 Sugars (cone . 7 ( 2 ml ) etc. Spray and heat the plate at 95° for 7 minutes ----~·------r---·------~------r------·--~ 2. Va nillin Vanillin crystalline (1 g ) + General H2so4 (cone. ) (lOOml) non--specific Spray and heat the plate .at rea.gent4ll- 80° for 5 minutes 1----+----·------t-----·------··------·---- 3. Su1fanj.l:i.c s ee page 9 Phenols acid ------·--- 4. 2,4-Dinitro 2,4-Dinitrophenylhydra zi ne Aldehydes phenylhydra (100 mg ) + C2H 0H (95%) and zine (100 ml ) + HCl 5(cone.) ·(1 ml) ketonesl3 ,___ __.______.__.. __ · ------~------·------33 Spray Spray Spray Spray He agent Reagent Reagent Reagent #1 112 11 3 11 4 b blEJ p 8 br Po p 0 i .. l p ····' r YA g ~ .-/ •; I• ' '• .• '~\ y gy dg b g y lb • • • • :• .• .• .• • • .:.' ,: "v • • l b = blue, p = pink, r = red, g = green y = yellow, bl = black dg = dark green, br = bro~m. gy = gray, lb = light blue Fig. 3 'rh:tn-layer Chromatogram of O:tl of Anemopsis with f our spray reagents 34 and. some traces still remained in the non-phenolic frac tion of the oil. Fig. 6 (page 37) shows the thin-layer chromato grams of fractions collected by fra ctional distillation (page 23). Ani saldehycle, the genere.l purpose spray reagent, was used, It \·ras ob served that although fine separation of components ·Nas not possible, 't'le did get 4 cuts wh~rein components were partially separated and con centrated. Although thin-layer chromatography did not yield any conclusive results, it i Spray Reagent Ill Spray Reagent 113 8 0 0 A 0 • '• .t : ( .... .: •• •. • ...,.. • • • 1 2 1 2 t 1 = Oil of Anemol!_si s 2 = Phenolic Fraction Fig. 4 Thin-layer Chromatogram of Phenolic Fracti on 36 r------Spray Reagent # 1 Spr ay Reagent 11 4 E3 0 ~ ...... J \ .• ,. 0 0 q q I. ••l•.. :,'I.. • • " ~ 1 2 1 2 1 2 = Carbonyl Fr action I<'i g , 5 Thin-layer Chromatogram of Carbonyl Fraction 37 Spray Reagent //1 8 0 0 0 8 • • • • 1 2 3 4 5 1 = Oil of An e mop s i ~ 2 = Fraction #1 3 -· Fraction // 2 4 = Fra ction /13 5 = l<,raction II 4 Fig . 6 Thin-l ayer Chromatogram of Fractions from Fractional Distilla tion of Oil of Anemop_si s 38 Ga s Chromatography Gas chromatoera ~hy ha s b ec ome an inv a lu~b le t ool in the field of ess ential oil resea rch. Separ ations of comryo nents \>Ihich formerly took da ys by t P·iious ch em ic £L l and phys ica l mea ns , or ~e re imposs i b le by these o l der methods , ma y n ow be a chievei in hours by gas chroma to gra phy . 25 Martin and Syng e45 introduced parti tion c hr oma t o gr a phy i n 1941. In this pa per the author s propo sed tha t a mobile pha s e n e ed not be only liqu i d , but c ould be a vapour ( ga s ). La ter in 194 9 , Martin a ni Ja~es 2 7 ela bor a t ed this i dea to introd'lC C " Gas Chromatoe;r a::)hy ''· As the t er m in1 ica t es , the mobile phase i n t h i s t echnique is a gas . Gas-J.iquid ch r om ~ t o g raohy (GLC) i s va r iously r e ferred t o as gas-liquid oarti tion chr o ~a t o grap hy , gas- partition chr:::>"!la t ogr a.phy , vapo11r phase chro:nat ogr aohy or va pour fract 8me try . Fi g . 7 (uage 39) s hows s c he:natica lly t he eq~ i nmen t usei for gas -liquid chroma togr aphy. The basic eq~ i pme nt c onsist s of a c o l u~n which ma y be a s tra i ght tube , co iled or U t J.be . The c -J l •nn i s nac' The st~ tlon a ry phase i s a liq uid o f low vol atility at the t e~pe r a t llre o f the ex :1e ri :nent . Di f fe r e~ t stat 1 on"l. r y '"lhases 39 BECORDER \¥ ;;:-r-:;;-:~:z-~~~~~.,;~~~~~ ,,:,--~-,...... , ..... ~.•...~~ ·'"w..u ~:u .-.~~. - .,_• .~ ·~ ';rl ~- ~f ~-~~~~~~\ : --,_,__ \ ,___,;'l.,...... l t...... W ISJt l ..-,~~- v ~~·\... lf~"""'-...... !IJ\y~ ''\-'. ~~~~-""""'-- ·=~ ~~ CAi1HIER GAS SUPPLY l \ BHIDGE J -!"'""" MANOI'!ETEH t COLU!I'JN \.· · - ..1~ ,r.. S S~SOR l,!Pq '. Jaf! . f~ I ,.d. ~ ·~&r'~; ·------SAMPLE CONTROLLEO TEM PERATURE PORT AREA Fig. 7 Schema tic ~i a g ram of Ga s-Liquid Chr o ma t og ra~ h . 4o may b e us ed r a n g ing from non-pola r t o pola r liqui1s . In the r ecent 1 ev e lopment o f c apilla ry col umn s 4 6 the c olumn wa ll i s coated with a s tationa ry pha se - n ~ inert support i s requ1re1. The s t a tio na ry p h ase a c ts as sel ective r e t a ria nt t o the c ~m ~o n e n t s of a v ol a t i l e samol e . The colu~n i s ma intained a t a p r eJe t ermi nel t emper a ture while a stream of ca rrier gas i s 9ass ed through the c ol umn . The carrie r gas is the . mob i l e p h~se a nd t ran s norts the v ol a tile c omoonents of the mixtur e through t he c olumn. By mea ns o f s ome s u ita ble device , a small sample of the vo l atile mixture t o b e a naly s ed is int r oduc ed into one end o f the l on g a nd narr ow c olumn . Th e c omJonents are a dsorbed o r absorbei b y the s t a tlonary ohase t o a n extent tha t the ir e ffe ctive speeds of flow thro ug h the col u.mn a lso differ . Thus the c orn ·')o !'l en t s · of the injected s am ple e~e rg e from the opposite end of t h e c o lu~n as s e-oar a t e h.an1s where they a r e det ected or " sen s ed " and s ub se q ~ ently r eco r~e d b y a sJi t abl e detec t or. The e f f iciency o f ga s chr oma tog r a ohy i s the r esult of two ma jor nr oc e sses ( i) sena r a tion o f the c o !'l s tituents of the Eamol e a nd (ti) sub sequent iet ectio n a n i me a s ur e ~ e nt s o f the s e c a n s tit ~en t s .2 6 ,4 6 The sena r a tio n powe r of the c o l umn, wh i c h i s the most v i t a l o r oc e ss , de ~ e n ls unon a n umb er of ~a rane t e r s , in c l ~d in g (1) q ua li t y a nd 41 quantity of stationary phase ( 2 ) particle size and n a t ure of inert solid sunport (3) un i f or mity o f packin g (4) dimensions of c o lumn (5 ) temoera ture (6 ) na t ur e , flow r a te a n i pressure of the c arrier gas and (7) properties of the co~ponents of the sample to be separat ed . The detector ind icates the nr esence and measures the amount of the c om ponents in the column e f fluent . A good dete ctor shoul1 have high sensitivity a nd s houl d have a l so fast a nd linear response t o the effluent gas . The "differential '' tyoes of 1etec t or s are :nost c om:nonly used.26 In this type of detector s ome prope rty of the effluent i s c ontinually c om0ared with that of t~e carrier gas . Thermal c on~uctivity, e l ectrical c oniuc tivity, dielectric constant and ioniza tion constant a r e some of the ohysica l nroperties wh i c h a r e used in "differential'' tyoe of detector s . The :ietector response is usua lly r e cor.iei by an aut c~a t ic r ecor1er. The presence of components of the sampl e in the e ffluent gas is in:i ica t ei by " peaks tl in the r ecor d ed chromatogr am . (Fig. 9 page 52). The choice of the s t ationary phase depends upon the na ture of the com ~ o s i t i on o f the sampl e mixtur e . The sta ti o n ~ ry ohase must h~ ve sufficient s olvent power for the c o:n oonents to be separat ed . If this is low the 42 c omponents may pass through the column ran i dly with noor separa tion. A certa in c omnatihility between the s t a t iona ry phase and the comoonent s of a mixture is a lso required . Thus if the compon ent s of a mixture are n on- pol a r a n on-pol a r stationa ry c hase will g ive a n ormal effi cien t separation; a Do l a r phase such as pol yethylene g lycol will efficiently separ a te pol a r compoun ds l ike alcohols a n d ph enols . Some stationary phases such as n hthalic est e rs , by virtue of t heir structures , ca n be used f or a wid e v a riety o f substanc es . Gas-Liquid Chromatogranhy of Esse~tj.al Oils Since 1 957 , more than 50 l i qui d sta tionary cha ses have b een intro~uc ed and used by va rious Wo r ker s i n the a nalysi s a nd investi gation of essentia l o i J.s . 25 , 47 Out of these 50 liquid c hases only a few have b een f aun ~ to be sati s factory f or all essential oi l s . It h ~s been pointed out tha t on a n on-nol ar station ary phase , there i s a linear r e lations hip between r etenti on time a nd boi l ing point f or ne:nber s o f any homo logous s e ries o f t er oene hydroca rbons and oxygenated t eroenes . Separations o f po l a r comnoun is like a lcnhols , ca r bonyls , esters a nd p he no l s are poor on n on-pol a r s t a tiona ry l iqu id phases , hen c e a nol a r o r f a irly no l ~ r liqui1 c hase should be used . FFA? ( moii f i ed c ar b o~a x )4 8 a nd Hy nr ose SP Bo49 a nd 43 various other· polar stationary lJ hases25 are most frequently used for resolving the mixtures of polar compounds. Ricciardi and his associates50 have studied the applica tion of gas-liquid chromatography with two different stationa ry phases alone or in series t o separate the comoonents of low boiling fractions of essential oils. By selecting two different suitable stationary phases, they were able t o achieve good resolution of closely related comoounds. Essential oils analysed by gas-liquid chromato graphy represent fairly wide ranges of component mixtures having varied pola rities and boiling points. If the analysis is ca rriei out isother~al ly, it is d ifficult to select conditions so tha t sufficient resol ution and good column efficiency are maintained during the entire analysis. Purnell51 has thoroughly discussed the paramete rs which contribute to the efficiency of total operation. He has shown that the selection of optimum column temperature is the most vita l f actor besides the stationa ry phase. If a lower c olumn temner a ture is selectei , thP. early ueaks will be sharp and well seoaratei but the late oeaks will be broad and the retP.ntion ti~es long. On the other han1 , tf a higher temoerature is use1, the late peaks will be brought closer to other oe a~ s and will be sharper . However , 4L~ resol~tion will be noor in the earl y r egion o f the chroma te- grams . The a"(nlica tion of the ·8r ogra mmeJ temperature technique to the analys i s of essentia l o ils has overcome these diffic ulties . Rasqtlinho48 has shown that programmed tempe r ature gas-·liq 1li 1 chromatogra')hy of essenti al oils achieves high r eso l ~ ti on an ~ column efficiency . He also has reported on the quali t a tive c haract erization of essential oils using programmed temoerature chr ~ma tography ani va rious stati onary phases . The author has reco~~ende d FFAP as a general ryurpose st~ ti onaTy phase for a majority of essential oi l s . C::: 2 A r ecent technique reportei by Scott~- uses flow o ro gra~m ing of the carrier gas . This technique i nv olves the increase (continous or stepwise ) !n the flow r a te of the ca rrier gas during the course of an a nalys i s . Zlatkls53 has a onlied this t echnique t o the a nalysi; o f vari ous essential o i ls . Gas - chro~ato graohy together with i nfra-red snec trosc opy has been empl oyej by Nigam a ni Levi54 , 55 to s epar a t e and i dentify many cJ osel y r e l ated sesquiteroene c om ~ounds . They also em oloyed a me thod of c oupl e t gas - liquid - thin-la y er chromatography f or s i multaneous ieter ~ i na ti on o f o i ~e rit o n e a nd pioerltono oxi 1e . 56 Attaway and hi s as s oclg t e s57, 58 have o ~ta i nei a v e r y high r esolution of 45 .. volatile components of Citrus oils by using a 50-foot long 4% C~rbowax 20 M column and low temperature. These authors isolated closely r el atei terpenes nresent in trace quantities in the Ci trus oils. The authors have also shown the usefulness of gas-liqui ·'i chroma togra9hy coupled with t hin-layer chroma t ogra9hy, infr a -red and mass spectroscopy. Bernahrd59 studied the gas c hr omatographic seoara tion of t er pene hydrocarbons utilizing caoillary columns with va rious stationary 9hases . He concluded that the stationary ? ~ase i s the most influential para~eter in the analysis of essential oils. Wahling and Sch>1 rtz6o ha ve described a method for the direct gas chroma togr anhic ana lysis of essential oil containing nl ant narts without stea~ distillation and hence perha?s gives a truer analysis of the a ctual composition of essential oils in such pl ants. Von Rud loff61 has also renorted t o have obtained an analysis of the vol a tile oil directly fro~ a single conifer neelle by gas chromatography . He also recommends apDlica tion of this metho1 f or direct analysis of other plant parts such as leaves a nd oetals. It i s expected that this method may be very usefuJ in an i nvestigation of pr oper time of collection of pl ant oarts , influence of 46 fertilizers and other factor s on quality a nd q uant ity of ess ential oil. Crippen and Smith62 ha v e described a pr ocedure for the sys teDa tlc identifica tion of peaks in gas chromatography. The author s h~v e a l so di s c ussei in detail different criteria used to deter mi ne whe t her mo r e than one comnonent is Dr esent in a oeak . Collection of fra ction s from the col umn eff luent of a gas chromat ogr a phy i s s till a problem. Many ela bor a te fr~ cti on collec tion ievtces a re described and and commercia lly availabl e . One of the most v ersatile and simplest tra poing techni ques i s t ha t of collecting a fra ction directly on infra-rei grade potassi um ~r om i ie , firs t ~escrib ed by Le ggon~3 Recently Millipor e Filter Cor poration (Millipore Filter Cor o . 3edf or d , Mass.,) has intro·"lllC Ad. a novel me thod of coll ecting a f r.9.c t ion for infra -red ana l ysi s 1irectly on a or e- mat ched filte r paper. It i s claimed tha t it is the mos t ef ficient method of collection of a microquant i ty of sa~ole for d irect i n fra-rej a nalysts. Adaptation of gas-liqlli 1 chr om a t o gra ~hy f or ana l ysis and investigation of e s s ential oils has increa s e1 s o mu ch during the l a s t 6 yea r s tha t, i n 1965 , The Es s e~ti a l Oil Ass0cia t i on of USA sugges t ed to their 47 members to use an official format for reuorting the results of ana lysis so that othe r l abor a tories could 64 reproiuce the same results very precise ly. Gas-liquid Chroma t ogr aDhy of Oil of Anemo psis It was ascerta ined by exnerime nts des cri bed earlier thqt the major nortion of the oi l of Mansa was com ~ os ed of the phenolic c ompounds ( ool a r) - methyleugenol and thymo l. He nce it was expected that a pola r stationa ry uhase woul d give good resoluti on. FFAP and Hyperose S?- 30, recommen ded by Rasquinho48 and Libe rty and Ca rtoni49 r espectlve ly, we re ther efcr e sel ec t ed f or the studies. 3oth these s t a tion ary phas es were f o und to be satisfa ctor y , Hy nerose SP- 80 y\elding slig htly b e tter r esolution in the earli e r r egions of t he chromat ogram ( s Pe fi g . 8 and fi g . 9 ). The sec ond mo s t critica l f a ctor a ft er the station3 ry ~ hase f or a chL eving gooi resolution of com- non ents a nd column e ffici encies i s the oper a ting t emuer a - ture of the column. Low colu~n t emnera tures were observed to g ive goo1 r esolution of the l ow boiling t erpen es in the e arly reg ion of the c hroma t ogr am , however , the hig h boiling ~ o l ~ r c ompounis thymol a nl methyleugenol l i d not give s ha r p neal<:s a nd ernr:rged out of the col umn a ft er a t,.s long time ( eg . at 150° thymol e~0rged after about 2 hours ). It was obse rvei tha t the most e f fic i ent seoaration was obtain?1 by onerating the c o lu~n between 180°-200° ( Fi g . 9 page 52). At these higher t e~ peratures faster r uns were obt a ined a t the sacrifi ce of resolution in the ea rly r egion w~erein low boiling t erpenes emerged . The Jow boiling fraction collec t ed fr o~ the Splnning Band Distilla tion column ( Fraction #J., ouge 19 ) was analysed at a t enperat~re of 110°. ( Fi g. 10 ~ag e 54). 'l'he high bo iling pol8r phenolic c J~npoun-ls Here absent in t hi s fraction hence gooi efficiency and r es~l ution c0uid be achievej at the J .~w te ~ne rat ur e . ?r~ ~~ammeJ t e~ · ,e rat~re oper~ tlon waul i be t he best s nlution f or the anal y s is of the total oil . However , the instr u me~t 1~ use did not ha v e 8.Ut0rn:=l.t ic nrogr·::t~!1Jled t e ·np er atu.r e c :mtr ols . The general c on1itions e ~~ loyel for the gas-l i quid c hro11a t o gra ::> hic ::tn9. l ys 1. s of the oil of f~l ~££~L~ are r eoort ei in Table 5 ( page 50). The forillat o f the t abl e c onforms to that sus ~ estei hy the Essential Oil Assoc i ation , USA ( EOA ). ~ny variati on s are iniica t ei on the chromate - gra:ns . R83Ults -::>f Gqs-Ltq•.tl 1 Chro'n:.=t t o€'ranhy Fi gures 8 ani q sh ~ ~ the chram R togra~ s of the t o t a l ail of !1an~n ·,..;i th ~?i\ ~ ' ani :J y n~ ras e S P--30 as the liquid s t a t iona ry ohases r espectively . FFA P shows a bout 20 neaks ani Hyoer ose SP-80 shows ab·: m t 25 -aeak s . The :>e a; ~ s 'II i th the Hy ner ose SP- 80 are s ha rper a nd bet t er r esolve1 , hence thi s s t a tiona r y nha s e was us ed f or a ll o ur a na lysis. The chroma t a gra~ of the oil of An e ~o u s i s can be conven i en t l y 1ivlded · i nto t hree r egions (Fig . 9 page 52) . The firs t 10 oeaks emerge i q ~ ic k ly within 5 mi nutes a nd constitute the early r e~l on. The next 10 pea ks , eTierging in 5 t o 15 minutes , con s t itut e t he mi1dl e r egion . The l a t e r egi on i s compri sei of 4 peaks . Table 6 - Re t ention Time of I1ajor Pea ks of Peak Retention time Pea k Retent ion tj.me in mtnutes i n minute s II --II .. ______... _____ 1 )8.25 7 7.75 2 22 .00 8 7.25 3 15.25 9 6. 65 4 11.25 10 6. 00 5 10.50 11 5.5 6 9.75 12 4.5 - -· 50 Table .5 - Gas-liquld Chromatographic Procedure r------·------·------Sample: Oil of Anemops is Californica Chromatograph: Autoprep Hodel A-700 ~------~------~------1 Column I>'Iaterial: Aluminium Length: 20' I. D. O.D. ]dl 4 Support: Chromosb lt1 60/80 Stationary phase 20% Hyperose SP-80 r------~------~---·~------1 Carrier Gas: Hellum Gas Flow Rate 75 mlnnin. Conditions: Column Temperature: Isothermal: 180° Progra.ramecl : - Inlet Temperature: 200° Inlet Pressure: 50 psi Sample size: 5 y.~ ~------~------~----·--~------~------·------Detector Type: Thermal conductlvity hot-wire type Temperature: 250° Filament current 150 Recorder: Sargent Hodel SR I'-1 Span: l mv Chart Speed: 1 11 / 5 min. Peak Principle components Retention time , min- t---~--+-No. ------·-----1--_..:::..:...::.:utes _____ . ______1. Thymol 38.25 2. Hethyleugenol 22.00 ~-~3·---~---~P~i~p~e~r~i~t~o~n~e;~------~------1~~~25 51 Sample size 2 ~~ Column Temp. 200° Region 1 J. )o ~ I IIi _Jit k.!W lAj\; ,_ ____ Time i n mi nute s - ·:-- Fig . 8 Gas-Liqui d Chroma togr am of Oil of Ane moosis using YFAP as a stationary pha se I ~ -";:, it. r~ I \~(\ ~ ~" Sample size 5 f Q_ ~ ~ ~ I I £ ..( ~ v' I ..:; !..., \ ~ ,:. I !(.!} ~ r~ 5 : :r \ ~ x~ <:)..! ·# I I~ -.. 1W fl v ' I I~ \...--1 '------1 ~· Time in minutes ~ 1..1\ l\.) Fig. 9 Gas-Liquid Chromatogram o f Oil of Anemopsis using Hyperose SP-80 as a stationary phase 53 Com parision of the r e t e ntion ti ~es o f the peak s 48 with Rasquinho ' s 1' Retention Master In -:'l.ex" a nd the actual chr o m~ to gra phy o f sampl e c omoound s gener a lly nresent in many essential oils showei that the early r e gion mi ght contain t erpen e hydr ocarbons of low boi ling point; the middle r egion pola r compounds such as oxygena t ei t er oenes , a nd the l a te r egi on phenolic c om - oo1.1n::ls . The f our fra ctions obtaineJ by rectifica tion of the o il of Ane~oosis (oage 2~ we re analyzed by gas liquid chr'J:na t ogr aphy . It l.ras observed that r> -st r t ial c once ntr8.t \. on of the c on s ti t11ents ') f the t otal oil ;,ras achieved ; Fr a ction ¥ 1 c onta ined the hy d r~c arbons which emergei in the early r egion o f the chroma togr am ( Fi g . 10) ; Fraction # 2 c onta i ned t he c om ~one nt s of t he mi::l::l le r egion ( Fig . 11); Fr a ction ¥ 3 showei ~ eaks c orresooniing to the c omnonents of the late reg ion and some c omoon ents of the ea rlier r egion (Fig . 12) ani Fraction ¥ 4 c ontained only the c omDon3nts of the late reg\.on ( Fig . 1}) . I'ie:1 ti flcat ion o f Con s tituents of Oil of A ne"'lO~)sis A Nell 't efine ·l sharo oeak of a gas chr::>mat:->gram i s due to the ~ re se nc e o f a s ingle c o ~ponent 1:1. the effluent carrier gas s trea·n . rJn:ler a 'ief i ni t P s et of 54 Column Temperature ll0° Sample size -1 J- ~ t Q) (/J § A (/J Q) p::: H 0 .p () Q) .p Q) A I Time in minutes "~ Fig. 10 Gas-Liquid Chromatogram of Fraction #1 from Fractiona l Distilla tion of Oil of Anemops i~ 55 Sample size 1 J~ l<1> (/) s::: 0 p. til rx:<1> ~ 0 .J..) () <1> .J..) .'1- <1> A J .),., ~ I Time in minutes ----~ Fig. 1 1 Gas-Liquid Chroma togram of Fr action # 2 from Fractional Distillation of Oil of __Anemo ....ps__ ls _ t Sample size 1. }At )/. ~ ~ )o .:;: i ! I \ I I \Jl Time in minutes 0\ Fig. 12 Gas-Liquid Chroma togram of Fraction# 3 from Fractional Distillation of Oil of Anemonsis I l I ! Sample size 1 JQ I t a> U) ~ 0 A U) a> 0:: F--1 0 ~ 0 :L. Q) <: ~ 0::: I j t-J J Time in minutes ~ Fig. 13 Gas- Liquid Chro~atogr~ of Fraction #4 from Fractional Distillation of \..1\ Oil of Anemopsis ---J 58 ooerational conditions, the r ptention ti~e is chara cteris tic of a certain co~pound.26 The time axis of a consta nt- sveed recorder chart shows the retention times of the peaks. Various methods can be used as aids in the identification of the comnounds ( peaks ): (l) the method of adding a suoposed comnound (2) the method of logarithmic nlotting (3) the use of two detect~rs giving different resnonses (4) running the effl11ent in the ~Tim e -Of-Fli ght '• mass s ry ectrophotometer (5) isola tion and senarate identification. 26 The two methods-1 and 5-were use~ to i~entify some of the c ~mpo nents present in the oil of Mansa. Methyleugenol (4-allylvera trole) has bee n shown to be the major constituent of the oil . Injecti~n of oure synthe tic me thyleugenol ga ve a neak with t h e sa~e retention t ime (22 min ) as that for neak 4 2 ( f i ~ . 9) under iientic ~ l c ondtti0n s . The fra ction c orres ~ on d in g t ~ the neak # 2 was con 4 ensed in a g l ass ca0illary atta ched to the effluent gas oort.65 Si~il a rly pure ~ethyl . eug~no l was senarately injected ani c on den sed from the effluent gas . The twJ liqui i samnle s Ga v e i 1enticq l infra-red so e ctru~ . (Fig . 14). Thus it was confir~ e d that n ea~ # 2 (Fig. Q) c orres p onds to me thyleugenol. Thymol was found to be on e of the cons tituents of • - • - -- ~ I 4000 3000 2000 1500 - -· -- - - -~eM-:] --. - ---fo oo · 900 800 700 ' I I I ~ I I I I I I I I I I I I I I I ' r I I I I 1 I • I I I '' t; I I .. 'I ' . I i I! . :I I I ... I I It • I ' I I I I I . .. I I . ''I I I. 'I . '. _..._ .. I I' 0.0 ...... I I ..,.....,'. . ~· ~ I I I I I I I I '. I I I I 0.0 -~ ~ I _,... I ! I_~ I I I !IT . ~ I II ~ I I I . I ':"\: I 'f"C. l I I I I I I I ' '1\ rv' I . I . 1'-i I I I. I I I I I I I l I I I I I I . .. ~'r~r · I I I I I I I 'I I I - I I I I I I . I I - I I I ...... - I . I I . I I • I I' . I I ' . I I I I I 1 I I I I I ' ' I I I I I I I I I I . o I . I . . I I . . . I 0 I I I I I . ' . . . I ...... 10 .1 0 I I I • I I . I I ' I .. I - I I . I . . .. . I • I I I I I I I I I I I I . I . I I I . '.I o I .. I I' I ~!L . I I I I I I I I ' '. ' . I I I I I 0 I I I I I I ' I . I 'I I I . I 0 l_,.. B . . I '',. I 'I ' I' I I . I .. . I I I I I I ' . ' I I I I 'I o I I u I .. . . ' 'I '. I' . ' ' ' ' . I . ' ' ' . : . ' . . ' ' ' : . j• I I ' ' • I . ' . : . ' ' I ~ . 20 '' : ' ' . ' .' .20 . : . ' . ' :( . . '.' ' ' . . ' : ' ' : : ~ .30 I I I I I I I I I I I I I I I I I I I . I I I I ! I I.• 30 I' ' I I I ) I I I I I w I . I I I I I I I I . . 'I I . I j 'I . I . I I I I I ' I I' I.· 40 3.40 I I I I I . ' I I I . I I o I I .. I HI'' : , . I I I I . ' '' ' ' : ' ' . . '. '' . ~.5 0 ...... ' ' '' 50 : .. .60 : 60 .70 ~ 70 I j I I I I I I II II I I II I I I I II I I l l I I II I I I Ill I I I 1111 I I I ll Ill I I I II I I I I I I 11 I I I I i I I I I I I I . I l I I I I I I I II I I I II I I I I I II 1.0 I I I I I I I I I I I ...... _.. ~ ...... L...-.i. . i.--1. ~ 1.0 I I I . . Liqu id Fi lm I ll Il l II I I I ll I] I I 00 Il l 111 I I I II llJl II I I II I I Ill II I II I I II I II I I Ill -I I I I l ii!Trll!rr, 00 3 4 5 6 7 ·.. . 8 . 9 . 10 11 12 13 14 15 ·WAVELENGTH (MICRO NS) A = !1ethyleug.ei.1ol B = Peak # 2 \..!\ Fig. 14 Infra-red Spectra of f1ethyl eugenol and Peak # 2 '-0 60 the oil (~age 11). Peak# 1 (Fig. 9) had the same reten- tion time ()8.25 min.) as tha t for pure thymol under identicql conditions. Further, the gas chro~at~gram of a sample of the oil to which pure thy~ol had been added, showed tha t the peak i l had increased in area. The third major constituent of the oil yields oeak # J (Fig. 9). Ga s-liquid chroma t ography showed that the non carbonyl fraction ( nage 9 ) was devoid of this neak. On the other hand, the ca rbonyl fra ction (nage 9 } gave a peak with the same r e t ention time (15.25 min.) as neak # J and the area of the peak was larger for the same sample s i ze tha n th3t of the oeak #3 for the t otal oil. Thus it was observed that the ~eak # J of the t ot a l oil was most l ikely due t o a ca rbonyl com ~ ound ani this c omno und concentrated in the ca rbonyl fraction. The ca r bonyl fra ction was therefor e used t o isolate a nd i dentify the ca rbonyl c omDound. The effluent carrier gas corresponding to peak # J was passed through a trap containing spectral grade carbon tetra chloride to dissolve the comoound Dresent in the effluent. The 1nfra-re ~ spectrum of this samnl e ini ica ted the presence of an ~- ~ unsatur a ted ket one (strong ca rbonyl stretching band at 1675 cm-1 }. Moreover, the o1or of a fraction condensed in a glass c a ~illary was reminiscent of 61 pepoermint oil. Thi s tnd. ic"l t e:i th~t the «-P-> unsat urated ketone mi ght be simila r to a constituent of pep0er~int oil. A survey of literature on cons tituent s of pepoer mint oil s howed tha t pi peritone might be s uch a c omno und . Gas chroma t ograohically uure pi oer itonA was obtai ned by collecting the major oea~ of a c ommercial samnle (K and K La hora tories , Inc. 121 Express Street, Pl a invi evr , tL Y.). Thi s nure oi neritone ha:i t he sgme retenti on time (15 . 25 mtn.) as tha t of the neak # J under i dentica l coniitions . Also , the infra -r e~ snectrum of oure pi peritone c ollect ed from ef f l uent gas was identica l t o that of the com~ound in the peak ¥ J (Fig . 15). Thus thiri major c onstituent of the oil of Anemoosi s was confirmed. to be pioeritone. The c oncentration of the other cons tituents of the oil of A..ne:noos i s \'la s found to be l ow , Isolation a nd definite 11entifica tion of these c ons tituents wo uld r equire r eneat e:i inj e~ tion of samol es ani collection o f fra ctions fro~ the effluent in orler to ob t ain sufficient s am~ l es f or e l emAn t a l, infra -red NMR a nd o ther a nalyses . Dtrect comparison of the r e t ention t i ~es of the peaks of the o il a nd those of known pure t est com noun js mi ght be of som ~ a id in i dentifica tion . Howev er , it was observ ed th~t thi s c ould only be a tentative l i entiflcation . Peak ¥ 12 ( Fig . 9 ), f or examJl e , had the sa~e r e t enti on time as pure lina l ool. The fraction corr es·lon1ing t o t his peak - ---.- ·------.. ------··--·-· -· - - 1 ·: __·4000 3000 2000 1500 CM- · 1000 900 800 700 : _;. l'r! rl r r r r I I I 1 I I I I I ! fliT ! I I I II I I 0.0 I I I I L •.. 0.0 ~-~ \lllllll l llll l llll l llll l llll l llf+lllll l llll l llll l llllll ll l l lllllllll l ll !l lll ll l l ll l l l ! ll l l llllll:! l ll! f l:!l: l I' rrrr ,I ,I ,I i 1IT T r i 1 1 ~ ~ i I I ~ ~ .10 " HfH n.. . ,... rr; B q=m I I 1't I ri I I I 'l l .40 " rT !ITTlf llrrTfiiTTTT ITT I 1 I I I I II 11 1 I 1 I IT r 1T 11 1! 11 10 1• O I I I I I j I I I I I I • - . Solve_nt CCl 0.1 mm . c~ll ' · • . . : · . I 1 : : . I : 4 00 ' ' I I IJ ll I l Ill I I I I l l l I I I I I I . ·1· l I . . · :1 I I \.1 I I I I ! I ~ I I i I I I I I I I I co . 3 . 4 . .· 5 .· . . 6 .. - .. 7 8 9 10 11 . 12 13 14 15 .- - ·.. ._ ' .: :~... ~:: - ,_ - -~ : -_ . . - WAVELENGTH (MICRONS) ...... -··- A= Piperitone B = Peak #3 0'1. Fig. 15 Inf r a-red Spectra of Piperitone N and Peak #3 W<'!S 8ol1 e c1 ed from i he e:fflu'.;nt port . 'j'br: :L r:.fr~1 - rcc1 spoc!·urn c:.n ·J }") [,8Ve ti1e s s. r~1e :r·et.·::ntion b.rr~s ( '9 . 75 ~i'in . ~·n d 0 . 51 min . ) 8S those of !:u:re oC ·-p:\.nene re~; pective l~r . '1' 1- tese c o:- ·. p cu::~ -:: s ~oul -:l ~1o t be c o nfirmc6 f0r ire J. gck of th< ~ t o.f u.s inc c.n inteunl st8.n -:~:: rc1 . 0 . 0 0 0 0 0 ....-. N (V) . 8 64 0 ~~-~-~ :~-=l=-L I ! I ! I I I I I ' ' l '"1 • - --- ·-H-++-H-+-HI-I-- 1-+, H,,H·+..r ++...,.jo: ...:..;;:._~IT.. ,._ ·-i·--· -·...-;-;-;- •., I .. ~- - - r~- ' '=< '' -- CO -"'~=' -- :J;..;~.... ~ . II I I I t . I ' ~ ,_ -.--L.L .,. I ' • I • o -(i>-- 1 - J-f-J-J- -1-t--t-t-t - J~t... : .. ::. ~- H ' II __ _jTI- lL -lT i I_ .LW 4-LW,-J+.~ ri ...- ~-; ~ - ~, r~ ~~'·l+ I 11 I I I : I• 1 I , • 0 . ll) 1 c./) I I I I( I I I I I 1: I li hi I 'I z ---:- r-+-~ - ~ ,; r- 1 1 - 1 1 I 1 11 : ~~T rr~-- ~- 0 - --H-1-1-- ~ ~- I I I I ~J~ : I I ! ~ r--. i) I Ill i ii''P 1·1 u - r-' I!' I I I II I II: § t : ~ I ~ -- I I I I ~ I I I I I : I I I I L . I H-t-+~,-H-t-++-t-t-t--~-H-t-rl-t-H--t ++-1----1--l- . !I L ~- -- --~r- 0 0 • I i 1, a, ! l1 :::E 0 u I 1.() ~.. -+-_ji-1--Hif---+--4-H-++-++-1'-H-+++-J-.L-++H+++I- -- I L~~ - ~ -- N >- 1 I I I I II,. ~ IT u z . ._ '~- _ ~L j_J_ I ! Lt+ 11· ~ _ I II w ::J -f--1r-:-++-1-+....J.-.+1 ++++-++-H-+++-I-++++++++-i-11 _ If---:-! I ,1 t 1 G l I i w Cot: - t--~- - ~ . ·1 --H--H- ~-+tt+ +~ ,Y ~--- ~-m~ ·-- u.. !.() - -- - ~ -~- -!I I I -~ 1'- -t- Tr 'd: ~ - , f-- -~-~--W I ! I I i I J 4 r-~Lr- __' ., -- M 'I lifT, I l ~r-r-.,fl ,, ,,,,: 1111, I' - _J._t--t-{ i - II I~ ~~:.~ .J ~ ~-~- _L ~~ I ~~:~ - ·t-J, ~ -- 0 I ! I I I ' ' ....1 -+-l I ' I I I I I I : II' i ..r I I I 0 1 ---~,--'.ll+- 1-+-+-,H - +Jd.-.__!tT.•-.,.,...-,-- L.!.!J L~-1 lJ ~-...'~- _LL..t W- J, . !J.! ~ --- _J..:L.- g 1 I I I ls i i I I I I I I I ! I I I ! I iII I 8~ I I I . -l~-~-~ !JT·- 1- ~-t- I I tl ! I ~ ·-l- ~- _!_!_ J_ _TI ~-~- J J -1 ~ ~~ - +~- . - ' I I ! I I I I ·I . ' 7.5 1 s.o 9.0 10.0 MICRONS 15.0 20.0 25.0 I I I I I I I I L_j__ i I I .I i 1 0 0 I I I ' I I I I ' I I I ' ' - I . i _ __j___ ~--- -l 0 0 · • 1----~ -- :_: -·- ·-· --- i-1 -i J: ·- ·1 .: -· : · · ~ i-:-L: ~--L j- il -l-~:.' ~ -~ i ~-: ___ , I :: · · i ,, - I · -~- i - . ;-! r:-l-~; ·' ~ -~- --:-: ;_~ -: ;-i·:-- :-; -· 1 '! : -l-l.. ;.. , '' 1··i j i i -'-~ ;-:-'+H--i-:_:__L_ ~-rj-~-'-:-+-- :....i-,- -j ! ~"-; h -r~---~------. - ! ....!.1 . ;.-J .. l-!- -- !-! - -+-! -~ - ~-!- - :-!-~.-- .L.l - ~+-!H - ~ - 1+!-+-l- !-LLf- -L!-Lr .. i 11 i : H- L-!-~. ~.--!- _ll-.J '-=f:-!-f- 1 ·-•.. !..J~.. L! !-! -.,---H:--_. I • I ' • ' I • ' ' •• ,. 0 • • • 0 1: I 0 I 1 0 0 0 1 I · ...... ___ ·--- · .,-· - ~~ ·:-- __.___ -·-.. -- . ; ·- r-r-• ~- -~--· - · -~ -~- ·--- -· - t-1-_._t-~ · -~-,.- :-t- · :- ~ · --1-·-·-: · '" 1--:---· - - ·-.-.-· ·•-··t-,- ; I I I ! i • I I • • I • I I I • I . I I . I I I . . I I I ' I I I I I • I I I I • I ' • I I ' 0 ' • 0 ' : 0 : I I J 0 I I 0 0 I I 0 0 0 40 I : I I l 0 0 I l_t' I 0 I I J 0 I J 0 I.J 0 : I : • : ..... _! ~-"-' ______....,!_ ------:---l-·-j -- --"-R+, -, j-n-:. i- ---~-- - ~· i- ___,_i,--~--- --+-",~,- --'-- - , -- ~- t·l-t-+~ · - ~-, -- -~ . . T---- . I ·------:_e--.-:..... _ I I - -.- · -~- ..J~--1- 1--1 I I ' I- . I ... . i I II I ....:---! . 1- J.....L.:_ ...... J-:- ....l....L I . ~-.---j- 1 , 1 • 1 • , ' 1 , , , 1 1 1 1 , 1 , ' r .J__j_j_J_H 1 L · , , • -I 1 _ _ _ rtt , t , 1 . ; ------, ·-1--L---, -"--1-;- -~ -.-•--t-+-j-· r-j"jj_' _! ____L[_j_ ...... , ' I IT I 1+ ,--1--t---l-; ------;--,-·- - -cr:: ·---·------~- :· ~-H---_:-- : -- · f----'- · -~--1..;-;-r!:! I lJ., l ! : --;-- -i :. ~ ---+r~.~- --- ,_ ... -~-~------~-,------10 .lO ts· - ____ _:_b-- ·--'-·· b----- I 0 ' ~.....;....~ :_ ' I I •· I I l I ' I '·..--+ ~-1--,----, -~ ·-'--'. J ___ ._;_j___ , 0 ' • I I I I I 0 ' ! . I *..L! I I I I I j I I I I ! .1 I • I ...._l_ ____ . -- ...... ! .. ~ ~ l. ~--~-_._ I I ____LLL.L.. :_:::_ __ -~~==-:-1 ·-::::= .....: -1 --- . =---::--:-_.:__ _1_-":_j-==-=~~~..:+~/K _j_ - ~- .• : --~· ~~Ji J J I =++:-~ _,_,_Ll__•_: ·,· ; ___ ;__ :...; .~ .. r:.,_: .!....J ....!...jl. ~-_:_I"± - 1\ . ~r\ j 1 . • , , •• ~- ! , , • , 1 , LL . 1 -r ! , , ·-1_ _ -1 ~ '-·- ---'--- ~!...... ! -- . ______·-·----·-··-·------...,- ·n ---:-.-r \--- ~- , ...--:--- .....:-iT-:-;-r · --~ 1 1 ~-.... ----J 1: · r~,, · 'I -r-, 111 . , , 1 • ' ---..- --·--·-----·------·-- -·-;-- j --.. ~ ~__rr=---1 ---'--- -L ~--_-•_ :.....;__4....;-+ . I ,'-i-f--\( ' I ;I---\\+I!...L l. • J ~ I --- +-'-4--H-: : ~·+ l i+1.+--· l- f-~·--:++-- --~--t--,-,----,~ --l : ~- ~' - ~ ! --t ------c------!-'-' -' !.__ --- ~ I I 1 g . r ..\ ..1_ I I : . .;...!...... !.... ~- - .. ..-!---!1-f---:----"--J----7--;-,·:. -.....,..-,--1 . . .--·-- t · ' --:--·7 1 I I I . \ ' I I ; I I I J ; I I I ' .I ...;·-----i-'~-'---,_''-I ------r-:------·1--- . -·- . - ., -I 1 I • • ...j...... :--- ··r-r--- .• 1 .r ..... ·=-r-..A. .. , -.--+--..--.-r1 · 1 ·--• .,-1 ·--- 20 U • 20 ------J--.!.-.- ~ _L__V~ -- \- ~ -~- ; ----!-1----t--1+:-J+-l-L_ t- L.....l-~\ ...... LL.....!---L-L_ L---1---L..{ -+~"""' --j--i-JLf-L-t-t·l- • '-·;-'-__; _ ____ ....L....-;----~- 1 · --·-- 1"- -±--. - --1-- -...... -- --'--~ -- -1---- -LI-:---LJ- ·-- ~ - ~ ~--'-· r-1 J...... L.----l-1-----'...... LLLf- \ -'-f-.=t -1--- - t----f--.-•------l~.,..- ) ·- , \ ------1 ;-; -· -•--·-· ~t--;--- 1 ·--r -1-Lr , ;-.l~~---:,_....._·. r--;---H-"-H -1--'. ±··+-L-t- :-t-+-- -c-t-- -H--:-~ -- ; !---;--ti-; j...., :-1----'-_r.:'j _ 7 ... . --·I .'.:....L... 1--- ·---l ti '-t- . .... ·- ---- L ___j__t - . ~--'---<-1-- · 1-' ---H--1--~ ' -l- -H- -tt----r--t"" . __ ,_ _j_!.._.;_j_l1 ------.. - ~- -·-· t--· . i •J I,,.. 1 1 -1 1 1 1 1 t t , : 1 I ~- I . _ _:_ [ If I I I j . . : • , j _ ___ f . , 1 1 ~ =-=-:jj -- ~ -=-- --- f -~-·- - ft_-~_---:-:: -:..; :=~= ::.-: ...l"':"I==!=J:_ ·. ·-·.- u_-r:-;:. -=-r:~t:t-\l+d_.., -h -!- _,_L±:;... ll:!-_-Wl-!- __ \. !-· , _L!-; I '. ~:~ ;_ +;.--:!----~- _:;- -r-f.= 0 ·- \ · --·-·- -··- ·1 • · - i -·,-: -· · · -.-:...... · ·- , - --- -,- ·---.r:-1--t-lr- --,-.-:· t-rt-;---· \ -~-- -, .. ,,,_-·: · ..-.,-i-- . ,_ \·- .. '""1'<:"" _ 1 .. ~----ii·-1--' ------,- . .. .L --1-- . --- ->-·-- -o-W-1.. -1----- ~'- ...J-,- ~-.....!- · • • - o-r---~--·------· 1 • ------,-- ~ ')0 1 ~ . 30 I , T 0 • ·~.: .)_ II l I II l : • I 0 • ' I I ,I ·j l ; I • I 1 i 0 ' ' • ! I 0 • ~ ) --)' r ·-l-t-"'o -· ---~-- '-;---1--·----- ,r --·t-('- ' ·- ~ -- ~: - -- - ~- ~----:-- -.-~---;- , ..- :-. -~~~~-~-r-~-:-~---T·· ·~ ------~-~----- .. . . --~- I : --- ·· · --· " ' -·-~-- - -~ ·-·-- · l . t . I ,\ { ---1 l.J. '-l ·-~ • ·- · J. -- ' --~-J....-'-1--1- . --L.L.l...j .. . I : I : ·!· '". 1.. : L...: .... - . ----· ....., ,-... ' I ~ I : I • • I J .. t ' I • I I I t\ • I i ' I t ' • - j I I • I \ I i I I I ' • • • 1 "o' -----·- ··~ ~ ------~-1· :-- · · ~.-J.. , •••• , u· :-·_t· -~v - t· ·-- ·-~-: -- -~-t-r iY ~_.·:---r- · -. ;· ...._,- '-1-'1 -r;-r-t.-t--' ,. ...r \ -: ...... ·.-~ ·-; · ·--~ i --i----:--~-----;· ~ 40 __ ,___ . -: ;---- · . -'------;-- -:....; ..:: . - . ·_· --·- \ t-:--;--:- ---:--:-:-- --_-:-i-11-:-:-t--;-}-H----r-,-i1 -,i I,.. _L_ ' ;\-+H :-! __ ;___ I-- -- ;··_;__ :·-140 • -- -- ... ---· ·--- ... : .. - j _LB.:_I __ J .. _:_,. l . . - " - -~--~------_,_:_.:_~! L~-: . ' . LI .I ...LL;~ .. --~-~tJI i - ~- -' _!__~-I l -~-,-~--I-~ -~~ I -~- _j 1 I I 1-~ .!: 1-·I ,• I I ----·-~~I ~· ...... -·-·-- ,. ···;-;-r;--;--·-; ·....--··- ·. il ;·· -.. ~ - t-·· ; ]"1_1_- --,- -i---- ~--,-I --!-t'"l_ ~ ..-1 . ~- -,- -I ... i f--~ ! ;· ; ; ' j : ., ,. ;- ... - ~ r----· ... ------· :----- · ·-j · -- · · ~ --.--;---1 - .-----,--·- J -•-· -· - ---·- 1- · -· :- \ 1 · 1 ':: . -- :--- .-i-, - :------r.o~ - J !~ ------·------· ·r·· --r-· ·J--·-- -·----~~ - ---n 1,!..~--~---. - · j-·-~ i>.~ ~+ :.....:....;....'----:_1 1 : ·· · -J . 50 . J\ 1-ft .... - -·- -- . ------·- - - --l--~- .L;. J .. : .. _,_;_!...:_ ~ 1·-11 1- ..1 .=-ct .J-...... ,... --1--] - I ~~ j ·~ . J-- . . : . I': : . : ·- . I· I .. I I l . I I I I "I ' I I I I f-t-·-J' I 0 I . I I ' • . - - · -·-- ... · -- - '-· - . -··-· -- ---~. ---- · - 1 "" •_ - · - - 1· •-1--- ..t ,_ .... --· -- - ,~-· I o-·r ' -- -t-- l - . - I\ . I . ...' ' ...... • ------L ·-~; •-·-1 r -~-1--L _; ____ _,_ , ___J_ •-1 ~- _ -1-L-L. 1:"'-~ · - . \ :_t _"_ -·t-.1-1 ~- -1 J_ -- -~. J ! . 1 I . . . i - ! . . 1 1 .6ol ~ -: .\ I t ==--= ~ ~-- ~-"" ·_:~:-~-:=:~~ -- -~-=:--~ ~~=:- -~-~~~ - ~;~ ~ ~-- t:-· ~ i~~ -;t~~ --·-~;:__ - -1-; . ;_ -i-LL_:: ·t ~'-, .~- ,\· ~ . . . : : ;- ~~ ~ .6o 1 .70 : -· I lr ; --~~~F.-~· ~~= - :~- ~= ~ :. :=~-~-:~: --~~~- ; . -::..~~~ .::!:::: :=r :_~- ~~~~f~= ~--~-- ;~ ~~l:~~ -~ :-+~.-~ =~:\' ~ . ·: ~ -- ; ~~~: ~ -~- :-~ .70 1 I . i! ;-::::::-:--~ 1- (7 •• 16B" ; Inf-a..:-ed:· Sh.e ctrum ·of:' -: ,_1 ::- "! l I ,. ; r I . H-1 I r--- · l t-; j : 1- : : 1 , - r- -- :-· : . i i - t ,. I ~~ p I ' : : ; i : . - ; i I i ~ : iljjlL_I·_i ij_ ; ; ; I ~-- -t:~;+t_ -:_. . _-' 1 Qj-_l.. ____l___ __t _·' ___I: ____ I "j , 'I ',I ,__:._~..;=r- ~ - '. I . I I , ', ~ --__ T:I -~ - Jl Q . Solvent ; CCl loe 1 mm· celi --- ~ r i--- -:- ·· -:-;. ,. :..,-t : I .\ · · · f l ~ -l _j-1 !J.. · ~ i -! i J -- ! 1 '\- ! __ - ; • , .... - -;~ • 1 · • · · 4 I · · · · T · .. ,.. . 1· ·- · !-r-1 - ---~ -- I· 1 ; I ·-·.!· 1 r-;--t-.l.r-1· , · -: -:-, · -1 ! ; 1 -;-; 1 · · · -· ------.. I I I o • 1··: · 1 • • 4'1 I ' ''• 1 '1'··•· L rJ.-l-1-_i__:r · o ;• · • 11 - :-,-- 1- 1 •· · • - · I- -- y- - -; - :- - -- ;. "" !- . . . :- ---::.:.~:-~t,...;-;: '" i:.. :..:-=--·-·~ ~-r-i :.;=-·:~:....:..: ...:.·~ ... ; I -~ ~ :-;; ~ ~,- ... :- co '- - - -..l-·------·------. - - '!,. __ .....-- _._...,..:, ...... ;.;;:.._ __::-:---=-=-...... co ... t""'\ I""' I""\ i ,j i) \.) 1200 1100 1000 900 800 700 600 500 400 FREQU::NCY (C-A ·1 ) 0\ \J1 66 The areas of the peaks wer e det er mined by cutting out each ma jor peak and we i ghing the pa per. The concentra tions of the three identified compounds are given in Table 7. Table 7 - Concentra tion of Ma jor Components of Oil of Anemop sis CONPOU ND CONCENTRATION % ~1ethyl euge nol 55 . 30 Thymol 13.57 Piperltone 5.19 ----·- ·-- · ~----.....------Tota l 74.06 ·-· . .. _..._ ____ .. ___ Thus it Ne.s obser ved. tha t thG above three compounds cons tituted about 74% of the tota l oil of Anemopsi s . The other 20 compounds contr ibute the r emainlng 26.% . OCH~ J.lethyleugenol Piporitone CHAP'l'ER VI SUHHARY The essential oil of fmemopsis Californica 't'las analyzed using a combination of separation methods. Gross isolation of closely related compounds 1·ras achieved 1-1i th the aid of fractional distillation. Functional group separation yielded a phenolic fraction from which pure thymol was isolated and confirmed by spot t e sts, melting point and preparation of cleri vatives. 'rhe carbonyl fraction was useful in the gas-liquid ch:romatographic analyses. Thin-layer chromatography Nas a convenient and useful tool in following the various separation steps. 'i'hese gross methods of separation 't'lere follm~red by gas-liquld chromatography. This sensitive method yielded the most s i gnlficant data. Hyperose SP-80 't•ras a sui table liquid stationary phase for the total oil. Gas chromato graphic separation coupled with infra-red analysis of the fractions collected from the effluent gas shm-1ed the presence of three components; methyleugenol, thymol and piperitone . Methyl eugenol had been previously reported 1-1hereas thymol and pipcri tone i'rere isolated for the first time from the oil of Anemopsis. These three components constitute about 74% of the oil of Anemopsis. CHAP'rEH VI I RECOMHENDATIONS FOR FURTHER RESEARCH The essential oil of Anem~ contains at l east 25 compounds. The i sol ation and characterization of individual compounds is a tedious task. However, pre fractionation of the oil by methods ·Nhich have been described, follm<~ed by gas-liquid chromatography, should yield results. The almost unlimited choice of stationary phases should ~ncourae;e future 't'rork on the isol ation Etnd. i dentification of these compounds , The h~rdrocarbon frac tion (Fraction /,!:1, page 2-3) , for example, could be analyzed on a rel atively non-polar liquid pha se such as DC-550 at low temperatures. An automatic programmed temperature arrangement should prove invaluabl e . If the peak #12 (Fig. 9) i s collected in a sufficient amount without the u se of a solvent: elemental analysis, NHR and. mass spec tra l analysis of the pure sample should yield data on its structure and identity. BIBLIOGRAPHY BIBLIOGRAPHY 1. Parsons, M. E., 'The Wild Flowers of California. The ir Names, Haunts and Habi ts ', ~ayot Upham and Co., San Francisco, 1904. pp 76. 2. Schneider, A., 'Phar macal Plants and Their Culture', Ca lifornia State Board of Forestry Bull etin 2 , Super intendent of State Printing, Sacramento, 1912. pp 66 . 3. Hore , H. , Cos pari, C. , and Husby, H. , 'The National Standard Dispensatory', Lea and Febiger, Philadelphia, Pa~ 1916. pp 1270. 4. Jepson, w. L., 'A Manual of the Flowering Plants of California ', Associated Students Store, University of California , Berkel ey, California 1923. pp 286 . 5. Nunz , P.A., and Keck, D. D., 'A California Flora', University of California Press, Berkeley, Cali fornia , 19 59. pp 114. 6. Kern, T. H., and Peebles , R. H., 'Arizona Flora', University of California Press, Berkeley, California, 19 51. pp 207. 7. Horton, W. J., and Paul, E. G.,~~ £hem . Soc~ , 72. 2264 (1957). 8. Pickering , G. B.,~ Qhe~, 29, 28 (1958) . 9. Childs , R. F., Dtssertatiol} Abstracts, 23, 3136(1963). 10. Childs , R. F ., and Cole, J. R., ![_. Am . Pha r m. Sci. , ~(5), 789(19 65). 11. Guenther, E., 'The Essential Oils', Vol I-IV, D. Van No strand Co., New York, 1948. 12. Schneider, F. L., 'Quantitative Organic t1lcroanalysist Cognition and Recognition of Carbon Compounds ', Academic Press , New York. 1964. 13. Stahl, E., 'Thin- Layer Chro~atography', Academic Press, New York, 19 65 . 14. USP XIII pp 574. 71 15. Cheronis, N.D., and Entrikin, J. B., 'Identification of Organic Compounds', Interscience Publi shers, New York, 1963. 16. Shriner, R. L., Fuson, R. c., and Curtin, D. Y., 1 A Labora tory Manual', 4th Ed., John Wiley and Sons . Inc., New York, 1959, pp 211. · 17. Allen, C. F. H.,~· Am. Che rn . Soc., 52, 2955(1930). 18. Brady, o. L., ~Chern. So~., 756(1931). 19. Pool, M. F., and Klose , A. A.,~ mn. Qil Chern. Soc., 28, 215(1951). 20. Sch1·rartz, D.P., and Pa rks, 0. VI., Anal. Ch~ .• 33, 1396 (1961). 21. Parsons, A. M., Ana lyst, 91, 297(1966 ). 22. Girard, A., and Sandulesco, G., Helv. Chim. ~ •• 19, 109 5( 1936). 23. Teitelbaum, c. L., ![_ • •Qr g. fhem., 22, 646 (1958). 24. Simonsen, J. H., and Owen, L. N., 1 '11 he Terpcnes ', Vol. 1-5, 2nd Ed., Universl.ty Press, Cambr idge, 194'7. 25. Burchfield, H. P., and Storrs, E. E., 1 Blochem1cal Applications of Ga s Chromatogr a phy', Academic Press, New York, 1962, pp 371. 26. Keulemans, A. I. t1., 'Ga s Chromatography', 2nd Ed., Reinhold Publishing Corpora tion, New York, 1960. 27. Ma rtin, A. J. P., and James, A. Y., Ana ly s ~, 7(, 915(1952). 28. Izmailov, N. A., and Shra iber, N. s., Farma zla, 3, 1(1938). 29. Stahl, E., Phar~ ie, ).1, 633(1956). 30. Heftmann, E., 'Chromatography', Relnhold Publishing Corporation, New York, 1961. 31. Guenther, E., Rogers, J. A., and Kulka , K., Anal. Che rn ,, )_2_, 39R( 1963). 72 32. Guenther , E., Ro gers, J . A. 1 Gilbertson, G. , and Koenig, R. T., Anal. Chern. , 37, I-I- 6R (l965) . 3J. Przybyl owicz, E. P. , Staudenmayer, '"· J . 1 Perry, E. s. 1 Baitsholts 1 A. D., and Tischer, T. N., ~ · Chromat os. 1 20 , 506(1965) . 34. Kirchner, J . G., Miller, J . JII. , and Keller, G. J . , Anal. Chern . , 23, 420 (1951). 35. Kirchner J . G., and f1iller. J. M. , Anal. Chern ., 26, 2002(1954) . 36. Randerath, K. , ' Thin-Layer Chromatography ', Ac ademic Press, New York, 1963. 37. Attaway , J . A. , Wolford, R. \{ o I and Edlmrds, G. J. t ~· Chern ., 21.. 74(1965) . 38. Attal·Tay , J . A. I Barabas , L. J o I and Holford, R. w. t -·Anal. Che rn . , 37 1 1289 ( 1965 ) . 39 . Barrett, C. B., Dallas, M. s . J., and Padel cy , F . B., Che~~ · Ind., (London) 1050(1962) . l-1- 0 . Horris, L. J . , Chern . Ind., (London) 1238 (1962). Jn. Gupta , A. s .. and sukdev, T., ~· Chroma~ ., 12, 189 (1963) . 42. Klouwe n, M. H., and Heide , R. , Perfuem Kosmetik 1 ~- 3, 196 (1962). 43. Christensen , G. M., Anal. Chem., .Jl+ , 1032(1962 ) . 44. Jaspersom-Schrieb , R. , Pha r m. Acta He lv., 36, 141(1961). 45. Martin, A. J . P. , and Synge , R. L. M. , Biochem. J . (London), 35, 1358(1941) . - · - 46. Dcsty , D. H., ' Gas Chromatogr a phy ' , Academic Press , New York, 1958 1 pp 36. 47 . Von Rudloff, E., Can.~ · Ch ern ., ~ . 631(1960 ). 48 . Ra squinho, L. M . A ., ~· Ga s Chromatog., 340(1965) . 49 . Libe r t i , A., and Cartoni . G. P. , ~· Chromato~ . • 2. 121 (1960) . 73 50 . Ricc i ardi, A. I . A., and ~ur ges , J. L., Rev . Fac . I n~ . Quim., 30, 37 (1961). - 51. Pur ne ll, H. , 1 Gas Chromatography ' , J. Hiley a nd Son s . Inc. , Ne'Vl Yor k , 1962 . 52. Sco tt, R. P . W., Na ture , 198 , 782(1963 ). 53. Zla t k i s , A. , Fenimore , D. c. , Ettre, L. s ., and Purcell, J. E . , J . Gas fhr Oip~t,o~ . , 75(1965). 54. Nigam , I . c., and Levi , L. , Can. J . Chern. , 40, 2083 "(1962 ). --- - _ _ , Can. J . Chern . , 41, 1726 ( 1963) . Ni gam, I. C. , Sa hast rabudhe , M., a nd Levl , L . ' Can. J. Chern . , 41, 1535(1963) . -- 57. At tal'my , J . A. , Pieringer, A. P . , a nd Ba r a b as, L. J . , Phytochem ,, ~ . 141(19 66). 58. _____, Phy_ toc hem. , ,2 , 1273( 1966). 59 . Bernhard, R. A. , Anal. Chern. , 34, 15'16 ( 1962 ). 60. Hahling , T ., a nd Shr a tz, E., Plant. Hc: d ., 13 , 218(1965). 61. Von Rudloff , E., !!· Gas Chroma tog . , 390 (1965 ) . 62. Crippen , R. C., a nd Smith, c. E. ,![. Ga s Chroma tog . , 37 ( 1965 ) . 63. Leggon, H. vi ., Ana l • .Qhem. , 33, 1295(1961 ). 64. Essentia l Oil Association of USA (EAO), J . Gas Chr o- ma tog .J 102( 1965 ). - - -- 65 . The Perkin-Elme r Corp . Norvra lk , Corm . 1 GC Nevlsl e tter ·' , Vol 2, No. 4 1966 . APPENDIX I The FFAP column was supplied by Wilkens Instrument and Research, Inc. (Wilkens Instrument and Research, Inc., 2700 Mitchell Drive, Walnut Creek, California). Hyperose-SP-80 column was prepared in the labora tory by the follOi'iing procedure: Twenty grams of Hyperose-SP-80 (Wilkens Aerograph) were dissolved in sufficient hot chloroform. To this solution, 100 g of Chromosorb W 60/80 mesh were added and the slurry was thoroughly shaken. The solvent wa s then removed in a rotary evaporator under water a spirator suction. The 20 foot aluminum column was filled with the prepared packing and the column was conditioned before use by heating at 190° overnight, while a slow current of nitrogen was passed through the column.