identification lichens

7/28/2019 Identification lichens

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JOURNAL ~OP~CI-IROMATOGRAPWY 23sCWROM. +$.7F .,., : : / ,.,, ,., j , , , ,( ,,, j .: , ,,,. .:; ::i,;, . 1 ;

. * . 1 ,.I, ,,.. ,, , ., , ; ; ; )I iA. STANDARDKZED METHOD FOR THE IDENTIFICATION OF LICHEN :,. PRODUCTS . : : .. , .:,,:. .a: ;. ., : 1: : 1

,/,: ., . : ,; ;,... ,:.c,:, ..>I ,,,.I ._. ,. / , , ,.;, /I

CWIC~TA DXLESERS~N AND EWR-DURKRWTINSSON s: 1 :Duke U+& ~bvsil y, Dur ham; N.C. a7706 (U.S.A.). 1 I, : (Rcd~ived~Se~tembdr 30th. 1969) ) ..,., ,, ,,. ,., , ...


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86;. C. E'..CULBERsoN,I. KRISTINSSON,.the worker surveying previously ,unstudied lichens, requires REV data for-all lichencompounds in the same solvent .systems. For, example, if the lichen thallus producescoloratiiih; With_,+-pheriylenediamine i (PD):, one :may choose:,a, solvent system recom-,mended.for PD-positive. substances, only to find that there are also PD-negativeconstituents present in the same plant and for which there are no Rr;, data for thatsolvent. It is then necessary to,select other solvent systems; hoping,by trial and,errorto find a suitable one. In spite of the,intensive interest in lichen chemistry+0 stand,ard-ized system has been presented. The method described in, this report- attempts.,&overcome (I), the hit-and-miss aspects of microchemical identifications, ,,(z),..the:djffi:,culties due to varying Rp values, and (3) the problem of incorporating and retrievingmicrochemical information. The system described was developed for use by: thelichenologists at .Duke University. While. the specific solvent systems and TLC platesused h&e been most successful in our laboratory, .the general plan of the method canbe modified and applied to any solvents used with any media.In essence the system involves the following. Chromatographic data from threestan,dard solvent systems for all compounds previously studied,are stored on punchedcards? The?ca~d~~,a;reke~~d~,not~to,specificR~~vdues in thethree~solutions~but:rathertd~R~,classes the limits of tihich &e,determined.by the R$ values of a control mixture.cousisteritly~used (as ~&marker :on:every :chromatogram. UnknoGns. are, identified, by,deterrW&ig their, I?$ classes in the. three solvent systems and :theu by sorting! thepunched- cards for the jkhown compounds:belonging to all the same R&lasses. !; :,I.::.;/;,,.These possibilities are tlieni narrowed do&i .by ctireful comparison of (a) actualR~$&his%vith respecttothe!R~ valuesof the control~marker spots; (b)colorreactions,(c) :appearance ,?oof,liespot ,.in shorti 8and- longwave UYI light; (d) solubility, 1and anyotherpertinentdata available::Thefinal identificationof the compoundis achieved byappropriate Iconfirmatory tests: ,:Punched cards made for compouiids that cannot. beidentified are added to the deck to permit the recognition of the same substances insp.ecies studied in, the future! ,, ;. ., (, ,. ,.I,, . ..(, ., ,,(, ; ., :MAT,ERIAI& AND ,METHODS ,:,! ,I_.;. . ,.Sozcyq& ,qjY: k;& j!&& L i ,,: ,., ,.-;:.,;? ,:,,, .. I, , .,.i ( :. . , ,.:a., ,,. ? ;, ;, . :: :: j :.,:, . ;,;; ,, , : , . (, ,.Ghvomato& ag5hic materials asd solvent systems ,,I.: /Ii, ,:; ,.f>$.:Cliromatograms,:were :developed in Brinkman tanks to a ,height of IO cm onMerckSilica! Gel:Faii. thin-layer plates. that ,had!been stored .in a desiccator over CaCls-NaOH ;,but not activated; .: The solvent systems are ; solvent: A : .benzene_dioxane-acetic, acid: 1go .:,25.: it; 238 ml) s;solvent B : hexane-ethyl ether&formic acid (5 :4:1,200 ,ml) ;.solvent CI toluen+acetic acid (85: 15, 240 ml) .,; .I: : . , ., 1 ., , ./ i rThe tbenzene;. hexane,. and toluene .uiere dried (CaCla); redistilled; and stored oversodiu,ni,2The; dioxiine and. anhydrous ethyl ,ether iwere 8passed through, columns :ofalumina: tb removeperoxides; The,,ether was stored,.'over sodium; The same, s&e&systems prepared without !these


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IbENTIIW%~~ON OP L~CWEN'PRODUCTS BY TLC 87were replaced when the Rp values of the controls deviated significantly from theirusual values. Atranorin and norstictic acid were selected for marker controls becauseof their ready availability and. because of their behavior in the particular solventsystems used. A mixture of these two compounds is available in a single extract ofmany common species, for example Parmelia perforata (Jacq.) Ach. in North, Americaand P. acetabulzcm (Neck.) Duby in Europe. :(Eighteen spots tiere applied to the plates z,cm from the bottom and g mm apart.On every plate the first, ninth, and eighteenth positions were used for controls. Thecontrols on a plate tiere spotted from the same solution to ehminate RF variation dueto concentration. . ., . . 1.. .,,. ,,, .Determi stat ioh of Ap classesThe developed plates were air-dried, examined under UV light (254 nm and366 nm), sprayed with 10% HPSOI, and heated at about IIO' until colors developed.The colors of,the spots were recorded as soon as the plates were taken from the oven.. In order to identify RF classes, first thecenters of the atranorin spots at positionsii and,:g,and ,at positions: g and: 18,are connected by a rule4 line in pencil. The same is

done for the norstictid acid. spots. (Usually spotsare slightly lo&& towards the centerof the plate, the effect being more pronounced at lower RF values, but sometimes thedirection of variation is reversed.)The distance between theorigin and the solvent front tias divided arbitrarilyinto:eight regions measured with respect to the RP valuesof the controls. ?he iimits,of the. eight .R p classes are shotin in Fig. I. All RF values for spots were recbrded and

,A+-solvent front Descrlptlon 0; RF Classes.

Fig. I . A copy of a plate run in solvent C showing the controls of atranorin and norstictic acid,at positions I, g, and 18, the horizontal penciled lines connecting the control spots, and the RFcl ssses of the spots of the principal compounds chromatographed. The number (or numbers)associated .with each spot indicate its Rp class and the diagram to the right of, the plate sum-marizes the Rp classes, The identities of the principal spots at each position on the plate are(I) atranorin (A), norstictic acid (N), and an unknown substance, an acetone extract of Pavmeliaperforata; (2) acetylportentol; (3) alectorialic acid (class 4) and barbatolic acid (class 3); (4) alec-toronic acid;, (5) anziaic, acid (classes 4-5) ; (6) atranorin;squamatik acid (class 3) (8) barbatic acid (classes 5-6) ; (9)

(7) baeomycesic acid (class 5) andatranorin (A) and norstictic acid, (N),the control; (IO) fumarprotocetraric acid and protocetraric acid (running together as a smglcspot) ; (I I) chloroatranorin ; (12 confluentic acid ; (13) atranorin (class 7) and a-collatolic acid(class ,5) ; (14) constictic acid; (I 5) cryptochlorophaeic acid ; (16) q-0-demethylbarbatic acid ;(I 7) djdy&ic acid ; aad (I 8). atranorin (A) and norstictic acid (N), the control:

.. J . Claromaiog., 46 (rg70)85-g3


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. C. F,. CULBERSON, W. KRISTINSSON~. .

J .~Chvomalog.,r 46 (1970) 85-93.


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IDEXUTIIVCAT~ON 6~ LICWEN PR~D~JCTS BY 'Il.C ._ gif'dhmxi ,by the valuesmeasured to the norstictic acid and atranorin lines at .the samehorizontal position (see Table I). :/,,, ., .: ;; I, ,fRecodhg in fovmatiovt 08 $wnched cards :

A punched card, prepared for every compound, is keyed for the Rp classes in,.the three: solvent, systems and for an alphabetic sequence, to the common ,chemicaJnarms.: Additional #data, and specific / RF values, always expressed :witli ,the, corre;:spending qvalues of the norstictic acid and. atranorin controls, are reoorded .dire&lyon the cards.' AS an example, ,Fig. 2 shows the punched cardfor lobaric acid, * :. ,

,,:; ,. ,.. : ,,.,, .I _. I .,. ;I ).

name 01 compoand color r&c::.. ,/, ,

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a ::.., /.a : ,,/,:I:(,;,: ,, ; ., . . ;., ,,., (,, ),\ ,.. .,, y ,I \ .: :I{: ;Fig. s. ,+n example of a punchod card for a, known compound showing the. information stored. ., 1: ). . :ci / ,.( , ,, ., .I denti f ication of compounds iti crzlde. extr kts of & he& for ckemotaxonomic sti~veys ,f, .I

*., 1 : Chemotaxonomic studies ontlichens are usually based upon chemical ,analysisof fragments of herbarium specimens. In our 1aboratory;the following procedure isused : cleaned plant material (I cm2 or less) is placed in a shell vial (Mercer Glass Works,Inc., N.Y. ; shell vials, short style, 112 dr, 35 x 12 mm) numbered in India ink. Thefragment is extracted three times by g-ro-min soakings in benzene at roomtemper;ature. The extracts are evaporated at about 50 on a microscope slide. The processesare repe,ated using ,acetone to extract the sample at elevated temperature &do), andthe %cetone extracts are evaporated on asecond slide. ,Usually ,many Specimensareanalyzed simultaneously,. and the .bennene and (acetone extracts of each sample iarechromatographed\ side-by-side on: the thin-layer. plate. *As usual,: ,at positions * I, :9;and, 18; the controls of atranorin and norstictic acid are included. ; .: . ; ,, I ; .

To spot the chromatograms, a drop or two of acetone,is flowed, over the residueon ,the slide,,and. .collected:ina micropipette:.,Three !plates may, -be, spotted isimul-taneously.: The Rp values of the ,spots are recorded and the .Rp classes :determined;The punched cards make possible the identification of, all previously studied &dpounds with the same Rp classes. Of the 104 compounds listed in Table I the maximumnumber of compounds with the same RF classes is six. The additional information onthe cards (Fig. 2) reduces the number of possibilities still further and suggests .the bestsolution or solutions in which to rechromatograph the unknown with appropriateJ; Chvomatog.; 46 (.zg7o) 85+3


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92 C; F.!CuLBERSON, H..,KRISTINSSONstandards; I f the compound is.riot identifiable, a punched card for. it is prepared andadded to the deck. l ., ., ,., . .:I ,, ; ,.,I ::;: ,RESULTS AND DISCUSSION . ., : :, .: *\ : ., ;(, ; Y,,. ,. ,,.:.- ,, , ,, j .!: . . . : ; y I,, ,

Table.eJ .summari&s, the.:.data:,obtained for the ,mosi -common. aromatic. and,triterpenoid!products~df lichens;.The compounds. are. arranged aticprdingi o increasingJ ?pclasses so $hat,. hose:with,.the,.most~simil~ RF -val.ueS, n,the ,thti+!esolvent systemsare grouped,,tog&her,. -It is urilikely: that, these RF valGes could. be reproduced exactlyinanother labor&ory,since oier:a period of some mbnths the Rp values of the controlsofatranorin and &stictic~acid~chan&e!slightly, But it is expected that the R I P lasseswhich depend only on,the relative order of spot& Gill be muchti&e con+ant. Iq,t:,$he dentification;,,pf.,upk~iq,wns -from. r!uc+]@ctracts df ,litihqs containingsever@comp,ound&t-inay be diffic&+b distinguish whi&sp&s bn,tl&chromatogramsrun in$ffereqt sol&fqq~ ,qq,diie to. the game compounds; Sometimes, one or morespots< w~~~.~~~~~~,&&l~dy ,other spots ii&Fe solv&ts; Colora&&~~with differentspray: r$~~~ts,can:~~~~~times $@p., Elution. of, band run .in one s&&t aiid &hro-ma~ogt~p~y,-ofth~~~~ted q$terial in $@a: .: .,!, : ; .; ( ,> , :1 1 i,, : I / ; : - :,; ; [ ; I, ! + ; , , : ) : ; .;j ,, : .._ ./ :, ,,; :,, ;, . i. : 1, L .I., ; : :,i / .; 1 : i, :a ,:.Y> ;: ,:. :.l.jc;:i; , :, ,,, ,. : _,, ; .,. : \ ( :. .I . !ACli4NOWLEDG~MENTS;.!~,..;:; ,':;,i+ ,::>; : .,: .: .j, , i , ,c, .: .( :,,: j ,: .i :,,.,.:::::.,,:.;!::_:;: ::;r , ,I ,;.:,,,, .~i:!,: !\ l,i,-,,,,,:,; i, ..,, - ._,:,; ,,,I:(. ,; ,. j ,!l!,,, . j ,;-,,~,~~ithank'~,Dr.:K.;A~oRAMu~T,~~ .E:dr,s&n$e ~fsElichen;c?~tainingyir,&sic:,acid.;~Dr;~M;_,~,E.,.H,~~~,~'~J~..fora&plbi,of,lichqnsotitaining. rhodophys,in md:e&ino-carpiea@d; lD&;.S.;:ti~;~~nhE~~forla~&ample. f-h&em&h&molic;acid:; .Dr; :S. ~-~UNECK.for .samples::of: acetylporfentol and planaic. :acid.;; Dr., .W:,.S. 6, :MMSS! for' &ples .ofseveral pultiinic acid, derivatives ; and, Dr.. C ., A.:WACNTMEISTER for amplds: of p&o-lichenic,arid:thiqphanic acids.):, ,.. I ..,. :.;..:-:,. ., II. +,, .:.;:,_, : ;,:I; ,,..I ,::, !,, ;,. .,;,-i c;.tr;.!:rlr;lisnvestigation was supported hy..a.grant. (G&&45) from, the I&i&ion ofGen&al:.Medical. Sciences,; Naii0na.l Institutes .of i,I: ,., ../ ,/ , , ;: ;, ,... ,:


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identification lichens

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