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JOURNAL

~OP~CI-IROMATOGRAPWY’

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A. STANDARDKZED
PRODUCTS
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METHOD

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IDENTIFICATION
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OF LICHEN
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CWIC~TA DXLESERS~N
(Rc’d~ive’d~Se~tembdr 30th.

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EWR-DURKRWTINSSON


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Duke ‘U+&~bvsily, Durham; ‘N.C. a7706 (U.S.A.).
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SUMMARY,.,

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,,., ,..” I,) A: pro&ure .for. the ,routine identification .of ,the .products of -lichen-forming fungi. by thin-layer. chromatography is described. Microextracts of plant ..fragments are chromatographed. in three solvent systems.: The, spots, of. unknowns are assigned to RF iclasses. defined :by the RF, values of marker, controls ,of two lichen substances (atranorin and norstictic .acid) chromatographed qnevery ,plate., The unknowns are tentatively identified. by sorting (by. Rp,,classes) .punched cards .summarizing microchemical .data. for: all. compounds: previously studied. .The preliminary identification is then confirmed by .additional : microchemical tests. The open&ded, system, can incorporate new and unknown compounds,as well as information from other chromatographic systems: Data obtained rby the standardized procedure are ,given ,for 104 products. ,’ ,._I. :
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INTRODUCTION

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The natural products of the lichen-forming fungi have become, .intimately involved in’ systematics and classification l. Some 300 specific. substances, have been reported from about 2,000 of the 18,000 described, species of lichen@. Approximately 100 of these compounds are aromatic products that have been frequently mentioned in the taxonomic literature. ,Today the lichen systematist ,faces the problem of learning to identify. these natural products without a set procedure to guide him. An important paper,,by .SANTESSON~records RR values for spots of some 80 compounds on thin-layer chromatograms ruu in a:variety of solvent systems. The compounds are grouped <by chemical type and by certain color reactions.and, Rp data are presented for chromatogranisrun in solvent systems.best suited to each group.:Several other papers devoted.to,the thinlayer chromatography (TLC) ,of. lichen substances a&o treat, specific groups of.compounds such as the aliphatic ‘acids4 and aromatic aldehydesa. Several chromatographic solvents havebeen: suggested in chemotaxonomic’studies where :a particular. system, was found to be’convenient,for distinguishing.a limited,number of specific compounds. While all of,this information is extremely v,sluable ,for confirmatory identifications, w ,/. .Clrvomatog., 46, (1970) 85-93

These possibilities are’ tlieni narrowed do&i . . s&e& systems prepared without !these <purificationsgave results sufficiently. .! ‘.error to find a suitable one..‘determined. .a..‘.classes the limits ‘of tihich &e.: /Ii’. ‘..re’ke~~d~. . .I.by ctireful comparison of (a) ‘actual R~$‘&hi”s%vith respectto’the!R~ values’of the control~marker spots..ether iwere 8 passed through.. In essence the system involves the following.’ . (.:. redistilled. . In spite of the. ‘..substances when .’ ~ : .arenot required.: it.:. one ‘:may choose:.METHODS .: ‘The solvent systems are .... . the hit-and-miss aspects of microchemical identifications. I.I. .’ :: :: j :. acetic..I: :”” / ...i : microextracts. . only to find that there are also PD-negative constituents present in the same plant and for which there are no Rr.:Punched ‘cards ‘made ‘for compouiids’ that cannot.:a. ‘. The system described was developed for use by: the lichenologists at . consisted. data for that solvent.virasmaintained constant.’ Ghvomato&ag5hic materials asd solvent systems :.:were :developed in Brinkman tanks to’ a . . 2The.” . ‘.f>$ i .‘.CULBERsoN. i <.: . if the lichen thallus produces coloratiiih.. and stored over sodiu.the:djffi:. I.:.:.intensive interest in lichen chemistry+0 stand.. While. 200 . (b)‘color’reactions.“? . deterrW&ig their..by trial and. :of... :“.elsewhere. .& overcome (I). ... . It is then necessary to... : . . similar: to those reported Ihere to ‘indicate ythat these precautions . three solvent systems’ and :theu ‘by sorting! the ! punched.for PD-positive. mended’. culties due to varying Rp values. are.had!been stored .’ ‘. the future! ./.Duke University. : i rThe tbenzene.solvent CI toluen+acetic acid (85: 15. thin-layer plates.'over sodium. and toluene .height of IO cm on MerckSilica! Gel:Faii. KRISTINSSON. /I’..> ‘. solvent: A : .+j f: S+dards.’ 3.‘by the’ R$ values of a control mixture.select other solvent systems. identified.-. requires REV data for-all lichen compounds in the same solvent . solvent system recom-. ‘.hexane.+-pheriylenediamine i (PD):.’ 238 ml) s. : I :’ : . I?$ classes in the. For.. that . . .‘ dioxiine and. ..the general plan of the method can be modified and applied to any solvents used with any media.ether was stored.. Sozcyq& AND ‘.:Cliromatograms.. . (.in shorti 8and.specificR~~‘vdues in the’three~solutions~but:‘rather td’~R~. (1!Thelevel’of the solutions in:the’tanks.‘:.’ . . .ecies .’ ’ : ... ..cards for the jkhown compounds:belonging to all the ‘same ‘R&lasses. and (3) the problem of incorporating and retrieving microchemical information.. ... be identified are added to the deck to permit the recognition of the same substances in sp.& ‘j’. With_. (. .longwave’ UYI light. (c) :“appearance . in. . cousisteritly~used (as ~&marker :on:every :chromatogram..uiere...: *.but not ‘activated..(.: ChLohaa#og. the specific solvent systems and TLC plates used h&e been most successful in our laboratory...I..unstudied lichens.: . The method ‘described in.ERIAI& ‘. 240 ml) . . C.’ L ‘.: .i ‘( .’ j!&& “. . . . ? ....qjY: &k.benzene_dioxane1 solvent B : ‘hexane-ethyl ether&formic acid (5 :4:1. 1 ‘. . example.ardized system has been presented.:. ::i . “. : ..?oof.... substances. ... of herbarium specimens..land the solutions JY. columns : of alumina: tb removeperoxides. hoping.not~to. The.~ 46.86.. anhydrous ethyl . E'.:pure ..25.dard solvent systems for all compounds previously studied.studied . Chromatographic data from three stan.attempts.in a desiccator ‘over CaClsNaOH .systems.are stored on punched cards? ‘The?ca~d~~. UnknoGns.. tlie’spot .(z). (+0j~8_+93! ... ..dried (CaCla). this report. The same. (d) solubility. . . . for’ routine ‘analyses. by.. 1and any other’pertinent’data available::The’final identification’of the compoundis achieved by appropriate Iconfirmatory’ tests: .’ : ’ ... ‘. the worker surveying previously . acid: 1 (go .I_ ‘.a..1 MAT.ml) .zthese:were available : and of ..ni.

first the’centers of the atranorin spots at positions ‘. (6) atranorin. (class . . (4) alectoronic acid. in North. aad (I 8). I.the spots were recorded as soon as the plates were taken from the oven.of ‘the. for example Parmelia perforata (Jacq. On every ‘plate the first. (I I) chloroatranorin . (16) q-0-demethylbarbatic (I 7) ..with each spot indicate its Rp class and the diagram to the right of. (2) acetylportentol. because of their behavior in the particular solvent systems used. the horizontal penciled lines connecting the control spots. (I 5) cryptochlorophaeic acid . atranorin (A) and norstictic acid (N). (9) atranorin (A) and norstictic acid. 46 (rg70)‘85-g3 .) Ach.5) . (14) constictic acid.) Duby in Europe. acid (classes 4-5) . . The controls on a plate tiere spotted ‘from the same solution to ehminate RF variation due to concentration. The colors of. and an unknown substance. The same is ‘done for the norstictid acid. the control.cm from the bottom and g mm apart. and eighteenth positions were used for controls.djdy&ic acid . 1. ‘. Atranorin and norstictic acid were selected for marker controls because of their ready availability and. sprayed with 10% HPSOI..A ..at positions: g and: 18. (3) alectorialic acid (class 4) and barbatolic acid (class 3). the control: . RF Classes . ‘In order to identify RF classes. ” ‘.. (13) atranorin (class 7) and a-collatolic acid acid .’ . . : (Eighteen spots tiere applied to the plates z.. A copy of a plate run in solvent C showing the controls of atranorin and norstictic acid. and the RF clssses of the spots of the principal compounds chromatographed.and . norstictic acid (N). spots. at positions I. The number (or numbers) associated . ?he iimits . . All RF ‘values for spots were recbrded and Descrlptlon +-solvent front 0. America and P. an acetone extract of Pavmelia perforata. (IO) fumarprotocetraric acid and protocetraric acid (running together as a smglc spot) . eight .) The distance between the’origin and the solvent front tias divided arbitrarily into:eight regions measured’ with respect to the RP values’of the controls. A mixture of these two compounds is available in a single extract of many common species. Claromaiog. (N). the effect being more pronounced at lower RF values. acetabulzcm (Neck. (8) barbatic acid (classes 5-6) . ( 12 ) confluentic acid . . (Usually ‘spots’are slightly lo&& towards the center of the plate.:g. examined under UV light (254 nm and 366 nm). I..Rp classes are shotin in Fig.. (7) baeomycesic acid (class 5) and squa’matik acid (class 3’) . and heated at about IIO' until colors developed.. Fig.’ ‘.are connected by a rule4 line in pencil. Determistatioh of Ap classes The developed plates were air-dried. J. but sometimes the direction of variation is reversed.IbENTIIW%~~ON OP L~CWEN'PRODUCTS BY TLC 87 were replaced when the Rp values of the controls deviated significantly from their usual values. ii and. ninth. and 18. the plate summarizes the Rp classes. (5) anziaic. The identities of the principal spots at each position on the plate are (I) atranorin (A). g.

.‘..~Chvomalog. W. C. J. CULBERSON. . KRISTINSSON~ . F.r 46 (1970) 85-93..

. 66 5 4 BOnilliC 43142. C+ Yellow* Yellow* Yellow+- 6 6 6 51131~67 52133868 51/32*67 51131.67 3~40/26.. i -. 5j. 66 a/31.I”3 / . pale streak* Pale*.‘.. 3 ‘.64 5313% 64 48l33. WBrown* . 39140. 3914ow 67 -3 4 Paludosic 38-39140s64-65 4-5 4 a-Collatolic . 57 48126. Yelloi*. Ct.a 0.66 Bro1VI.57 55bts 55 52!25.65 45-46127s 61 33130.69 53134..’34143.. 65.71. C+ pigment..55/26.56 37/25. 57 i . stenosporic Perlatolic Sphaerophorin ~Microphjlllinic 4013%68 Unkndti triterpene( ?)” 47143. 57 5oii6.! . . 57 38/26*57’ 34ii8.*+ . . 70 42-43142t70 42143I67 4=4~/40. ~~Akctorialic‘. 61 bray green‘. KC+ Amiaic 4 4 : r is II 4 3 8 4-0-Demethylbarbatic Divaricatic Difffactaic BdXltiC St?kitiC 6 :-5 5-6 5-6 5-6 Homosekikaic Didyinic Imbricaric -.‘ 67 $5 5 k6 2-3 3 3 5 5-6 6 6 6 Yellow*.65 53138..-:6 : -. . . : D . 67 . -. 56/31._.66 Norlqbaridone . i _.65 :.65 Shl 5W 56125. 71 :. (Ct) Yellow pi@ent Pink or pale brown*. K+ KC.. ::.’56 45197B55 49127. Thiophanic hr& . (1: :. 57 37125157. _ 36-37143.190) d? . 60 33125s57 3*31/24.. KC+ Br&nish*. K+ Green&*.-Olive&k . : ‘~OrstiCtiC 4 o&uIlic~ -:.66 48131867 47-48131866 47130865 50131-32866 50/28.66 puiple*: Kt. 57 37120. .67 41/3!% 67 44msi W%-39.’57-58 32125.61 3.57 45-46127157 46-47127. 69 5-6 4 Yellow+.. (h@tfed oft p...64 40131.. greenish*. .65 43/29..:a... 5 unknown ~ivith I+lic~ 6 6 6 6 6 6 6 5 6 38-j9/4oa 67 3914% 70 41143.65 5 4 Glomelliferic 39/4o. 5$39. .. 3*44/38166 3-6 . 56125157-58 Brownish purple* Yellow* . . :5-6.67 42-43140867 .67 23-24i31164 28/29.59 47126.4..64.38-39142. 43-44126856 49/!6. C. 66.. 54 48127. UVYellow*. KC+. Brightlavender*. 04 42130.&@t&ol ursolic * . KC+.&yanic _. . . 5-6 2 5-6 fj!. . 7’ ’ Ento&@ :. 5I-52132867 . KC+ Broa*i (Kt). 57 :36/28. 6 W39./. 70 Orcinol 43/3&a ‘.c+ Br$s$K+. _ I . 53 ..64 *35/3o* 66 z r” 5 Yellow* Brownish* Yellow+.:57 .57 38126s57 33126. -.. ‘..58 o-15/23.63 4 4 4 4 4 4 4 4 4 6-7 7 7 7 4 4-5 4-5 5 5 5 7 5 : 5 5 5 5.54 38/?5* 53 38127. K+ .WYellow pigment. KC+ Yellow* . 54 w/q.:.6o 35125. :: ./l 42131.:5..64 4013r..66 5?52/29. __._.l’ 3 VJ z Yellow-brown+ Pinkish @y* .-” 5 . 34 4‘. . W+ + + -. .40/49... .‘6 c.tii%x 67 Cryptochldrophaeic 4ji@s 67 Confluetitic :_ 4&47/4oa 67 Planaic 4514os64 P/40. 71 3 _I $5 . 43i24.:-:. .+ Yellow*.Evec 33142.- : . 64 i6-$31.70 56-57134s 70 5$-5613j 69 4Q/30. 24130. . - _.5747-48/?515Q-57 43125. j-6 : 4x/26.. 36/32.5d : fj+ S-6 6 6 Emodin 2 6 6 ‘. 35143.I-4 _ Strepsih . C-f blue-green Yellow* Yellow* Yellonr* Yellow* Pale*. .. : Greenish* g5 c :. 44143867 4ol39. 64 18-&j/27. C+ Brow@. KC+ c5 0 5 Ytillt+ : Metichkkphaeic’ . .j7120.&4.57-5f 25i3h 64 3oi28. e$+-~~.56 33-34126.::. 6g .J ‘3 ~-“-~. .: : . (ct) ’ z s z 8 -4-0-Methylgyrophoric _: :. 66 45/32*67 44133. (Ct).i3 2 42/27.. C+ green Yellow*.57 23131. Rhodophyscin 44/4o. PD+. PD+ Pigment.67 4714oB67 67 52140.i.~cT”~qJup8JuA~ ‘I :.

. . . 4C. .go.KRISTINSSON... u/.I i /. H.F.< CULBERSON.

’ : *.’ .... is keyed for the Rp classes in the three: solvent. always expressed :witli . ‘.' AS an example.ckemotaxonomic sti~veys ‘. Fig...at positions * I.and. and specific / RF values. .. atranorin controls.C .by the 6~ LICWEN PR~D~JCTS BY 'Il. -be. .. .’ .:. .. . ‘. : .Fig.the following procedure is used : cleaned plant material (I cm2 or less) is placed in a shell vial (Mercer Glass Works.is flowed. 2) reduces the number of possibilities still further and suggests . spotted isimultaneously. . . . Chvomatog.. . .f::.I _‘..chemicaJ narms.a : . 2 shows the punched cardfor lobaric acid.Rp classes :determined.ated ‘using . the controls of atranorin and norstictic acid are’ included. . 35 x 12 mm) numbered in India ink. known compound showing the.IDEXUTIIVCAT~ON f'dhmxi ...... ).’ 1 : of compounds iti crzlde. ature.. y ::‘. ....f.‘.zg7o) 85+3 . .’ and.: . . all previously studied &d pounds with the same Rp classes.‘. a drop or two of acetone. .Y... 18. “. information stored. “’ I To spot the chromatograms.’ *As usual. The additional information on the cards (Fig. name 01 compo and color r&c ::.’ /“.bennene ‘and ( acetone ‘extracts ‘of ‘each sample iare chromatographed’\ side-by-side on: the thin-layer./. ‘a . . I. Inc.‘.‘. and the %cetone extracts’ are’ evaporated on a’second slide. A punched card. extrkts of &he& for .. . .’ . .analysis of fragments of herbarium specimens...’ .the slide. asuggcstcd contr0l species .\ ..many Specimens’are analyzed simultaneously.dire&ly :... plate’.’ .“: :I{: . 112 dr.‘. short style.. 46 (. :9.the same /...f .the best solution or solutions in which to rechromatograph the unknown with appropriate J.I’ \ ..: Additional #data. 1: ... to the common . ..‘.J .Three ! plates may.: 1 :c.+n example of a punchod card for a. -‘:.: .. .Usually .spots are recorded and the . ‘. Of the 104 compounds listed in Table I the maximum number of compounds with the same RF classes is six. are reoorded . shell vials.I ).:I:(./.: : . I . corre.‘. In our 1aboratory. . .. The extracts are evaporated at about 50” on a microscope slide.’ . systems and for an alphabetic sequence. The fragment is extracted three times by g-ro-min soakings in benzene at roomtemper. ). visible ‘:ligfit :. and ‘the .( ..collected:in’a micropipette:. The punched cards make possible the identification of.: The’ Rp values of the .: spending’ qvalues of’ the norstictic acid and.acetone to extract the sample at elevated temperature &do). : ‘.’ ‘. (..: . prepared for every compound.. . on’ the cards..: . acid and atranorin lines at . The processes are’ repe. over’ the residue on . . .‘. s.. ._ gi horizontal Recodhg valuesmeasured to the norstictic position (see Table I). / . . . . ..:. N. * infovmatiovt 08 $wnched cards . .I Chemotaxonomic studies’ ontlichens are usually based upon chemical .the.. i Identification .. .

.: ‘. carpiea@id.I: s:: > i.92 C...upk~iiq. .A~oRAMu~T..I... are.: .’ ‘. i ! + .‘:*..: ‘1 :‘i’?.I.:I .... 1 i’.in.. .most~simil~ RF-val..arid:thiqphanic acids..' .lis investigation was supported hy. ~~nhE~~forl’a~&ample. aromatic.the .: .. .. i I.j .. and triterpenoid!products~df lichens. Naii0na. y I.z:!‘:. Sciences.’ ” %.:“..” I..hew:F. :.litihqs containing sever@. H. cr!uc+‘]@ctracts df .... .. i. systems and by: the. :MMSS! for' +&ples ..tr.:1 .. ‘.data:. .~~ithank'~..:ti~.. . derivatives ..of several pultiinic acid. i : ‘the (Mational.. substance.. :. :.. .6.the.“. and.:.qq. ..‘. ! c..obtained for the .. : :.-i c.in md:e&ino- ACli4NOWLEDG~MENTS.‘i.1I”_ i::._’ ! :. .. of-h&em&h&molic..ueS.’ :(.tl&chromatograms run in$ffereqt sol&fqq~ .: ::....“: ” .. ‘.be varied in n&n? w&% &nd i’t ‘can: b~::. : .i :...::.:. :>a:’ .can:~~~~~times $@p.. :‘ac .(. .. first with that . .’ : .other spots ii&Fe solv&ts. ! f ./ .’ (G&&45) ‘from.::!\ l. :I: i..’ .‘i. .i!*.] 1..:. .‘... ... .. cqntinugl a$dition of card’s f6~~:eey. new i&de&s of lichen&oey in our ltibcratory ha\ie been able to identify . Y’. RESULTS AND DISCUSSION : *\ ‘: . c?q$u&ls.. ..f j :: .~M.:.:. from.’ i. f:.. i . I :.‘.. ‘Th.i I+ :.o!vent. ” Table.‘. :S.o i~l. the ‘game compounds. 3 The ‘syste..thti+!e . ‘.....c?~taining :yir.~'~J~..tog&her. ttheir.b~~~e~e’i’~~d:~hen:j. .“’ \ ( ’ ‘:.lichqns cotitaining. .partiqdar uriideneified substance...of <Health:.1 (GB-$o@Q .t:. a band run .of.I: .$$ a simple ‘acetone extract. .acid:.i !z.‘. ...I ....:‘-:..!. of. the double extract&’ of the.repprt qsqn ..!::_‘: i. up Iwhen the i :.&sic : . : .‘.... .~.:.KRISTINSSON standards. : :. be reproduced exactly inanother labor&ory. and..S..is enctiuntbred i in ‘other ..~... and: one.a...jc.method &yes meaning to the designation “unidentified stibstance’ ’ and !issures $h&t.~~~~~~.. :. :acid. a s&n$e ~fsElichen..>i. ... I..Dr.~_z$#.. l’!.be’ cleared. . .for sa&plbi..... .L~. ‘.. II. I :. 1’. recognized again.._. ..‘-. . .I’ .W:...from . rhodophys.:.: :. ( .I ....ound&t-inay be diffic&+b distinguish whi&sp&s bn.The compounds..h $Fetdxiei often allovris the identification of :&pounds :‘wbtild be @bscu&c$..~~ . ‘7 : .!~. F. arranged aticprdingito’ increasing J?p’classes so $hat..I L .:I ..‘.....’ . . ..‘)‘.‘_ .i./ ‘.&&l~d by . j i . Colora&&~~with different spray: r$~~~ts’. ‘. Ii. *I . But it is expected that the RIPclasses ” which depend only on. .’ . . I.‘...< ) r .. If the compound is.H. : .?‘$riy . ^.!:rl’r.those:with.Eopndation.l.~.e. .. 1 : ‘. .’ .. : ....:. it is prepared and l .:< . Sometimes...Dr.. Dr.t&*ek st?r@&l . !./ .):. Elution.the relative order of spot& Gill be much’ti&e con+ant.~~&@ti~. . :’ ‘. species. . . ‘.‘.. ~-~UNECK..+xp.. . lD&...~~~....in one ‘s&&t aiid &hroma~ogt~p~y.. ...::‘>. : .! :(“...’ ... ’ i ..Medical.‘diie to.‘..y I. ‘.._.f”anv. lichenic.’ . .I ::. ../. . these RF valGes could. .grant.i / . .. is’ finally. artimattc :li&en substsinces. I . ..:I. * ‘“‘T. . for .E. the.Y>. ‘.. i .characterized.I”.wns Y-from....~Dr. .be.solvent systems ‘are grouped. i.! .I ..eJ ... ... .. -It is urilikely: that. ... .v:‘:\.: ‘.i:_i.: j ... : :..‘:::::.“‘~i:!.. ..idd& . I .. one or more spots<w~~~.:.E:dr.fhis .j .solvents’ &ually solve the mbs& difficult.....:. .:.. ‘I ..l Institutes .. ! . i : ... p’.-of’th~i~~~ted’ q$terial in $@<. .. .first chromtitographic plates..: .riot identifiable. ! . .“. : (’ : I ’ / :..:.summari&s. .by. :. A. Iq.mosi -common.:... [ . j .:.pf. ..:K. Dr. . .comp.: . of ::the%ommdti..:./ .‘.:.-... C . .. .. :. .‘_....‘. .j..c. ’ .I .:i..” ‘_‘.. ....cJ’:p~nchdd :$ards desq&&+ .: i.:.S..‘.. .acid..: : . will..Science. ‘. +.‘since oier:a period of some mbnths the Rp values of the controls ofatranorin and &stictic~acid~chan&e!slightly... : ) : .. ..’ ‘...( :.if it ./.. a punched card for. . Atid :fixially.r:.1: :. .samples::of: acetylporfentol and planaic.’ : .:.... ‘&+$&&icountere$ i+ surveys. I. L.!CuLBERSON. sample.. :. :.: :!. the I&i&ion of Gen&al:. : ....: added to the deck. . .: WACNTMEISTER for samplds: of p&o.$heidentification...“.:.“. . i ‘. . I .’ . . Using Ithis’method. 1 .. allowing : all such j records i to. j .

967) 1162. Chvoma@5. L! CULBERSON..i’gGg. I W.Notih : Czkolina I?ros’s. BENDZ. 3 J.. 19: (1963) 2254.9. Taxon. : .3 2&‘i. ! ‘.~ Acta Chcm. * I? ’ i i ’ ’ : . ?I (x. SANTESSON. .: CVLBERSQN.’ *.gk6j “. 46 ji97p)..” 5 J.” :’ !““’ ’ ! 85. J. Stand. C. . SANTESSON.’ 2 4 G.. Acta iShbm. I?. Acta Cl&em.IDENTIFICATION REFERENCES OF LICHEN: PRODUCTS BY TLC .I. Cha&?l Hill. J. SANTESSON'AND Ir:T~r3tiii. ‘Univerpjty’ of .~~S&d. ‘r&i’: _i 93 . SC&&. 18 (1969) 152. .’ ‘.