JOURNAL

~OP~CI-IROMATOGRAPWY

23s ::
. *

CWROM. +$.7F ,,. .:; ::i,;,

.,.,
. 1
;

/
.
1

,.,, ,., j
,,..

,
,,

, ,(
, .,

, ;

,,, j .:
; ;

,
)I i

,.I,

A. STANDARDKZED
PRODUCTS
,/,:

METHOD

FOR THE
: : ..

IDENTIFICATION
, .:,,:.

OF LICHEN
;. .,

:,.,
1 ,,,

.
.,

.a:
,:.c,:,
, ,.;,

: 1:

:
..>I

.
,.

:
,

,;

;,...

.I

._.

/I

CWIC~TA DXLESERS~N
(Rcd~ived~Se~tembdr 30th.

AND

EWR-DURKRWTINSSON

1 I, ) ,

s:
: . ...

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

.,., ,, . ,, ,.,
1969)

:.

<:,

:,.
. :,

,_

,
:. ,, :

(,

).,,,

SUMMARY,.,

;,

;,;:

.,

., ,.

,. ,,

,,., ,.. 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. :
,. ;
INTRODUCTION

1 1 .,

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 chemotaxonomicstudies where :a particular. system, was found to beconvenient,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

86;.

C. E'..CULBERsoN,I.

KRISTINSSON,.

the worker surveying previously ,unstudied lichens, requires REV data for-all lichen compounds in the same solvent .systems. For, example, if the lichen thallus produces coloratiiih; With_,+-pheriylenediamine i (PD):, one :may choose:,a, solvent system recom-, mended.for PD-positive. substances, only to find that there are also PD-negative constituents present in the same plant and for which there are no Rr;, data for that solvent. It is then necessary to,select other solvent systems; hoping,by trial and,error to find a suitable one. In spite of the,intensive interest in lichen chemistry+0 stand,ardized 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 retrieving microchemical information. The system described was developed for use by: the lichenologists at .Duke University. While. the specific solvent systems and TLC plates used h&e been most successful in our laboratory, .the general plan of the method can be modified and applied to any solvents used with any media. In essence the system involves the following. Chromatographic data from three stan,dard solvent systems for all compounds previously studied,are stored on punched cards? The?ca~d~~,a;reke~~d~,not~to,specificR~~vdues in thethree~solutions~but:rather td~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! the ! punched- 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) actual R~$&his%vith respecttothe!R~ valuesof the control~marker spots; (b)colorreactions, (c) :appearance ,?oof, tliespot ,.in shorti 8and- longwave UYI light; (d) solubility, 1and any otherpertinentdata available::Thefinal identificationof the compoundis achieved by appropriate Iconfirmatory tests: ,:Punched cards made for compouiids that cannot. be identified are added to the deck to permit the recognition of the same substances in sp.ecies ,, ; ,. ., (,studied ,. in, the future! . .. I,, ,.
(, ., ,,(, ; ., :
. L , i <, ::i ; : I : : , 3.1 MAT,ERIAI& ;. Sozcyq& AND . ,METHODS . j!&& ,:,! ,I_

,qjY: &k;&

j,+j f: S+dards; consisted, :of,:pure ,substances when .zthese:were available : and of .i : microextracts; of herbarium specimens.elsewhere. . : .: . ~ : ,,>

, I, , .,.i ( ;, ,: *,.-;:.,;? ,:,,, ,.. ,.:a., ,,. ? . . ,.,

;, . :: ::

j :.,:,

;,;;

,,

. (,

,.

,,I.: /Ii, , Ghvomato&ag5hic materials asd solvent systems :; ,.f>$ i .:Cliromatograms,:were :developed in Brinkman tanks to a ,height of IO cm on MerckSilica! Gel:Faii. thin-layer plates. that ,had!been stored .in a desiccator over CaClsNaOH ; ,but not activated; .: The solvent systems are ; solvent: A : .benzene_dioxane1 solvent B : hexane-ethyl ether&formic acid (5 :4:1, acetic, acid: 1 (go .:,25.: it; 238 ml) s; . , ., 1 ., , .. 200 ,ml) ;.solvent CI toluen+acetic acid (85: 15, 240 ml) .,;: .I: : / . : i rThe tbenzene;.hexane,. and toluene .uiere,dried (CaCla); redistilled; and stored over sodiu,ni, 2The; dioxiine and. anhydrous ethyl ,ether iwere 8 passed through, columns : of alumina: tb removeperoxides; The,,ether was stored,.'over sodium; The same, s&e& systems prepared without !these <purificationsgave results sufficiently, similar: to those reported Ihere to indicate ythat these precautions ,arenot required. for routine analyses, ,. /I, .. . (1!Thelevelof the solutions in:thetanks.virasmaintained constant;land the solutions

JY.: ChLohaa#og.,~ 46, (+0j~8_+93!

IbENTIIW%~~ON

OP L~CWEN'PRODUCTS

BY TLC

87

were replaced when the Rp values of the controls deviated significantly from their usual values. Atranorin and norstictic acid were selected for marker controls because of their ready availability and. because of their behavior in the particular solvent systems used. A mixture of these two compounds is available in a single extract of many common species, for example Parmelia perforata (Jacq.) Ach. in North, America and 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. The controls on a plate tiere spotted from the same solution to ehminate RF variation due to concentration. . ., . . 1.
. ,,. ,,, .
.

Determistatioh of Ap classes The developed plates were air-dried, examined under UV light (254 nm and 366 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 positions . ii 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 center of the plate, the effect being more pronounced at lower RF values, but sometimes the direction of variation is reversed.) The distance between theorigin and the solvent front tias divided arbitrarily into:eight regions measured with respect to the RP valuesof the controls. ?he iimits ,of the. eight .Rp classes are shotin in Fig. I. All RF values for spots were recbrded and

Descrlptlon +-solvent front

0;

RF Classes

,A

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 RF clssses 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 summarizes 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 Pavmelia perforata; (2) acetylportentol; (3) alectorialic acid (class 4) and barbatolic acid (class 3); (4) alectoronic acid;, (5) anziaic, acid (classes 4-5) ; (6) atranorin; (7) baeomycesic acid (class 5) and squamatik acid (class 3) ; (8) barbatic acid (classes 5-6) ; (9) atranorin (A) and norstictic acid, (N), the control; (IO) fumarprotocetraric acid and protocetraric acid (running together as a smglc spot) ; (I I) chloroatranorin ; ( 12 ) confluentic acid ; (13) atranorin (class 7) and a-collatolic acid acid ; (class ,5) ; (14) constictic acid; (I 5) cryptochlorophaeic acid ; (16) q-0-demethylbarbatic (I 7) ,djdy&ic acid ; aad (I 8). atranorin (A) and norstictic acid (N), the control: .. J. Claromaiog., 46 (rg70)85-g3

C. F,. CULBERSON,

W. KRISTINSSON~ ..

J.~Chvomalog.,r

46

(1970)

85-93.

.J

~--~.~cT~qJup8JuA~ I :.. .:a,,*+ ;/l 42131. 04

42130,65 :. -

_, ,.j7120;:57

j-6

4x/26,57 45-46127157
46-47127. 57 :36/28, 61

bray green; KC+ Yellow*

.i3

2
42/27,58 o-15/23, 54 w/q, 57 i .. .
24130, 66

45/32*67 44133.67 23-24i31164 28/29,64 *35/3o* 66

z
r 5

Yellow* Brownish* Yellow+; C+ Brow@; Ct, KC+, PD+ Pigment; pale streak* Pale*; C+ green Yellow*; PD+, K+

KC;, (ct)

z s z
8

-4-0-Methylgyrophoric _: :. .34143; 6g .. . . . __. ;-:6 : -.- 5 ..::. .Evec 33142.71. i _.! ;6 c..:-:.. .:5. (1: :- .&yanic _. 35143. 71 3 _I $5 .. i -; 3 ;:. -.-Olive&k .. _ 36-37143, 71 :, ; :5-6._.- 4. ~~Akctorialic. .38-39142, 7 Ento&@ :.; 3*44/38166 3-6 ..I3 / ..I-4 _ Strepsih .: : .40/49.61 3., 34 4, : ~OrstiCtiC 4 o&uIlic~ -:. 3914ow 67 -3 4 Paludosic 38-39140s64-65 4-5 4 a-Collatolic . 39140.65 5 4 Glomelliferic 39/4o, 66 5 4 BOnilliC 43142. 69 5-6 4 Yellow+;c+ Br$s$K+, . Yelloi*; KC+
Amiaic

4 4 :

r is II 4 3 8

4-0-Demethylbarbatic
Divaricatic Difffactaic
BdXltiC St?kitiC

:-5 5-6 5-6 5-6

Homosekikaic Didyinic Imbricaric -. 56125157-58

Brownish purple* Yellow* ; C.+ Yellow*; C+ Yellow* Yellow* Yellow+-

6 6 6 51131~67 52133868 51/32*67 51131, 66. 5I-52132867 ;&4;/;; 64 i6-$31. 64 18-&j/27,63

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. 5
unknown ~ivith I+lic~

6 6 6 6 6 6 6 5 6

38-j9/4oa 67 3914% 70 41143, 70 42-43142t70 42143I67 4=4~/40,67 42-43140867 . 44143867 4ol39.67 41/3!% 67 44msi W%-39,67

3~40/26,57 38126s57 33126.54 38/?5* 53 38127, 57 48126,56 45197B55 49127, 54 48127, 53 .55/26,59 47126, 57 5oii6,57 55bts 55 52!25,57-5f 25i3h 64 3oi28,64 40131.65 43/29,65 45-46127s 61 33130.64. 36/32,66 48131867 47-48131866 47130865 50131-32866 50/28,66

Bro1VI.l

3 VJ z

Yellow-brown+ Pinkish @y* ; C-f blue-green Yellow* Yellow* Yellonr* Yellow* Pale*, KC+ Br&nish*; UVYellow*; C+ pigment; greenish*; K+ Green&*; KC+ Broa*i (Kt), (Ct), KC+

c5 0

Ytillt+

: Metichkkphaeic .. Thiophanic hr& ,&@t&ol ursolic * . e$+-~~;

stenosporic Perlatolic Sphaerophorin ~Microphjlllinic 4013%68 Unkndti triterpene( ?) 47143. 70 Orcinol 43/3&a . Rhodophyscin 44/4o,66 Norlqbaridone ;tii%x 67 Cryptochldrophaeic 4ji@s 67 Confluetitic :_ 4&47/4oa 67 Planaic 4514os64
P/40,67 4714oB67 67 52140; 67

$5 5

k6

2-3 3 3 5 5-6 6 6 6

Yellow*. : Greenish*

g5 c :. ;a 0, .5d

: fj+

S-6 6

6 Emodin

2 6 6

;; 5-6 2 5-6

fj!.

W39,65 53138; 67 .. 5$39.65

Shl 5W 56125,57-58 32125, 57 37120. 60 33125s57 3*31/24,56 33-34126; 57 38/26*57 34ii8,6o 35125, 57 37125157. 43-44126856 49/!6,56 37/25,.5747-48/?515Q-57 43125, 5j. 43i24.57

23131.64 4013r; 66 a/31. 65. 56/31.66 5?52/29,64 5313% 64 48l33.69 53134,.70 56-57134s 70 5$-5613j 69 4Q/30.66

puiple*: Kt, KC+, (Ct) Yellow pi@ent Pink or pale brown*; WBrown* . Brightlavender*;WYellow pigment; K+ ; W+ + +

-.

::

.,i. .,

,_., -. . ::.
: D

_
I

., ,-

: ,, .. (h@tfed

oft p.190) d?

go,

.I

/,

u/.

4C;, .F,.< CULBERSON;

H. ,KRISTINSSON,

IDEXUTIIVCAT~ON
f'dhmxi ,by the

6~ LICWEN

PR~D~JCTS BY

'Il.C ._ gi

horizontal
Recodhg

valuesmeasured to the norstictic position (see Table I).

acid and atranorin lines at .the same

/,,, : ., .: : ;; I, ,f

,. 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 ,chemicaJ narms.: Additional #data, and specific / RF values, always expressed :witli ,the, corre;: spending qvalues of the norstictic acid and. atranorin controls, are reoorded .dire&ly :. , on the cards.' AS an example, ,Fig. 2 shows the punched cardfor lobaric acid, *

infovmatiovt 08 $wnched cards

,,:;

,.

,..

.,,

,, .I

_.

.,.

;I

).

name 01 compo and color r&c ::.. ,/,

, -:. ,,
visible :ligfit

:. ; .:,

,,

1:

,:

,, .,.

. , ,J ; ,,f::.;,

asuggcstcd contr0l species

, .. a ,/,:I:(,;,: ,, ; ., . .,;., ,,., (,, ),\ ,.. .,, y ::.., /.a : , ,I \ .: :I{: ;

Fig. s. ,+n example of a punchod card for a, known compound showing the. information stored. .,: 1 :c,, ., ). . / ,.( , : *. . i

Identification
, 1 :

of compounds iti crzlde. extrkts

of &he& for ,ckemotaxonomic sti~veys ,f,

.I

Chemotaxonomic studies ontlichens are usually based upon chemical ,analysis of fragments of herbarium specimens. In our 1aboratory;the following procedure is used : 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. The fragment 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 processes are repe,ated using ,acetone to extract the sample at elevated temperature &do), and the %cetone extracts are evaporated on asecond slide. ,Usually ,many Specimensare analyzed simultaneously,. and the .bennene and ( acetone extracts of each sample iare chromatographed\ 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 residue on ,the slide,,and. .collected:ina micropipette:.,Three ! plates may, -be, spotted isimultaneously.: The Rp values of the ,spots are recorded and the .Rp classes :determined; The punched cards make possible the identification of, all previously studied &d pounds with the same Rp classes. Of the 104 compounds listed in Table I the maximum number of compounds with the same RF classes is six. The additional information on the cards (Fig. 2) reduces the number of possibilities still further and suggests .the best solution or solutions in which to rechromatograph the unknown with appropriate
J; Chvomatog.;

46 (.zg7o) 85+3

92

C; F.!CuLBERSON, H..,KRISTINSSON

standards; If the compound is.riot identifiable, a punched card for. it is prepared and l ., ., ,. , , . .:I ,, ; ,.,I ::;: added to the deck.
RESULTS AND DISCUSSION
: *\ : . ., ;(, i : ; 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 aticprdingito increasing J?pclasses so $hat,.those:with,.the,.most~simil~ RF-val.ueS,in,the ,thti+!e .solvent systems are grouped,,tog&her,. -It is urilikely: that, these RF valGes could. be reproduced exactly inanother labor&ory,since oier:a period of some mbnths the Rp values of the controls ofatranorin and &stictic~acid~chan&e!slightly, But it is expected that the RIPclasses which depend only on,the relative order of spot& Gill be muchti&e con+ant. Iq,t:,$heidentification;,,pf.,upk~iiq,wns Y-from. cr!uc+]@ctracts df ,litihqs containing sever@,comp,ound&t-inay be diffic&+b distinguish whi&sp&s bn,tl&chromatograms run in$ffereqt sol&fqq~ ,qq,diie to. the game compounds; Sometimes, one or more spots<w~~~.~~~~~~,&&l~d by ,other spots ii&Fe solv&ts; Colora&&~~with different spray: r$~~~ts,can:~~~~~times $@p., Elution. of, a band run .in one s&&t aiid &hroma~ogt~p~y,-ofth~i~~~ted q$terial in $@<.t&*ek st?r@&l ,solvents &ually solve the mbs& difficult..~~&@ti~. Atid :fixially, the double extract& of the, sample, first with that ,,b~~~e~ei~~d:~hen:j.~.h $Fetdxiei often allovris the identification of :&pounds :wbtild be @bscu&c$.$$ a simple acetone extract. . ..; , :, I. 3 The syste- ,cJ:p~nchdd :$ards desq&&+ ,fhis ,repprt qsqn ,be varied in n&n? w&% &nd it can: b~::,+xp,~_z$#,by, .idd& ,hew:F,o!vent. systems and by: the. cqntinugl a$dition of cards f6~~:eey, c?q$u&ls. and, &+$&&icountere$ i+ surveys. Using Ithismethod, new i&de&s of lichen&oey in our ltibcratory ha\ie been able to identify ,,:,fanv.; of ::the%ommdti, artimattc :li&en substsinces. from; ttheir,first chromtitographic plates. Th;e.method &yes meaning to the designation unidentified stibstance and !issures $h&t,?$riy ..partiqdar uriideneified substance., will..be, recognized again, ,if it ,is enctiuntbred i in other ; species; allowing : all such j records i to.be cleared; up Iwhen the i :. I ., I .I . : , .,, :. :(._ ! :. substance. is finally;characterized,;, ; : ;,. 7 : ; ., I
. , 1 i,, i ! + ; , , : ) : ; .;j ,, : ( : I / :; : - :,;; ; [ ; i I, : .,!, ; :>a: ; .; ( ,>i,,:1 , i ; : :,i / .;] 1.: i, :ac ,.Y>.;: ,:. :.l.jc;:i; , :, ,,, ,. : .._ ./ ; :, ,,; _,, ,::>; : .,: - ._,:,; :,, ;, . i. : . , \ ( :. .I .,. ,: : , ,c, .: .( :,,: j ,: ,., j ,; 1 .I L .. !

.i :,, .: .j, , i ., ,;.: .:::::.,,:.;!::_: i;: ::;r:, ,I ,,,, ^.~i:!,::!\ l,i,-,,,,,:,; i, ..,, ,,,I ,! :(. l!,, , . j i ,;-, ,~,~~ithank'~,Dr.:K.;A~oRAMu~T,~~ ,.E:dr, a s&n$e ~fsElichen;c?~taining :yir,&sic : ,acid.;~Dr;~M;_,~,E.,.H,~~~,~'~J~..for sa&plbi,of,lichqns cotitaining. rhodophys,in md:e&ino-

ACli4NOWLEDG~MENTS;.!~,..; f:; ;,' ,:;,i I+

:,. :, i . p; :I: i; ;; i,,).. ,:,; ! f ; 1; *I ;: 1 :i?,f j :: .,,; Ii./, ..I,,,1: :,, ,; ,, , ,,. . . L,:,:,I: s:: > i,I: ,,o i~l,,v::\;L~.i !z,z:!:..:*,,:, ,.,../ ,/ , , ;: :!,i!*,,, . ! ,,i..,i:_i;j ;;y I;

carpiea@id; lD&;.S.;:ti~; ~~nhE~~forla~&ample. of-h&em&h&molic;acid:; .Dr; :S. ~-~UNECK. for .samples::of: acetylporfentol and planaic. :acid.;; Dr., .W:, .S..6, :MMSS! for' +&ples .of several pultiinic acid, derivatives ; and, Dr.. C ., A.: WACNTMEISTER for samplds: of p&o,::, !,, ;,. .,; lichenic,arid:thiqphanic acids.):, ,.. I ..,. :.;..:-:,. ., II. +,, .:.;:,_, : ;,:I; ,,..I ,-i c;.tr;.!:rlr;lis investigation was supported hy..a.grant. (G&&45) from, the I&i&ion of Gen&al:.Medical. Sciences,; Naii0na.l Institutes .of <Health:, and: one.1 (GB-$o@Q .from .:.: ,,;..: :., : :,, 1 : , .I. i : the (Mational,;Science;.Eopndation. .., ! c,, I; I,.; I :.;:I ,,,, ,, * T,I.:;,; ,;1I_ i::,.
,:,., ,,,,, ,,/;, I :, i ; ,,,:; ..< ) r , I. ;,,:< ,.. ..;.:,; ,,i;.., ./ , ;;;;

IDENTIFICATION REFERENCES

OF LICHEN:

PRODUCTS

BY

TLC
.igGg. r&i: _i

93
,.I. , ! , ,

4 G. BENDZ,

I W. L! CULBERSON, Taxon; 18 (1969) 152. C. I?.: CVLBERSQN, Univerpjty of ,Notih : Czkolina I?ross, Cha&?l Hill; 3 J. SANTESSON, Acta Cl&em. SC&&, ?I (x.967) 1162.
J. SANTESSON'AND Ir:T~r3tiii, Acta iShbm,~~S&d.~ Acta Chcm. Stand.. 19: (1963) 2254. *

I? i i

: , *. : ! !
85;9,3

2&i;gk6j

,.

5 J. SANTESSON,

: , J. Chvoma@5, ,.

46 ji97p),