High-Speed Liquid Chromatography of Lichen Extracts

Author(s): Chicita F. Culberson

Source: The Bryologist, Vol. 75, No. 1 (Spring, 1972), pp. 54-62
Published by: American Bryological and Lichenological Society
Stable URL: http://www.jstor.org/stable/3241522 .
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 High-Speed Liquid Chromatography of Lichen Extracts'

CHICITA F. CULBERSON2

Abstract. High-speed liquid chromatographyis a recently devised method

for the analysis of compounds that cannot be studied by gas chromatography,
either because these are not sufficiently volatile or are too unstable at elevated
temperatures. Preliminaryresults indicate that many aromaticsecondary lichen
products are not amenable to gas-chromatographicanalysis. Constructionof a
simple high-speed liquid chromatographthat will effectively analyze crude ex-
tracts of lichen fragments is described. This method should permit 1) rapid
quantitative estimations of substances in crude extracts, 2) isolations of pure
substancesfrom small amountsof complex mixtures,and 3) separationsof com-
pounds difficult to resolve by thin-layerchromatography.

Today thin-layer chromatography is the most general method for surveying the
occurrences of secondary lichen products (Culberson & Kristinsson, 1970). It is suc-
cessfully augmented by mass spectrometry for detecting and identifying many lichen
pigments (Santesson, 1969). For some taxonomic and for many physiologic studies,
however, these methods are not adequate. In most areas of phytochemistry other than
lichenology gas chromatography provides an invaluable means for rapid quantitative
analyses and for easy separations of pure components from mixtures. Coupled to a
mass spectrometer, the gas chromatograph becomes an excellent tool for rapid identifi-
cations as well. In 1965 Shibata and his coworkers (Shibata et al., 1965) described the
analysis of the lichen triterpene zeorin and some of its laboratory derivatives by gas
chromatography of the free substances and of their more volatile trimethylsilyl ethers.
Although the results appeared promising, the method was never pursued for the analysis
of the other naturally-occurring lichen triterpenes. More recently, the biosynthesis of
protolichesterinic acid was studied by gas chromatography (Bloomer et al., 1970a, b).
But these appear to be the only reports on the use of gas chromatography for analysis of
secondary lichen products.
The fact that gas chromatography has found little use in lichen studies is to be
explained by the thermal lability and low volatility of most of the well-known secondary
products of lichens. As a matter of fact, even in other fields where gas chromatography
does play an important role, it is often necessary to convert compounds to more volatile

1 This investigation was supported in part by grant GB-25346 from the National Science
Foundation. Equipment for the construction of the high-speed liquid chromatograph was
purchased through grant GB-27365 from the National Science Foundation. Instrumentation
for gas chromatographywas made available in the Duke University Phytotron through grant
GB-7153 from the National Science Foundation. I thank Dr. C. Lcchmiiller of the Department
of Chemistry, Duke University, and Dr. W. A. Dark of Waters Associates for valuable advice.
2 Department of Botany, Duke University, Durham, North Carolina 27706.

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1972] CULBERSON: HIGH-SPEED LIQUID CHROMATOGRAPHY 55

derivatives before these can be analyzed. It has been suggested that perhaps as many
as 80% of all existing natural products are not amenable to analysis by gas chroma-
tography. Recently, to meet the need for a good method to analyze compounds of low
volatility or low thermal stability, high-speed liquid chromatography was developed
(Bombaugh et al., 1970; Felton, 1969; Kirkland, 1969). The method has been suc-
cessfully applied to a variety of compounds, and it is certain that this technique will
provide new impetus to the study of secondary natural products that are difficult to
volatilize. The present report describes the first application of this method to the analy-
sis of some typical lichen compounds, with special emphasis on substances for which
gas chromatograms could not be obtained.

MATERIALS AND METHODS

Gas Chromatography.-A Packard7300 Series Gas Chromatographwith a flame ionization

detector was used in this study. Chromatogramswere run on a 2-meter column of 3% SE-30
on ChromasorbW at an N2 flow rate of 40 cc/min.
Attempts were made to chromatograph1) the free depsides lecanoric acid and perlatolic
acid, 2) trimethylsilyl derivatives of lecanoric and perlatolic acids, 3) trimethylsilyl derivatives
of hydrolysis products of lecanoric, evernic, barbatic, divaricatic, and perlatolic acids, and 4)
trimethylsilyl derivatives of the complex fatty acids caperatic acid and roccellic acid and of
an unknownfatty substance in CladoniasubcariosaNyl.
Trimethylsilyl derivatives were prepared by dissolving the substances in an excess of
N,O-bis( trimethylsilyl)-acetamide (BSA) (Klebe et al., 1966) in a capped vial. The resulting
solution was injected directly onto the column after allowing a few minutes for reaction to
occur. Chromatogramsof hydrolysis products of depsides were prepared either from pure
samples of the phenolic acid units or from mixtures obtained by microhydrolysisof crude lichen
extractswith cone. HSO4 by the usual method (e.g., C. F. Culberson,1967).

The High-speed Liquid ChromatographySystem.-The high-speed liquid chromatography

apparatusused in the present study is diagrammedin Figure 1. The pump is a Milroy D Con-
trolled Volume Pump, Model DB-117R, with an explosion-proofmotor (Milton Roy Co., Box
12169, St. Petersburg, Fla. 33733). This pump delivers a constant-volume, pulsing flow of
solvent at pressures up to 70 kg/cm2 (1000 psi). The pulses are dampened somewhat by
restrictors (R1 and R2) packed with silanized glass wool. The detector for aromatic substances
is a Model UVM-254 Ultraviolet Detector (Savant Instruments,Inc., 221 Park Ave., Hicksville,
N.Y. 11801) with an 8-al flow cell and with full-scale ranges from 0.64 to 0.02 optical density
units. The detector for analysis of aliphatic substances is a Model R-401 Refractive Index
Detector (Waters Associates, 61 Fountain St., Framingham, Mass. 01701). The detectors
can be used in series or alone and with either flowing or trapped reference solvents. The UV
detector can be operated simply with air in the reference cell. A dual pen recorder, Model
DSRG (Sargent-Welch, 35 Stern Ave., Springfield, N.J. 07081), plots signals from the detectors
against time.
All tubing and Sweglok connections are constructed of 316-stainless steel. The column
used in the present study is a 1-meter precision-bore,316-stainless steel tube, 0.25 in., o.d. X
0.083 in. (2.1 mm), i.d., with a 2> stainless-steel fritted disk at the outlet and a 10> disk at
the inlet. The injector port is a Model 420045 Liquid ChromatographInlet (Precision Sam-
pling Corporation,Box 15119, Baton Rouge, La. 70015). The column was packed with Corasil
II (Waters Associates) by the dry-packingmethod.
To operate this chromatographicsystem, the solvent in the reservoir (Fig. 1) is warmed
and evacuated briefly to remove dissolved gases, which would interfere with the performance
of the detector. Subsequent to this initial degassing, the magnetic stirrer effectively maintains
the solvent at a desirable, slightly elevated temperature. The pump is turned on and the valve
(Fig. 1) is closed until the gauge registersthe desired pressure. The flow rate can be measured
by timing the collection of a known volume of effluent from the detector. Although this rela-
tively simple apparatus has provided very satisfactory results for the present study, complete
high-speed liquid chromatographysystems, some of very sophisticated design, are now available
from several manufacturers (e.g., E.I. duPont de Nemours, Nester/Faust, Varian Aerograph,

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56 THE BRYOLOGIST [Volume 75

I-- WA STE

DETECTOR

COLUMN
GAUGE

VALVE
INJECTOR 't
PORT -
J

-+
R

SRESERVOIR
~> STIRRER

MPUMPZ
?;?;?::?: ?4-

FIGURE1. Diagram of the basic elements of a high-speed liquid chromatograph,which

operates to pressures of 70 kg/cm2 (1000 psi). The column and tubing are of stainless steel.
RestrictorsR1and reduce the pulses from the pump.
R_

Waters Associates) at costs from $3,000 to over $20,000. The minimum cost to construct a
high-speed liquid chromatographwith a UV detector is about $2,000 without the recorder,
and an inexpensive single-pen recorder could be used with such a single-detector system.

High-pressure Liquid Chromatographyof Some Lichen Products and Extracts.-Solvent

systems for elution of compounds to be detected by absorption of UV light at 254 nm must
be relatively transparentto light of this wavelength. The usefulness of a number of systems
incorporatinghexane, iso-propyl alcohol, iso-propyl ether, and acetic acid in various combina-
tions and proportionswas tested against a series of pure samples of known lichen products and
then against some crude lichen extracts.
For rapid analysis of lichen samples, fragments were extracted in vials and the extracts
evaporated on slides just as lichen samples are usually extracted for TLC or microcrystaltests.
To achieve partial separationsby differential solubility, the lichen is extracted first with benzene

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1972] CULBERSON: HIGH-SPEED LIQUID CHROMATOGRAPIIY 57
at room temperatureand then with hot acetone. Samples of pure lichen compounds and crude
residues from lichen fragmentsmust be redissolved in a solvent that does not absorb at 254 nm
before injection onto the column. Although it is best to use the same solvent that is being
employed to elute the column, many lichen compounds had very low solubilities in these mix-
tures. For these slightly soluble compounds better results were obtained using chloroform or
iso-propyl alcohol as injection solvent. The volumes of solution injected ranged commonly
from 1 to 10 and the samples were injected directly onto the column with the carrier liquid
/l, the system.
flowing through

RESULTS AND DISCUSSION

An exploratory study of the usefulness of gas chromatography for the identification of

some secondary lichen products showed that this method could be used successfully
for some complex fatty acids, but that the characteristic aromatic esters, of special inter-
est to lichenologists, could not be chromatographed. Figures 2-3 show gas chromato-
grams of the trimethylsilyl derivative of caperatic acid at 1900 and at 2150. Perhaps a
thermal reaction accounts for the great decrease in retention time and increase in peak
symmetry observed for this relatively small increase in temperature. Under similar con-
ditions, the tricarboxylic acid roccellic acid gives a sharp peak in 14 min. at 1900 and in
5 min. at 2150. But the unknown fatty substance in Cladonia subcariosa Nyl., which
also produces atranorin and norstictic acid (W. L. Culberson, 1969), behaves like
caperatic acid although with shorter retention times.
Bloomer and his coworkers (Bloomer et al., 1970a, b) have detected protolichesterinic
acid by first treating the extract to convert all of the fatty acid to lichesterinic acid
which was then methylated with diazomethane and chromatographed at 2600. Partial
decomposition of the methyl lichesterinate occurred at this high temperature. If proto-
lichesterinic acid was methylated directly, two peaks of about equal size were obtained
by chromatography. Although the method was found to be extremely sensitive and
useful for these biosynthetic studies, used alone it could not distinguish lichesterinic
acid from protolichesterinic acid or the natural occurrence of the methyl esters. The
results of the present study suggest that gas chromatography of trimethylsilyl derivatives
should be a most useful technique for lichen fatty acids.
Attempts to gas chromatograph the typical lichen depsides lecanoric acid and perlatolic
acid failed completely, however, and only decomposition products could be detected
even when the compounds were first converted to their trimethylsilyl derivatives. Silyl
derivatives of some phenolic acid units that can be obtained by hydrolysis of lichen
depsides did chromatograph readily. Figure 4 is a chromatogram prepared by micro-
hydrolysis of evernic acid and silylation of the crude products. The small peaks at 19.0
and 32.0 min. are probably the 2-hydroxylsilyl derivatives since these products decrease
rapidly with time as the peaks at 31.0 and 46.5 min. simultaneously increase. By analogy
with the known behavior of salicylic acid (Choby & Neuworth, 1966), the 2-hydroxyl
would be expected to silylate less rapidly, and the fully derivatized product should be
less volatile than the 2-hydroxytrimethylsilyl esters. Figure 5 illustrates the good sepa-
ration obtainable for homologous units, which suggests that this method would be
useful for studying joint occurrences of homologous depsides.
The results of the gas chromatographic studies are summarized in Table 1. It is
clear that, as expected, most lichen extracts cannot be analyzed directly by gas chroma-
tography since this method does not allow separation of the labile aromatic esters, the
very substances of particular interest in systematics because they include the secondary

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58 THE BRYOLOGIST [Volume 75

2 4
R=CH3

1
6CH3
RO3 s COOSiMe3 RSiMe
R = SiMe3
OSiMe3

CAP

0 10 2 120 130 140 150 0 10 20 30 40 50

3 5

QCOOSiMe3
CH-O OSiMe3

CAP R=C3H7

R=C5H11
R-CsHII

0 10 20 30 40 50 0 10 20 30 40 50
TIME IN MINUTES

FIGURES 2-5. Gas chromatogramsof trimethylsilyl derivatives of the fatty acid caperatic
acid and of phenolic acid hydrolysis products from depsides. - 2. Broadened peak for the
trimethylsilyl derivative of caperatic acid chromatographedat 190t. - 3. Sharp peak for the

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1972] CULBERSON: HIGH-SPEED LIQUID CHROMATOGRAPHY 59

TABLE 1. Retention times for gas chromatographyof some trimethylsilyl derivatives on

a 2-meter column of 3% SE-30 on ChromasorbW at the temperaturesindicated.

Temperature Retention
Trimethylsilyl Derivative (oC) Time (min.)
Caperatic acid 190 143.0
Caperatic acid 215 34.0
Roccellic acid 190 14.0
Roccellic acid 215 5.0
Unidentified substance from C. subcariosa 190 57.0
215 16.0
Evernic acid A ring 130 31.0
Evernic acid B ring 130 46.5
Barbatic acid A ring 130 46.0
Barbatic acid B ring 130 64.5
Divaricatic acid A ring 150 19.0
Perlatolic acid A ring 150 38.0

products unique to lichens. These compounds can be successfully separated by high-

speed liquid chromatography.
High-speed liquid chromatography utilizes small-bore columns (1-3 mm i.d.) packed
with specially designed supports through which a solvent is pumped at a relatively high
speed, compared to conventional gravity-flow or low-pressure column chromatograms.
Pressures of the order of 35-70 kg/cm2 (500-1000 psi) are generally required to
maintain the desired flow rate, and separations of the components of mixtures are
achieved in minutes as compared to hours or days required for normal column chroma-
tography. The need for high pressures and detection of minute amounts of substances as
they leave the column requires some special features in the design of the apparatus.
Figures 6 and 7 are high-speed liquid chromatograms of pure samples of perlatolic
acid and lecanoric acid. The runs were completed in less than 10 minutes. In both
cases the compounds were collected and identified (TLC) as the undecomposed
depsides. All orcinol-type depsides and depsidones tested give excellent peaks as do
usnic acid, the dibenzofuran didymic acid, the anthraquinone ;parietin, and the xanthone
lichexanthone. Even tridepsides like gyrophoric acid and 4-O-methylgyrophoric acid
are readily separated. Similarly, p-orcinol depsides like atranorin, barbatic acid, and
diffractaic acid and the depsidone hypoprotocetraric acid chromatograph very well. The
carrier solvents employed in the present study were less well suited to the analysis of
highly oxidized p-orcinol depsidones, which are much less soluble in organic solvents
and tend to chromatograph too slowly under the present conditions. These initial tests,
made with pure samples of lichen products, established that the typical aromatic lichen
products chromatograph without decomposition. Subsequently, a number of crude
lichen extracts were tested.
Figures 8-10 are high-speed liquid chromatograms of crude lichen extracts. By

trimethylsilyl derivative caperatic acid chromatographedat 215?. - 4. Trimethylsilyl deriva-

tives of the products from a microhydrolysisof evernic acid (T = 130?). - 5. Trimethylsilyl
derivatives of homologous phenolic acid units represented by the perlatolic acid A-ring unit
(R = C5Hll) and the divaricatic acid A-ring unit (R = C3H7) (T -= 1500').

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60 THE BRYOLOGIST [Volume 75

6 7 8 Ev

LEC
PER ATR

LEC

I,

T T
.032 As .032 As I As
.004
I•

0 2 4 6 8 10 0 2 4 0 2 4

9 10

BAR CR

.016As
I I
.008 As .008 As

Us
ATR

DEM

0 2 4 0 2 4 6 8 10 12 14
TIME IN MINUTES

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1972] CULBERSON: HIGH-SPEED LIQUID CHROMATOGRAPHY 61
adjusting the composition of the carrier solvent, reasonable elution times were obtained
for a variety of compounds. The solvent systems containing mixtures of hexane, iso-
propyl alcohol, and acetic acid eluted the compounds in much the same order that they
would be observed if they were chromatographed on SiO2 thin-layer plates with hexane-
ethyl ether-formic acid (5:4:1 v/v). 4-O-Methylated compounds are easily separated
from their corresponding demethyl derivatives with which they so commonly occur.
Thus lecanoric acid is readily distinguished from evernic acid in the extract of Parmelia
taylorensis Mitch. (Fig. 8) and barbatic acid is separated from 4-O-demethylbarbatic
acid in P. physcioides Nyl. (Fig. 9). 4-O-Demethylbarbatic acid has previously been
reported from a single species of Ramrnalina(Huneck et al., 1968), but it also occurs in
many species of Parmelia and Cladonia that produce barbatic acid.
The separation of homologous compounds is more difficult, just as it is by the usual
TLC methods. Figure 10 illustrates the partial separation of the homologous meta-
depsides cryptochlorophaeic and paludosic acids from Ramalina paludosa B. Moore,
completed in just 15 minutes. The original separation of paludosic acid from crypto-
chlorophaeic acid by gravity-flow chromatography took four days (C. F. Culberson,
1967). It seems probable that the great flexibility of solvent systems and column
packings that can be used for high-speed chromatographic separations will provide good
combinations for even the most difficult separations.
Although thin-layer chromatography will doubtless remain the simplest reasonably
reliable method for routine identifications of secondary lichen products, high-speed
liquid chromatography should be a very useful adjunct with applications to the study
of nonvolatile compounds comparable to those of gas chromatography to studies of
volatile ones. High-speed liquid chromatography may be particularly useful for rapid
quantitative measurements of compounds in mixtures and for separations that are diffi-
cult or impossible by TLC.

LITERATURE CITED

BLOOMER, J. L., W. R. EDER&W. F. HOFFMAN. 1970a. Biosynthesisof (+ )-protolichesterinic

acid in Cetraria islandica. Jour. Chem. Soc. (C) 1970: 1848-1850.
- , - & . 1970b. Some problems in lichen metabolism: studies with the
mycobionts Cetrariaislandica and Cladonia papillaria. THE BRYOLOGIST
73: 586-591.
BOMBAUGH, K. J., R. F. LEVANGIE, R. N. KING & L. ABRAHAMS. 1970. Considerations for a
simplified high-performanceliquid chromatograph. Jour. Chromatogr.Sci. 8: 657-663.

FIGURES 6-10. High-speed liquid chromatogramsof lichen products and of crude lichen
extracts. - 6. Lecanoric acid ( Lec): A solution (1041) in iso-propyl alcohol chromatographed
with a flow rate of 1.4 ml/min, of hexane-iso-propylalcohol-acetic acid (100:6:1 v/v). - 7.
Perlatolic acid (Per): A solution (841) in iso-propyl alcohol chromatographedwith a flow
rate of 1.4 ml/min. of hexane-iso-propylalcohol-acetic acid (100:6:1 v/v). - 8. The residue
from an acetone extract of Parmelia taylorensis: A solution (541) in chloroform chromatographed
with a flow rate of 1.2 ml/min. of hexane-iso-propyl alcohol-acetic acid (100:6:4 v/v).
Atranorin(Atr), evernic acid (Ev), and lecanoric acid (Lec) are separated. - 9. The residue
from an acetone extract of P. physcioides: A solution in iso-propyl alcohol chroma-
(2.5/l)
tographed with a flow rate of 1.1 ml/min. of hexane-iso-propyl alcohol-acetic acid (100:4:2
v/v). Atranorin (Atr), barbatic acid (Bar), and 4-O-demethylbarbaticacid (Dem) are sepa-
rated. - 10. The residue from an acetone extract of Ramalinapaludosa: A solution (10,1) in
chloroformchromatographedwith a flow rate of 1.2 ml/min. of hexane-iso-propylalcohol-acetic
acid (100:2:3 v/v). Atranorin (Atr), usnic acid (Us), cryptochlorophaeic acid (Cr), and
paludosic acid (Pal) are separated.

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62 THE BRYOLOGIST [Volume 75

CHOBY, E. G., JR. & M. B. NEUWORTH. 1966. Trimethylsilyl derivatives of salacylic acid.
Jour. Org. Chem. 31: 632-634.
CULBERSON, C. F. 1967. The chemical constituents of Ramalina paludosa. THE BRYOLOGIST
70: 397-405.
& H. KRISTINSSON. 1970. A standardized method for the identification of lichen
products. Jour. Chromatogr.46: 85-93.
CULBERSON, W. L. 1969. The chemistry and systematics of some species of the Cladonia
cariosagroup in North America. TiHEBRYOLOGIST 72: 377-386.
FELTON, H. 1969. Performance of components of a high pressure liquid chromatography
system. Jour. Chromatogr.Sci. 7: 13-16.
HUNECK, S., G. FOLLMANN & J. SANTESSON. 1968. 4-O-Desmethylbarbatins~iure, ein neues
Depsid aus Ramalina subdecipiens Stein. Zeitschr. fiir Naturforsch. 23b: 856-860.
KIRKLAND, J. J. 1969. Techniques for high-performanceliquid-liquid and ion exchange chro-
matography with controlled surface porosity column packings. Jour. Chromatogr. Sci. 7:
361-365.
KLEBE, J. F., H. FINKEINER & D. M. WHITE. 1966. Silylations with bis(trimethylsilyl)acet-
amide, a highly reactive silyl donor. Jour. Amer. Chem. Soc. 88: 3390-3395.
SANTESSON,J. 1969. Chemical studies on lichens. 10. Mass spectrometry of lichens. Ark.
fdr Kemi 30: 363-377.
SHIBATA, S., T. FURUYA & H. IIZUKA. 1965. Gas-liquid chromatography of lichen substances.
I. Studies on zeorin. Chem. Pharm.Bull. (Tokyo) 13: 1254-1257.

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