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ANALYSIS OF LICHEN SUBSTANCES

Overview | Spot Tests | Common Spots | TLC Overview | TLC Procedure | Literature


OVERVIEW
Lichenologists have been interested in identification of lichen substances ever since the Swedish lichenologist NYLANDER (1867) found that morphologically similar lichens could easily be distinguished with a simple spot test of KOH or hypochlorite solution. Soon other substances were discovered which gave useful spot test reactions. Spot test reactions are still commonly used in lichen identification because they are very simple and inexpensive. 

The color reactions indicate the presence of closely related substances in a lichen thallus. They are, however, not very specific and it is usually not possible to identify lichen substances only with spot tests.

Microcrystallization techniques, the controlled re-crystallization of extracted lichen substances and observation of these crystals under the microscope, soon became a means to distinguish substances more accurately. The technique was were very popular before thin layer chromatography (TLC) became more readily available. TLC is a relatively simple and inexpensive technique and today it is probably the most widely applied method. TLC allows to identify almost any lichen substance, however, it is not especially sensitive to low concentrated substances which may frequently not show on a TLC-plate. In addition to TLC high performance liquid chromatography (HPLC) thus became another, often routinely employed and highly sensitive technique. It allows not only to record the presence of a substance but also its concentration. 

Although these methods allow to distinguish between specifically between various lichen substances, they do not reveal any aspects of the molecular structure of a substance. Lichen chemists have used a wide array of methods such as mass spectrometry, x-ray analysis and various spectroscopy techniques to investigate molecular structures of lichen substances. These techniques are quite elaborate and not necessary for species identification. However, with knowledge of the molecular structure and the study of biochemical pathways it becomes more and more evident which substances are closely related and thus often derived from simple changes. This information is obviously important to evaluate taxonomic value of a lichen substance.

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SPOT TEST

General | Recipes | Procedure |
Common Spots

 [back to overview]

Spot test reactions are a quick and inexpensive way to screen for lichen substances. However, concentrations of secondary metabolites in a lichen thallus may vary considerably and are thus not always revealed with a simple spot test. If expected color reactions fail it is always sensible to test if the reagent still reacts result with a test specimen. Old spot test solutions become unreliable and should be replaced. If in doubt whether a certain substance is present more sensitive techniques such as TLC need to be employed.

The most commonly used spot test reagents are Potassium hydroxide (KOH or simply K), Sodium Hypochlorite (commonly household chlorine bleach, C) and p-phenyldiamine (P or sometimes abreviated Pd). KOH is a strong base and should therefore used with caution. Chlorine is highly reactive with most organic and inorganic molecules and even though it is still widely used to bleach paper or clothes it should nevertheless also be handled with caution. p-Phenyldiamine was widely used to dye hair and develop photographs, however, it soon was replaced when tested on animals where it indicated to cause cancer.

[Spot Tests]

Other less frequently applied reagents are iodine and nitric acid. Iodine reacts blue with the starch-like polysaccharide isolichenin and in some species an I+ blue medulla can be diagnostic. Iodine is also routinely used to study the ascus apical structure an important character in delimitation of lichen families and genera. Nitric acid is commonly used to detect specific pigments in the margin of apothecia.

Spot Test Recipes

K: A 10% solution can be made by dissolving 70g potassium hydroxide pellets in 200 ml water. The solution stores almost indefinitely.

C: A saturated solution of freshly dissolved Calcium or Sodium Hypochlorite in water will remain reactive for less than a day. Commercially available bleach has a preservative and therefore usually can be stored a few months.

P: A solution of P can easily be made by dissolving p-phenyldiamine crystals in a minute amount of ethanol. This is usually the safest way to handle the carcinogenic substance. If one is less concerned the following recipe will give a rather stable solution which will last a few months: 
Steiner's Solution: Dissolve 1g p-phenyldiamine, 10g sodium sulfite in 100 ml water with 2 ml detergent (any commercially available dishwashing liquid will do).

I: Reactions with iodine vary quite considerably with the concentration. Without pre-treatment with K one might observe a reddish rather than a blue reaction. A reasonable concentration for routine studies is the follow recipe:

Lugol's Iodine: Dissolve 0.5 g iodine crystals with 1.5 g potassium iodine in 100 ml water.
N 50% Nitric acid. Extremely strong acid, handle with care!!!

[Spot Tests]

Spot test procedures

If you are concerned about the preservation of your herbarium specimens and especially the paper packets you should not use spot tests directly on the lichen. 

1. Remove a small piece of lichen from your specimen.

2. Place the sample on a white filter paper.

3. Add a minute amount of spot test reagent with a very thin capillary tube or a dissecting needle.

4. Observe color changes quickly, but check again later: 

 C commonly gives a pink to bright red, very rarely a copper-green color reaction. It usually reacts very fast and soon fades, a good way to check again is to apply K first and then quickly to ad C. This test is usually referred to as KC and mostly gives you a stronger, though sometimes quite different color reaction (e.g. Pertusaria amara reacts C+ reddish and KC+ violet). 

CK the application of first C and then K gives usually very different results. It is, however, rarely used.

[Spot Tests]

K mostly gives a deep yellow to dark red reaction. Another common color reaction is K+ purple. A very weak reddish or faintly brownish reaction is usually regarded as K-. K usually reacts much slower than C and often it is also indicative whether the reaction is K+ yellow without changing to red or whether you'll eventually get a red color reaction. So - wait and see!

P (or Pd) commonly gives a yellow, orange to red color. According to the concentration of the substance in the thallus and the age of the reagent it will take some time for a positive reaction to develop.

5. Discard the tested sample and remember to take precise notes which color reactions you observed! Annotate your specimens!

[Spot Tests]


COMMON SPOT TEST REACTIONS

Spot test reactions of common lichen substances can be found here.

THIN LAYER CHROMATOGRAPHY

General | Recipes | Procedure | Literature

 [back to spot tests]
[back to overview]

General

In thin layer chromatography lichen substances are first extracted from a small sample of the lichen specimen. This extract is then spotted on a glass or aluminum plate coated with amorphous silica gel. Different solvent systems are applied to "run" the plate in a sealed glass tank. The plate is placed in a tank which contains a small amount of solvent. Capillary action draws the solvent through the amorphous silica coating. The lichen substances are carried along according to their affinity to the various solvents. When the solvent front on the plate has reached a certain height plates are removed from the tank and left to dry. The solvent evaporates and some of the lichen substances may already be visible as faint spots. Concentrated sulphuric acid is then applied to the plate. The plate is left to dry again and afterwards placed in a laboratory oven. At 110°C characteristic color reactions of the lichen substances with the sulphuric acid will develop. Many spots which might previously not have been visible will now show up.

[TLC]

There are several diagnostic features which will help to reveal the identity of the spots, i.e. the lichen substances. First of all the distance traveled in a particular solvent is characteristic for each substance. The distance traveled by a substance can be expressed as the Rf-value (retention value). Absolute values may vary considerably according to slight variation in experimental setup. One common method to overcome this variation is to divide the TLC plates into classes of Rf-values. Control specimens which contain substances with well known Rf-values like norstictic acid and atranorin are thus used to "calibrate" the plates (see Fig. 1 & TLC-reference plate). Another way to overcome experimental variation is the calculation of relative rather than absolute Rf-values:

Rf-values are characteristic not only for the specific lichen substances but they also differ according to the solvent used. Atranorin for example has a relative Rf-value of 75 in solvent A but a value of 78 in solvent B' and 79 in solvent C. Norstictic acid has a relative Rf-value of 40 in solvent A but a value of 32 in solvent B' and 30 in solvent C. A standardized procedure to distinguish lichen substances would therefore not only use one but preferably two or even three different solvents.

Apart from Rf-values, color is an important diagnostic character. The color of the substances usually is considerably different under visible light and UV-light. Many lichen substances have a very characteristic fluorescence under UV-light. 

[TLC]

A few substances will already show up on a TLC plate before treatment with sulphuric acid ("charring"), in fact almost all substances are visible as dark spots under short wavelength UV light. Under long wavelength UV-light some of these substances may show a characteristic fluorescence which may change drastically after treatment with sulphuric acid. 

Recipes for various solvents

A toluene / dioxane / acetic acid (180 : 45 : 5)

Dioxane in solvent A is hygroscopic and absorbs water over time. The solvent thus rapidly deteriorates and accurate Rf-values can only be recorded with relatively fresh solvent.

 

B hexane / diethyl ether / formic acid (130 : 80 : 20)

This solvent has largely been replaced by B'. B will deteriorate in less than 6 hours whereas B' can be used up to four or five days.

 

B' hexane / methyl tert-butlyl ether / formic acid (140 : 72 : 18)

 

C toluene / acetic acid (170 : 30)

For most lichen substances C provides the best discrimination. It is also very stable (several weeks) and therefore is the most common solvent routinely used.

Solvents E and G are mostly used only to discriminate substances which will otherwise not be resolved very well:

 

E cyclohexane / ethyl acetate (75 : 25)

Solvent E discriminates between non-polar derivates of lichen compounds and substances which have very high Rf-values in A, B, B' and C. It needs to be prepared fresh daily.

[TLC]

 

G toluene / ethyl acetate / formic acid (139 : 83 : 8)

Solvent G is very stable and discriminates well between substances with very low Rf-values in A, B, B' and C.


THIN LAYER CHROMATOGRAPHY  

- Procedure -

Preparation | Extracting | Spotting | Running | 1st Examination | Sulphuric Acid

2nd Examination | Final Evaluation | Tip

[back to spot tests]
[back to TLC overview]

[back to general overview]

Preparation of the plates: Use only soft pencil !!!

 1) Draw a line two cm above the bottom of the plate. This lower line represents the "start line". You will spot the extracts of lichen substrates on this line.

2) Tick this line in 1 cm distances and number each tick from 1 to 19. Number 2 and 18 will be your controls, thus write C underneath.

3) Draw a second line across 12 cm above the lower line. This upper line marks the solvent front, i.e. how far you will let the solvent run up the plate.
4) Mark the plate above the solvent front line with a plate number, date, your name and the solvent system you are going to use.

Extracting Lichen Substances:

 

1) Label nineteen Eppendorf micro centrifuge tubes (1-19). Mark # 2 and # 18 also with C to indicate that they are your control substances.

2) Place the Eppendorf micro centrifuge tubes into a rack. 

3) Fill out the TLC record sheet and mark your specimen packets:

- Write plate #, solvent system and date on top of the form.

- write specimen # and preliminary identification (if any) into the rows indicating each specimen.

 

[TLC Procedure]

 

- Write the tube numbers, date and solvent system you are using on the specimen packets from which you are going to take a sample

4) Carefully remove a small sample of your lichen (with forceps or scratch a sample of the rock substrate) and place it into the labeled tube. If you have several thalli growing in close proximity be extremely cautions not to include even tiny thallus fragments from adjacent thalli!!!

5) Place a tiny amount (one or two drops) of acetone in each sample tube. Your specimen should barely be covered with acetone.

Spotting Extracts on the Plates:

For each lichen sample...

After two to three minutes you can use a capillary tube to spot your acetone extract onto the TLC plate: 

- Just immerse the capillary tube into the sample tube. Acetone extract will be drawn into the tube by capillary action.

- Carefully spot the capillary tube onto the mark on your TLC plate. Don't let too much extract be drawn onto the plate. The smaller your spots on the plate are the less likely they will float into each other when they run up the plate.

- Repeat the procedure until you can clearly distinguish a spot of lichen substance on the plate

 

[TLC Procedure]

 

Running the Plates:

1) Smear a very small amount of Silicone Grease along the rim of the tank to make an airtight seal.

2) Fill the solvent glass tank with your solvent about ½ cm.
[If you are using solvent B or C, the plates will show more consistent results if they were primed first. For this procedure insert your plate into a TLC tank saturated with 60% formic acid for 5 minutes (solvent B) or in 60% glacial acetic acid for 10 minutes (solvent C). During this procedure the plates must not get into direct contact with the liquid formic or glacial acetic acid. Place them onto some support above the level of the acids. The vapor in the sealed tank will thus evenly penetrate the silica on the plate.]

3) Carefully insert the plate and cover the tank with the lid.
4) Apply a little pressure to seal the lid tightly onto the tank.

5) The plate will run between 12 to 15 min until the solvent front reaches the top line.

6) Take out the plate. Don't let the front run over the top line.

7) Let the plates dry (solvent evaporate).

 

1st Examination of the Plates:

1) Spray with water to make fatty acids visible, circle and cross fatty acid spots.

2) Let the plate dry.

3) Examine the plate under visible light and circle each spot you can see. Mark the color.

4) Examine the plate under long wave UV and underline each spot. Mark any conspicuous color (e.g. UV+ bright white).

5) Examine the plate under short wave UV and indicate each spot with parentheses.

 

[TLC Procedure]

 

Treatment with conc. Sulphuric Acid & "Charring":

1) Brush the plates with conc. Sulphuric Acid and let them dry.

2) Place the plates into the oven at 110°C for approximately 8 min.

3) Check color development. The color should appear bright and well developed but not burned.

4) Take the plate out and let it cool down.

2nd Examination of the Plates:

1) Examine the plate under visible light and observe the color.

2) Examine the plate under long wave UV and indicate conspicuous colors, halos and quenching.

Fig. 1: The final TLC Plate
Final evaluation of your information:

With all the information you can now start to find out which substances you have. The form I have provided for you allows you to enter which spots you found on each Rf-class. Thus you will have to divide your plate into Rf-classes according to your control substances atranorin and norstictic acid. You can then compare all your information with the table from WHITE & JAMES (1985) to figure out which spot represents which substance.

[TLC Procedure]

Another way to evaluate the information is to measure, calculate and record the relative Rf-values for each spot. If entered into the program WINTABOLITES (download here) (Fig.2) you can then use these Rf-values together with your data on visibility before charring, color in visible and UV-light to let the program generate some suggestions on possible substances. However, the program is quite sensitive to what information you enter and thus not always generates good suggestions. A very valuable function however is the huge database packed with data on lichen substances. You can access this database under "Data - View Record" (Fig. 3).


Fig. 2: WINTABOLITES Input Form
Fig. 3: The WINTABOLITES Database ("Data -View Record")

Tip:

With time colors of the spots on the TLC-plates will slowly fade. A good way to store them for some time is to put them into conventional polyethylene sheet protectors. This also allows you to touch the plates without getting into contact with sulphuric acid. The strong acid in the plates can easily diffuse into paper and slowly "eat" it away. Within the sheet protectors you can also scan in the plates for a more permanent records of the colors.

[TLC Procedure]

Store the Eppendorf micro centrifuge tubes with your lichen specimen. Then you can repeat your TLC analysis with different solvents at another time.

Once analyzed specimens should be annotated with the information about the Substances found. Place an annotation slip into the packet, e.g.:


[TLC Procedure]


LITERATURE

[back to spot tests]
[back to TLC overview]

[back to general overview]

Historic Reference:

Nylander, W. (1867): Hypochlorite of lime and hydrate of potash, two new criteria in the study of lichens. - Linnean Society's Journal of Botany 9: 357-365.

General Methodology:

WHITE, F.J. & JAMES, P.W. (1985): New Guide to microchemical techniques for the identification of lichen substances. - Bulletin. British Lichen Society 57: 41. 

CULBERSON, C. F. & KRISTINSSON, H.-D. (1970): A standardized method for the identification of lichen products. - Journal of Chromatography 46: 85-93

CULBERSON, C.F. & JOHNSON, A. (1982): Substitution of methyl tert.-butyl ether for diethyl ether in the standardized thin-layer chromatic method for lichen products. - Journal of Chromatography 238: 483-487
HUNECK & YOSHIMURA (1996): Identification of Lichen Substances. - Springer, Heidelberg, 492 pp.

An excellent update to the White & James Guide to microchemical methods is the following reference:

ORANGE, A., JAMES, P.W. & WHITE, F.J. (2001): Microchemical methods for the identification of lichens. - British Lichen Society. 101 pp.

You can buy this guide directly from the British Lichen Society.



[TLC Literature]

The following books compile most substances so far known to occur in lichens:

CULBERSON, C.F. (1969): Chemical and botanical guide to lichen products. - Chapel Hill (University of North Carolina Press) 1-628

CULBERSON, C.F. (1976): Supplement to chemical and botanical guide to lichen products. - The Bryologist 73: 177-377

CULBERSON, C.F.; CULBERSON, W.L. & JOHNSON, A. (1977): Second supplement to chemical and botanical guide to lichen products. - St. Louis (Missouri Botanical Garden) 1-400 p.

A computer program to interpret TLC-plates:

MIETZSCH E., LUMBSCH, H.T. & ELIX, J.E. (1994): WINTABOLITES (Mactabolites for Windows). - Users manual and computer program, 2nd ed. (Universität Essen) 54p.

[TLC Literature]


© Frank Bungartz, 2001 - Lichen Herbarium, Arizona State University

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