Colors of Orcein Dyes from Evernia prunastri via AFM

Chemistry and Colors of Orcein Dyes

9-Mar-22 Gevan R Marrs

Background

The basic reactions and chemical structures reported in various sources for creating “orchil” or orcein dyes from lichen acids with an “amonnia fermentation method” (AFM) are remarkably consistent over the last 70 years (for example, see Figures 1-4, listed chronologically).

Figure 1. Musso 1960.

Figure 2. Beecken et al 1961 / 2003.

Figure 3. Upreti et al 2010

Figure 4. Rather et. al. 2018

These diagrams uniformly show the lichen acid depside lecanoric acid hydrolzed to orsellinic acid, which is then decarboxylated to orcinol, which then goes through condensation reactions with oxygen and ammonia to produce orcein (aka orchil). I have previously written about how differing lichen acids, like evernic acid found in Evernia prunastri, can also be converted by this pathway to purple ocein dyes. (Gyrophoric acid  is readily converted to lecanoric acid and orsellinic acid via hydrolysis, so then follows the pathways shown above.)

Here, however, I want to discuss further not the lichen acid precursors, but the orcein dyes that result. 

Orcein

Orcein is not a single compound, but instead a set of related compounds and isomers of certain molecular formulas. Again, there is great consistency in reported compounds found in orcein.  Upreti et al 2010 shows this set of 8 compounds (Figure 5.)

Figure 5. Orcein deriviatives, Upreti et al 2010.

The Wikipedia entry for “orcein” (as of March 2022) shows, without citation, these same 8 compounds.

Figure 6. Wikipedia entry under "orcein", source not cited.

A website called “StainsFile” – with information on orcein stains for biochemistry, shows the same 8 compounds (without citation of source). Figure 7.

Figure 7. StainsFile orcein "homologues".

But in my digging into the historical source of elucidation of these structures, it seems that key was the work of Musso et al in the 1950’s, published as a host of papers under his authorship (with co-authors sometimes), but assembled and published together (in German) in 1961, Beecken et al. This work was later translated into English, and re-published in 2003. The work is ground-breaking (IMO) as they not only use a host of methods to deduce the structure of orcein compounds, they also verify their hypotheses by synthesizing the compounds from precursors. While they do not publish a single figure of the 8 compounds shown above, as good chemists they describe the structures:

Figure 8. Eight orcein compounds (3 chemical formulas, 2 with 3 isomers and 1 with 2 = 8 compounds) described by Beecken et. al. 1961 / 2003.

But Beecken et al 1961/2003 also provide a very useful table of these compounds, including two other very interesting types of information. Figure 9 shows this table, and it includes 1) The quantitative proportions of different compounds, at least in their sample of orcein (for which I can find no mention of the source), and 2) the colors of solutions of these different compound groups, as well as how that shifts with pH.

Figure 9. Table 1 from Beecken et al - quantities and colors of orcein compounds.

Note in their Table 1 that the same 8 compounds (for main components) are named as in sources shown in Figures 5-7 above. I believe this is because later researchers used this fundamental work as correct. But the important interpretations for our craft dyeing work are as follows.

Quantitatively, about 85% of the orcein compounds give the classic orhil dye “purple” aka violet or deep violet in alkaline pH, but shift to red tones in acidic pH. But the other 15% are different, giving “red-brown” at alkaline pH, and “deep-violet” in acidic pH.

In my experience to date with AFM dyes from Evernia prunastri, I do not get the “classic” purple-to-red shift as pH is lowered.

My Hypotheses

It seems to me that given the different colors produced by orcein Groups 1 & 2 above, compared to Group 3, that if the proportions of differing groups (or even in the un-named “secondary components) varied, then the resulting dye colors may well be different, and accordingly any color shifts from pH adjustment might be different. 

I have previously written about how there are different lichen acid precursors in Evernia prunastri compared to classic orchil dyes (Rocella, Umbilicaria, etc.) – it seems plausible that any resulting orcein dyes from Evernia prunastri could have a different mixture of actual orcein compounds compared to historical orchil lichen dyes.

When I tested wool dye samples at varying dye bath pH levels with a 45-day AFM ferment of Evernia prunastri I found relatively little color shift in the range of 9 to 10.2, but quite a shift to gray at very high pH (13, along with severe fiber damage…). At lower pH values (5.5 and 4) I see a shift to orangish-brown tones. This might match most closely to Group 2 behavior shown in Figure 9. But with nearly opposite color shifts for Groups 2 and 3, the specific mix of orcein compounds may make the pH color shift unpredictable.

Figure 10. Changes in wool dye sample colors with pH for 45-day Evernia prunastri AFM dye.

My Conclusions

I think that any purplish orcein dyes created from AFM of Evernia prunastri may well contain a different mixture of the specific orcein derivitives, compared to historically prominent purple orchil lichen dyes. Accordingly, it is not surprising that the dyeing results with changes in pH for Evernia prunastri AFM dyes differs from that typically reported for orchil dyes (purple at high pH shifting to red at low pH.)

References

1960 Musso, H (1960). "Orcein- und Lackmusfarbstoffe: Konstitutionsermittlung und Konstitutionsbeweis durch die Synthese. (Orcein and litmus pigments: constitutional elucidation and constitutional proof by synthesis.)". Planta Medica. 8 (4): 431–446. doi:10.1055/s-0028-1101580.

1961 / 2003 H Beecken, E-M Gottschalk, U v Gizycki, H Kr mer, D Maassen, H-G Matthies, H Musso, C Rathjen & Ui Zdhorszky (2003) Orcein and Litmus, Biotechnic & Histochemistry, 78:6, 289-302, DOI: 10.1080/10520290410001671362

2010 Upreti, D.K., S. Joshi and S. Nayaka. 2010. Chemistry of common dye yielding lichens of India. ENVIS Forestr Bull., 10 (1): 122-133.

2018 Luqman Jameel Rather*, Salman Jameel, Showkat Ali Ganie and Khursheed Ahmad Bhat, Lichen Derived Natural Colorants: History, Extraction, and Applications, Handbook of Renewable Materials for Coloration & Finishing pp. 102-114, Scrivner.

 

Chemistry of Evernia prunastri depsides and conversion to orcein dyes

 Lichen Acids Chemical Structure and Conversion to Orsellinic Acid

5-Mar-22 Gevan R Marrs

Background

Each of the three main 3 articles I have that show the 3 reactions converting lichen depsides to orsellinic acid, then on to orsinol and finally orcein dye compounds portray the starting depside as lecanoric acid. Lecanoric acid is mentioned as a common acid found in those lichens well-known for orchil dyes, purples and reds, and a C+R spot test reaction. Since Evernia prunastri does not contain lecanoric acid, but three others, I wanted to compare structures lichen acids of known “orchil productive” lichens with Evernia prunastri lichen acids to see if I can deduce how to facilitate purple dyes from Evernia.

Approach

I surveyed various sources to find the chemical structures of the main dye producing lichen acids, then sought references to specifici lichen species (or genera) that contained those lichen acids. Then I tried to speculate and/or find information to support ways in which Evernia lichen acid(s) can create orcein. The Appendix shows the full collected set of data.

Data

Lecanoric acid – C+R, well-known to create orsellinic acid, then orcinol leading to orcein – pH sensitive red and purple dyes.

Found in: Ochrolecia Rocellichen acid Parmelia (some) Punctelia rudecta Usnea subclavata Parmotrema (some) Umbilicaria (some spp. incl mammulata, americana and deusta) Flavopunctilia soredica

Gyrophoric acid – C+R, well-known to create orcin leading to orcein – pH sensitive red and purple dyes

Found in: Ochrolecia Lasillia Umbilicaria (some spp incl mammulata, americana and deusta) Xanthoparmelia

Lichen Acids in Evernia prunastri

Evernic acid – C-, not shown (that I have found yet) creating orcinol leading to orcein – NOT pH sensitive pinkish to purplish-brown dyes.

Found in: Evernia prunastri

Note that Evernic acid is Lecanoric acid with a methoxy group replacing hydroxy (O-CH₃ replacing O-H) on terminal ring.

Usnic acid – C-,– Brown and some Yellow dyes

Found in: Evernia prunastri Usnea Cladonia Lecanora Ramalina farinacea and R. pollinaria Parmelia Lobelia oregana, pr pulmnaria Flavoparmelia caperata (brown dye) Flavoparmelia soredica (brown dye)

Not obvious to me how this would readily generate Orsellinic acid. I don’t think any of the other listed species (e.g., Usnea spp.) generate reds or purples with AFM, so this doesn’t seem to be likely a source of purplish dyes via AFM for Evernia prunastri.

Physodic acid – KC+R to O -  ? dyes

Found in: Evernia prunastri

I’m really unclear how the carbon chains would be stripped to give a kind of depsidone structure….

Reaction comparisons for different depsides

Classic depsides -> orcin reactions are Lecanoric acid hydrolysis to two Orsellenic acid molecules, these are then decarboxylated to 2 Orcinol molecules. For example, Rather et al 2019 shows:

Conclusions

So it seems to me that:

1.     Lecanoric acid is the prototypical, successful precursor to the classic purple and red orchil dyes.

2.     Gyrophoric acid can have one ring hydrolyzed off the tri-cyclic depside, creating one molecule Orsellinic acid and leaving one molecule Lecanoric, which can then hydrolze to two more Orsellinic acid molecules. So in a sense Gyrophoric acid is like “super-Lecanoric”. Upreti et al 2010 says explicitly that this happens. (Page 126). 

3.     I don’t see why one cannot split off via hydrolysis on Evernic acid one molecule of Orsellinic acid, leaving one with ? (methoxy-Orsellinic acid[1]? Oh, it is called Everninic acid per Upreti 2010). Kind of a “half-hearted” Lecanoric acid, as it only produces one molecule orsellinic acid on first hydrolysis. I’m not sure what can transpire if the everninic acid is decarboxylated. Oh - The Wikipedia entry for Orsellinic acid states: “This (orsellinc acid) is also produced when everninic acid and ramalic acid is boiled with barium hydroxide.” Upreti et al 2010 also states that indeed this happens (p. 126). They show:

By chance a reference was recently posted (Mosbach and Schultz 1971) that states specifically there is an enzyme that does convert everninic acid to orsellinic acid! “

“Orsellinate decarboxylase, catalyzing the decarboxylation of orsellinic acid (2,4-dihydroxy. 6-methyl benzoic acid) to orcinol has been isolated from the lichen Lasallia prustulata. The substrate specificity of the enzyme has been tested: only orsellinic acid, 3-chloro-, 5-chloro. orsellinic acids, homo-orsellinic acid (2,4-dihydroxy-6-ethyl benzoic acid) and everninic acid (4-methoxy-2-hydroxy-6-methyl benzoie acid) were decarboxylated.”

This does not necessariliy mean that the conditions of our AFM is an enzymatic decarboxylation, but it means the reaction can occur, so could go on to produce orcinol and then orcein.

My hypothesis is that the relative difficulty that craft dyers seem to have with Evernia AFM compared to C+R lichens may well be the added difficulty of achieving conversio of everninic acid to orsellic acid.

References

1971 Klaus Mosbach and Joachim Schultz, Studies on Lichen Enzymes Purification and Properties of Orsellinate Decarboxylase Obtained from Lasallia pustulata, Avdelning för Biokemi, Kemicentrum, Lunds Universitet (Received March 18/ July 22, 1971)

2018 Luqman Jameel Rather*, Salman Jameel, Showkat Ali Ganie and Khursheed Ahmad Bhat, Lichen Derived Natural Colorants: History, Extraction, and Applications, Handbook of Renewable Materials for Coloration & Finishing pp. 102-114, Scrivner.

2010 Upreti, D.K., S. Joshi and S. Nayaka. 2010. Chemistry of common dye yielding lichens of India. ENVIS Forestr Bull., 10 (1): 122-133.

Full PDF of this article.

Appendix Database of Lichen chemical precursors and dye colors by AFM (yellow highlights are species accessible to me)

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[1] everninic acid ; Synonyms: everninic acid;orsellinic acid 4-methyl ether ; 

 

[2] Upreti 2010 and others show Orcinol as a hydrolysis product of a depside precursor in lichen. Whether Shaheen reference suggests detection after hydrolysis of a precursor is unknown. Since the lichen genera cited are lichens with either lecanoric or gyrophric acid, it seems they (Shaheen et al 2019) may be describing an intermediate. Orcinol is clearly a precursor to ammonia-fostered production of orchil / arkil / orcein purple dyes.

Gevan’s Ammonia Method For Developing Lichen Dye from Evernia prunastri

 

Gevan’s Ammonia Method

For Developing Lichen Dye from Evernia prunastri

Last updated 16-Feb-2022 Gevan R Marrs Revision 1

Overview

This “recipe” is intended to provide sufficient dye liquid to dye 50g wool to a medium to dark shade (depending on dyeing time and temperature) within a reasonable time (1 to 6 hours) and temperature (130 to 180°F), all within a reasonable dye development time (4-6 weeks). All quantities can be adjusted scale for different amounts of wool, or different starting weights of Evernia prunastri.

Ingredients

·      50 g of dry Evernia prunastri

o   If it is fresh (wet) use double the weight.

o   Insure it is 90% or more Evernia prunastri and not the “looks-similar Ramalina farinacea

·      500 ml (~2 cups) Household Ammonia solution

o   This quantity assumes that your “household ammonia” is typical and somewhere between 2%  and 4% ammonia. If you suspect or know it is something different, use the table below to adjust ammonia:water proportion to get correct total dye solution and ammonia %.

o   Make sure there is good ammonia content, either by ph paper, ph meter, specific gravity, or…smell test (just be careful about sniffing ammonia vapors).

·      500 ml (~2 cups)  ml water

o   Tap water probably OK, if in doubt buy distilled water– it’s cheap.

Method

1.     Use a glass jar with a snug-fitting lid (think canning jars) with a capacity of  2000 ml (2L) (that is, 2 quart). 

2.     Place weighed amount of lichen in jar and “smoosh” it down if it is dried to better allow liquid to cover lichen. (I would NOT grind the lichen – it diminishes access to chemicals in the early stages.) 

3.     Measure out volume of water (first) and pour over lichen.

4.     Then measure out ammonia solution. Do this without delay[1], keeping the jug closed as much as possible, and quickly add the ammonia solution to the dye jar. Cover the jar without delay.

5.     Shake it well. It is helpful to shake it every couple hours for the first day, and perhaps daily after (but I don’t think that is crucial after a couple days).

6.     I would NOT open the jar to give it fresh air. I believe the loss of ammonia is worse that any limited oxygen. 

7.     Keep the jar in a warm location if feasible – 68-70°F. The trouble of a warm-water bath (say 90°F) is not worth the minor gain in dye development time. Cooler probably slows it, but above 50°F probably still goes OK.

I have no evidence that sunlight is useful or needed. Mine have developed in near darkness and seem just fine. A sunny location may favor warmth as cited in 7 above.

Expectations – is it progressing?

You should see, within hours, the dye development solution turning a rich brown. If it is not, go to troubleshooting. With about 10-15 days you should see considerable reddish-brown developing, progressing to dark purplish (aka “grape juice” stage), and eventually on to almost black-purple so dark you cannot see into the jar.

Scaling the recipe (note these don’t need to be exact, so conversions are rounded)

Weight of wool to dye, grams (WOF 1:1)	Weight of Lichen to use, dry, grams / ounces	Weight of lichen to use if wet, grams / onces	Needed size of jar for dye development	Amount of 2%-4% HH ammonia solution, ml / cups	Amount of water, ml / cups
50 g / 1.75 oz	50 g / 1.75 oz	100 g / 3.5 oz	2 liter / 2 quart	500 ml / 2 cups	500 ml / 2 cups

If you want to use a smaller jar (1 liter or 1 quart), cut the numbers above in half. If you want a gallon (4 liters) double the values above.

Adjusting the ammonia:water amounts if different ammonia strength

The target is to have 1 to 2% ammonia in the dye development solution. Here are a few amounts of ammonia solution and water to get the target total 1 liter (1 quart) of dye development solution.

Jug ammonia %	3% (2-4%)	5% (4-6%)	10%	25% (gasp)
Proportion of water:Ammonia	1:1	7:3	8.5:1.5	94:6
mls water	500 ml or 2 cups	700 mls or 3 cups	850 mls or 3.5 cups	940 mls or 4 cups
mls jug ammonia	500 ml or 2 cups	300 mls or 1 1/3 cup	150 ml or 2/3 cup	60 mls or ¼ cup

 

Figure 1. Example of what a 2-quart dye development bath should look like for proportions. This solution is just one hour old. The pH should be above 10.5 or there is likely a problem with ammonia.

The next day the solution should be quite dark brown:

Successful Dye

Reddish tones should start to develop with 7-10 days, turning to grape-juice purplish within 15 to 20 days.. Here’s what my best dye (so far) looked like after 19 days.

Troubleshooting

My dye development solution is not turning brown with the first day or so. What’s wrong?

I would suspect first that maybe it’s not Evernia prunastri, but Ramalina farinacea instead. Study how to distinguish them. 

Secondly I would suspect ammonia too low (which will mean pH too low as well). Open the jar and fan air from above the jar to your nose[2]. If you don’t smell ammonia it means you had weak household ammonia, or it was lost somehow. I would do the fan-and-sniff test on jugs of ammonia until you find one that smells strong. Pour off the old dye development solution and re-dose (but only if it hasn’t yet darkened – otherwise you’re pouring off extracted dye precursors.)

My dye development turned brown but is not developing red tones even after a couple weeks. What’s wrong?

I would expect loss of ammonia from too much exposure. If you can check pH, it should still be above 11 or it needs re-dosing with straight ammonia solution. Leave headspace for air, even if you have to up-size to a bigger container. This might salvage a “stuck” development bath.

Glossary

Dye Development – the process by which dye precursors from the lichen are extracted and converted to orcein dyes by soaking in an ammonia solution.

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[1] Ammonia gas evaporates “quickly” from open containers – like within an hour at 68°F. So it’s not like seconds, but you should strive to keep that ammonia in the jar.

[2] Caution! Ammonia gas is very soluble in aqueous solutions, like in your eyes and respiratory tract. Never stick your nose right over a container of ammonia solution – always carefully fan air to your nose to minimize exposure.

Gevan's AFM Recipe for Evernia on Google drive (for pdf download).

Oxygen Role in AFM Lichen Dyes

 Oxygen in the AFM for Lichen Dyes

Background

·      Assume that the reactions shown by Upreti et al 2010 are correct. That is:

·      Reaction 3 (Rx 3) requires both Ammonium hydroxide and Oxygen, that is, O2 dissolved in the dye solution.

·      O2 is not very soluble in water – only about 9 mg/L at 20°C. That is about 0.009% (much lower than the NH4OH likely present)

·      Accordingly, increasing O2 in solution should help.

·      This suggests leaving significant “head space” – air inside the AFM vessel.

o   Upreti et al 2010, for example, specifies only filling the vessel 1/3 with liquid, perhaps for this reason.

·      Many anecdotal suggestions for AFM specify:

o    daily shaking of solution in order to assist O2 dissolution. It may help – certainly can’t hurt.

o   Removal of the lid occasionally to allow new air / O2 into the head space. Makes sense if the O2 is being depleted.

Oxygen Tracking

A “thought experiment” may suggest the relative importance of shaking, opening, or bubbling air through AFM solution.

·      Assume you have a 2L container. Add 1L distilled H2O at 20°C. Close container and agitate for “a while” until equilibrium solubility is achieved.

o   At that point there should be about 9 mg of O2 in the water.

o   In the headspace prior to dissolution there should have been 1L of air

§  Air is about 21% O2 and 78% N2 by volume. Since the molecular weights of each are similar, say there is 20% O2 by mass.

§  Air weight per liter according to web calcs, is about 1.225 g, or 1,225 mg. If 20% is O2 by weight, then it contained 245 mg of O2. 

·      Therefore….less than 4% of the O2 in the original 1L of air in the jar went into solution at equilibrium. That is, 96% is still in the headspace. Until that is consumed by the reaction, no more O2 would be depleted from the headspace air. 

My Conclusion

Opening a jar daily to allow “fresh air” with O2 is not really necessary to keep O2 in solution. On the other hand, NH3 in solution is quite volatile (hence the smell when the jar is open) and daily opening allows gaseous NH3 to escape, and eventually diminishes the NH3 in solution (although by my thinking we have a considerable excess of NH3, compared to oxygen, so probably not an issue.)

In my experiment I have two open jar treatments – one with bubbling air. I will try to detect a diminishment of NH3 (by lack of odor) and may supplement additional NH3 at some point.