The G.E. reagent is 3:1 acetic acid to glycerol

24 July 2023

Recently I've been trying microcrystallization, a technique for identifying lichen chemicals, where extracts of lichen chemicals are crystallized using various reagents. Once applied, the crystals are allowed to form (think a "crystal growing kit" from your childhood, but much smaller!) and then are viewed under high powered microscope (usually 100-400x).

Hypogymnia inactiva extract; G.A.W. (1:1:1 = glycerol/ethanol/water by volume). The pretty colors come from polarized light filters placed perpendicular to each other before and after the sample ("cross polarization"); the only light that is visible in this configuration is that which has been diffracted by the crystals.

3:1 or 1:3?

One of the most common reagents is G.E., which is made up of glycerol (a.k.a. glycerin) and glacial acetic acid. When reading about the G.E. reagent I noticed a confusing discrepancy; some sources gave it as 3:1 glycerol:acetic acid, and some as 3:1 acetic acid:glycerol. After some searching, the earliest reference to the G.E. reagent I could find was in a paper by Yasuhiko Asahina, who pioneered the technique in general, in 1936 (Mikrochemischer Nachweis der Flechtenstoffe II). This paper says G.E. is 3:1 acetic acid:glycerol, which, as far as I can tell, is the correct formula, and the one lichenologists used.

Excerpt from Asahina's 1936 article "Mikrochemischer Nachweis der Flechtenstoffe II", introducing the G.E. reagent as 3 parts acetic acid and 1 part glycerin.

I believe the 1996 book Identification of Lichen Substances by Siegfried Huneck and Isao Yoshimura to be the earliest example of this error. Another book with the error, from 2001, Microchemical Methods for the Identification of Lichens by A. Orange, P.W. James and F.J. White, cites Huneck. It feels like an easy mistake to make; G.E. is intuitively mostly the G (glycerol), not the E ("ethanoic acid", the systematic name for acetic acid).

Tragically, Huneck's book has a large number of photos of microcrystallization, most of which using G.E. The formula for G.E. is only given once in the book, so it's unclear whether they followed it when taking the photos, or if it was just a typo.

Why it matters

Microcrystallization as a technique has long since fallen out of favor due to thin-layer chromatography, another analytical technique capable of identifying small amounts of substances and requiring less subjective interpretation of results. However, unlike thin-layer chromatography, microcrystallization is easy to do, requires less hands on time, and doesn't require the use of dangerous chemicals.

For these reasons, I believe microcrystallization could have a place for separating species which differ only in chemistry. However, without a high quality and reliable source of images to compare with, microcrystallization might fall out of favor.

Although the error seems limited to these books, they are both frequently referenced (Google Scholar says 1,277 citations of Huneck and 2,262 citations for Orange). It's troubling to think that readers may reach for one of these books when preparing G.E., with confusing results when their crystals don't form as expected, or, if they do catch the error, that they might be discouraged by the conflicting information. Getting to the bottom of the issue for me required tracking down the Asahina article, which is in German and from 1936.

I want to help this situation by providing resources about microcrystallization online. Although Huneck is immensely valuable, the photos often lack the lichen source, as well as when the photos were taken relative to applying the reagent (some crystals form very slowly, some very quickly). A way of querying photos of microcrystallization results by taxa, reagent, extraction method, etc. would do a lot for the usefulness of the technique.

References and resources