# Microcrystallization

> Mediated Wiki article. Canonical URL: https://mediated.wiki/source/Microcrystallization
> Markdown URL: https://mediated.wiki/source/Microcrystallization.md
> Source: https://en.wikipedia.org/wiki/Microcrystallization
> Source revision: 1249358009
> License: Creative Commons Attribution-ShareAlike 4.0 International (https://creativecommons.org/licenses/by-sa/4.0/)

{{Short description|Analytical technique used in lichenology}}
[[File:Norstictic Acid Crystals.jpg|thumb|right|Crystals of [norstictic acid](/source/norstictic_acid), obtained by adding [potassium hydroxide](/source/potassium_hydroxide) solution to an apothecial section of the [crustose lichen](/source/crustose_lichen) ''[Aspicilia cinerea](/source/Aspicilia_cinerea)'']]
'''Microcrystallization''' (or '''microcrystal test''') is a method for identifying [lichen metabolites](/source/lichen_product) that was predominantly used before the advent of more advanced techniques such as [thin-layer chromatography](/source/thin-layer_chromatography) and [high-performance liquid chromatography](/source/high-performance_liquid_chromatography). Developed primarily by [Yasuhiko Asahina](/source/Yasuhiko_Asahina), this approach relies on the formation of distinctive [crystal](/source/crystal)s from lichen [extract](/source/extract)s. Although now superseded by modern [analytical methods](/source/analytical_chemistry), microcrystallization still holds importance for compound purification and analysis using [X-ray crystallography](/source/X-ray_crystallography).

==History==

Between 1936 and 1940,<ref name="Asahina 1936"/><ref name="Shibata 2000"/> Japanese chemist and lichenologist Yasuhiko Asahina published a series of papers in the ''[Journal of Japanese Botany](/source/Journal_of_Japanese_Botany)'' detailing the microcrystallization technique.<ref name="Huneck 1996"/><ref name="Mitchell 2011"/> This simple and rapid method allowed for the identification of major metabolites in hundreds of lichen species, contributing significantly to [taxonomic](/source/taxonomy_(biology)) research.<ref name="Mitchell 2011"/> The technique was introduced to western lichenologists in a 1943 publication by [Alexander Evans](/source/Alexander_William_Evans),<ref name="Evans 1943"/> and was used regularly until more advanced techniques such as [thin-layer chromatography](/source/thin-layer_chromatography) and [high-performance liquid chromatography](/source/high-performance_liquid_chromatography) were introduced and integrated into laboratories. Decades of research on the [secondary metabolite](/source/secondary_metabolite)s of lichens culminated in the publication of ''Identification of Lichen Substances'', a 1996 work by [Siegfried Huneck](/source/Siegfried_Huneck) and Isao Yoshimura, that summarized analytical data for hundreds of lichen molecules, including images of microcrystals.<ref name="Olivier-Jimenez et al. 2019"/> Ultimately, the microcrystallization method had limitations, as it was unable to detect minor components or analyze complex mixtures of lichen substances.<ref name="Le Pogam et al."/><ref name="Nash et al."/> Despite these drawbacks, microcrystallization played a crucial role in the study of correlations between lichen chemistry, [morphology](/source/morphology_(biology)), and geographic distribution.<ref name="Nash et al."/>

==Procedure==

To perform microcrystallization, a small piece of lichen is extracted using [acetone](/source/acetone) or other [solvent](/source/solvent)s, [filtered](/source/filtration), and [evaporated](/source/evaporation) to yield a [residue](/source/Residue_(chemistry)).<ref name="Le Pogam et al."/><ref name="Huneck 1996"/><ref name="Galun et al."/> The residue is transferred to a [microscope slide](/source/microscope_slide), and a drop of microcrystallization reagent is added before capping with a [cover glass](/source/cover_glass).<ref name="Le Pogam et al."/> Commonly used [reagent](/source/reagent)s include GAW (H<sub>2</sub>O/[glycerol](/source/glycerol)/[ethanol](/source/ethanol) 1:1:1, v/v/v) and GE ([acetic acid](/source/acetic_acid)/glycerol 1:3).<ref name="Le Pogam et al."/> Slides using GE or GAW are gently heated and then allowed to cool, promoting the crystallization process.<ref name="Le Pogam et al."/><ref name="Huneck 1996"/> Once formed, crystals are best observed under [polarized light](/source/polarized_light) with a 200–1,000-fold magnification.<ref name="Le Pogam et al."/>

This method requires basic laboratory equipment, including a [microscope](/source/microscope) equipped for polarized light, [test tube](/source/test_tube)s, [pipette](/source/pipette)s, a micro [spirit-lamp](/source/spirit-lamp) or micro [Bunsen burner](/source/Bunsen_burner), [spatula](/source/spatula) or [scalpel](/source/scalpel), and microscope slides and cover glasses.<ref name="Huneck 1996"/> Lichen substances can be identified based on the distinctive shape and color of their crystals.<ref name="Le Pogam et al."/><ref name="Huneck 1996"/>

==Identification and interpretation==
The process of crystal identification involves comparing them to images of crystals in different solvents found in published sources. Although the shape of the crystals depends on the solvent and, to a certain degree, the substance concentration, it is usually possible to recognize the fundamental crystalline forms. Care should be taken to differentiate between undissolved substances, which might be crystalline but lack a characteristic shape, and recrystallized substances. Microcrystal samples cannot be preserved for long, as they start to degrade within hours or days.<ref name="Orange et al. 2001"/>

Distinguishing between [gyrophoric acid](/source/gyrophoric_acid) and [lecanoric acid](/source/lecanoric_acid) using thin-layer chromatography can be challenging. However, if one of these substances is known to be present, a microcrystal test can help differentiate them. In the GAW solvent system, lecanoric acid forms long, curved crystal clusters, although the results can be inconsistent, especially in the presence of other substances. Gyrophoric acid, when present in the GE solvent system, may manifest as small, fine crystal clusters or rounded aggregations of tiny crystals. Lecanoric acid in the GE solvent system produces needle-like crystal clusters, but these are not as well-formed as in GAW. These tests can help distinguish ''[Punctelia borreri](/source/Punctelia_borreri)'' (which contains gyrophoric acid) from ''[Punctelia subrudecta](/source/Punctelia_subrudecta)'' (which contains lecanoric acid).<ref name="Orange et al. 2001"/>

When two substances generate similar-looking crystals, their [optical properties](/source/optical_properties) can be used to differentiate between them. Certain crystals alter the [polarization plane](/source/plane_of_polarization) of [transmitted](/source/Transmittance) light, and when rotated between crossed polarizers, they alternate between bright and dark every 90°. The [extinction angle](/source/Extinction_(optical_mineralogy)) is the angle between a specific [crystal axis](/source/crystal_structure) and the filter's polarization plane when the crystal appears dark (in extinction). For instance, this method can be employed to distinguish between [perlatolic acid](/source/perlatolic_acid) and [imbricaric acid](/source/imbricaric_acid), which both form long, straight crystals in the GE solvent system but exhibit extinction angles of 0° and 45°, respectively, in relation to their long axis.<ref name="Orange et al. 2001"/>

==See also==
* [Spot test (lichen)](/source/Spot_test_(lichen))

==References==
{{Reflist|colwidth=30em|refs=

<ref name="Asahina 1936">{{cite journal |last1=Asahina |first1=Y. |year=1936 |title=Mikrochemischer nachweiss der Flechtenstoffe (I) |journal=Journal of Japanese Botany |volume=12 |pages=516–525 |language=de}}</ref>

<ref name="Evans 1943">{{cite journal |last1=Evans |first1=Alexander W. |year=1943 |title=Asahina's microchemical studies on the Cladoniae |journal=Bulletin of the Torrey Botanical Club |volume=70 |issue=2 |pages=139–151 |doi=10.2307/2481365 |jstor=2481365}}</ref>

<ref name="Le Pogam et al.">{{cite book |editor-last1=Upreti |editor-first1=Dalip Jumar |editor-last2=Divakar |editor-first2=Pradepp K. |editor-last3=Shukla |editor-first3=Vertika |editor-last4=Bajpal |editor-first4=Rajesh |first1=Pierre |last1=Le Pogam |first2=Gaëtan |last2=Herbette |first3=Joël |last3=Boustie |year=2015 |chapter=Analysis of Lichen Metabolites, a Variety of Approaches |title=Modern Methods and Approaches in Biomonitoring and Bioprospection |series=Recent Advances in Lichenology |pages=229–261 |publisher=Springer India |isbn=978-81-322-2180-7}}</ref>

<ref name="Nash et al.">{{cite book |last1=Elix |first1=J.A. |last2=Stocker-Wörgötter |first2=E. |chapter=Biochemistry and secondary metabolites |editor-first=Thomas H. |editor-last=Nash III |title=Lichen Biology |edition=2nd |publisher=Cambridge University Press |location=Cambridge, UK |year=2008 |pages=118–119|isbn=978-0-521-69216-8}}</ref>

<ref name="Huneck 1996">{{cite book |last=Huneck |first=Siegfried |last2=Yoshimura |first2=Isao |title=Identification of Lichen Substances |publisher=Springer Berlin Heidelberg |publication-place=Berlin, Heidelberg |year=1996 |isbn=978-3-642-85245-9 |oclc=851387266 |page=47}}</ref>

<ref name="Mitchell 2011">{{cite journal |last=Mitchell |first=M.E. |year=2014 |title=De Bary's legacy: the emergence of differing perspectives on lichen symbiosis |journal=Huntia |volume=15 |issue=1 |pages=5–22 [136–137] |url=https://www.huntbotanical.org/admin/uploads/02hibd-huntia-15-1-pp05-22.pdf}}</ref>

<ref name="Galun et al.">{{cite book |editor-last1=Galun |editor-first1=Margalith |last1=Galun |first1=Margalith |last2=Shomer-Ilan |first2=Adiva |chapter=Secondary Metabolic Products |volume=III |year=1988 |title=CRC Handbook of Lichenology |publisher=CRC Press |location=Boca Raton |page=134 |isbn=978-0-8493-3583-9}}</ref>

<ref name="Olivier-Jimenez et al. 2019">{{cite journal |last1=Olivier-Jimenez |first1=Damien |last2=Chollet-Krugler |first2=Marylène |last3=Rondeau |first3=David |last4=Beniddir |first4=Mehdi A. |last5=Ferron |first5=Solenn |last6=Delhaye |first6=Thomas |last7=Allard |first7=Pierre-Marie |last8=Wolfender |first8=Jean-Luc |last9=Sipman |first9=Harrie J.M. |last10=Lücking | first10=Robert |last11=Boustie |first11=Joël |last12=Le Pogam |first12=Pierre |title=A database of high-resolution MS/MS spectra for lichen metabolites |journal=Scientific Data |volume=6 |issue=1 |year=2019 |pmid=31780665 |pmc=6882832 |doi=10.1038/s41597-019-0305-1 |page=e294 |bibcode=2019NatSD...6..294O}}</ref>

<ref name="Orange et al. 2001">{{cite book |last1=Orange |first1=A. |last2=James |first2=P.W. |last3=White |first3=F.J. |year=2001 |title=Microchemical Methods for the Identification of Lichens |publisher=British Lichen Society |isbn=978-0-9540418-0-9 |pages=40–43}}</ref>

<ref name="Shibata 2000">{{cite journal |last=Shibata |first=Shoji |title=Yasuhiko Asahina (1880–1975) and his studies on lichenology and chemistry of lichen metabolites |journal=The Bryologist |volume=103 |issue=4 |year=2000 |doi=10.1639/0007-2745(2000)103[0710:yaahso]2.0.co;2 |pages=710–719}}</ref>

}}

==External links==
*[https://www.youtube.com/watch?v=2IoUzuuf8Ww Microcrystal Tests for Lichen Substances], YouTube tutorial presented by Yoshihito Ohmura, [National Museum of Nature and Science](/source/National_Museum_of_Nature_and_Science)

Category:Lichenology
Category:Crystallography

---
Adapted from the Wikipedia article [Microcrystallization](https://en.wikipedia.org/wiki/Microcrystallization) by Wikipedia contributors ([contributor history](https://en.wikipedia.org/wiki/Microcrystallization?action=history)). Available under [Creative Commons Attribution-ShareAlike 4.0 International](https://creativecommons.org/licenses/by-sa/4.0/). Changes may have been made.