A methodological approach for the simultaneous quantification of glycerol and fatty acids from cork suberin in a single GC run

Abstract

Introduction Suberin, as part of plant protective barriers, is one of the most important natural polymers after cellulose and lignin. For a full elucidation of suberin structure the quantification of glycerol, fatty α,ω‐diacids and ω‐hydroxyacids, the major building blocks of suberin, is of primary importance. Glycerol is often lost in the most used analytical procedures or rarely determined by deficient or too laborious techniques. Objectives Propose a simple, accessible and reliable methanolysis work‐up procedure for an accurate and simultaneous quantification of glycerol and suberin fatty monomers in the same GC run. Material and methods Cork from Quercus suber L. was depolymerised by methanolysis. Glycerol was derivatised to an organic soluble form before the suberin monomers recovery in water/organic solvent partition. Gas chromatography flame ionisation detector (GC‐FID) response factors were determined for glycerol, ferulic acid and one for each fatty monomer substructure. Additionally, 1,2,4‐butanetriol and methyl nonadecanoate were used as internal standards. Results The proposed experimental approach allowed the glycerol and all the fatty suberin monomers in the same GC run to be quantified accurately. Glycerol represented 30.6 area%, 14.2 mass% and 38.4 molar% of suberin and the COOH/OH groups ratio was 0.6:1 in the proposed experimental approach in contrast with 0.10 area% and COOH/OH ratio of 3:1 in the most used protocol. Furthermore, ω‐hydroxyacids/α,ω‐diacids mass ratio was 1:1 as opposed to an area ratio of 1.5:1. Conclusion The proposed work‐up procedure revealed to be a reliable analytical tool for the complete analysis of suberin allowing the future knowledge to grow towards a better understanding of suberin structure throughout its range and variability.

The Making of Plant Armor: The Periderm

“…Lignin is also an abundant fraction of phellem; it consists in an aromatic polymer composed mainly of guaiacyl (G) and usually fewer syringyl (S) monolignol units, compared to the lignin found in xylem or phloem (36,43,93). In cork oak phellem, lignin is enriched in ferulic acid (93,105) while in potato phellem, ferulic acid was also identified forming ether linked amides, such as feruloyltyramine (116).…”

Accessing suberin from cork via ultrafast supercritical hydrolysis

“…S3, † while Table S4 † summarized the identities and quantities of the major suberin acids determined from suberin samples via conventional method and ultrafast supercritical treatment. Only the major peaks are presented herein, following NIST and related literatures 15,18 and does not involve full evaluation of other molecules detected that cannot be assigned. The compounds were detected as their corresponding methyl ester/trimethylsilyl derivatives.…”

“…Moreover, there exists an inter-monomer ester linkage between the hydroxyacids and diacids ( polyaliphatic constituents), an ester or ether crosslink between the polyaliphatics and polyphenolic part and a C-C, amide and ether bonds connecting the polyphenolic part to the rest of the cell wall components. 13 Thus, the isolation of suberinic fatty acids from cork follows a sequential and laborious train of removing extractives, followed by suberin recovery through alkali methanolysis 14,15 or alkali hydrolysis, 16 then acid hydrolysis for cellulosic fractions, leaving lignin behind. Alkaline hydrolysis is a harsh process, where ω-hydroxyfatty acids with epoxy and hydroxyl containing derivatives are obtained.…”

Cutin extraction and composition determined under differing depolymerisation conditions in cork oak leaves

“…25 This cutin composition clearly differs from the general composition of suberins which have α,ω-diacids as a major lipid component and ferulic acid as the major aromatic component, and specifically of the cork oak suberin composition containing α,ω-diacids followed by ω-hydroxy acids. 26,27 The cutin depolymerisation extracts also included a small proportion of terpenes (1.7% of all compounds) and a minor content of alkanes (0.1%) that correspond to some residual cuticular waxes, probably of intracuticular nature, that were not completely removed by the dewaxing solvent extraction.…”

“…For instance, glycerol has three hydroxyl groups, thereby allowing formation of three ester bonds, while alkanoic acids only allow one ester bond, diacids two ester bonds and ω-hydroxy acids allow two ester bonds (one with an hydroxyl group the other with a carboxyl group). The polyester macrostructural building has been addressed with a similar approach for suberin 27,28 but, to our knowledge, not for cutin.…”