EGU General Assembly 2023
© Author(s) 2023. This work is distributed under
the Creative Commons Attribution 4.0 License.

A new solid phase extraction method for purifying plant sugars for compound-specific hydrogen isotope analysis

Selina Hugger, Meisha Holloway-Phillips, Ansgar Kahmen, and Daniel B. Nelson
Selina Hugger et al.
  • Department of Environmental Sciences - Botany, University of Basel, Basel, Switzerland (

As plant organic compounds are formed, their hydrogen isotopic composition (δ2H) is influenced by the hydrology of the environment as well as by the plant metabolism, and hence contains information about both. Until recently, cellulose has been the only plant carbohydrate routinely measured, because it persists in tissues over long time scales and can therefore be used to understand time-integrated signals preserved in tissues like tree rings. However, knowledge gaps about how environmental and metabolic signals are imprinted in the cellulose δ2H value currently limit the interpretation of cellulose δ2H variation with respect to plant metabolism. Measuring δ2H values of biosynthetic intermediates like sugar molecules from fresh tissue offers a possibility of filling some of these knowledge gaps to better understand the cellulose δ2H signal.

Measuring δ2H values in carbohydrates is complicated by the presence of hydroxyl hydrogen. This is because this hydrogen pool can isotopically exchange with surrounding liquid water or water vapor (e.g., during sample processing and analysis), obscuring the primary plant hydrological and metabolic information contained in the isotopic values of carbon-bound hydrogen. The contribution of exchangeable hydrogen can be accounted for using dual equilibration techniques, but in most cases, these permit only analyses of bulk leaf extracted sugars. Thus, another difficulty lies in obtaining individual sugar compounds from plants. An alternative to dual equilibration is therefore to derivatize the plant sugars prior to analysis with hydrogen of known isotopic composition to form new compounds that no longer contain exchangeable hydrogen, e.g., by acetylation. Acetylation makes sugar compounds amenable to gas chromatography (GC), so this technique also allows for compound-specific analyses of multiple compounds in the same sample, such as different sugar types. GC analysis also has the advantages of requiring smaller sample amounts and providing better assurances of sample purity than are possible for bulk sample measurement approaches.

Here, we present results on streamlining our acetylation approach to facilitate rapid and cost-effective purification from sample matrices of water-soluble plant carbohydrate extracts. We acetylate whole carbohydrate extracts, and then compare recovering sugar acetates from the sample matrix by liquid-liquid separation with recovery by reverse-phase solid phase extraction (SPE) using a C18 sorbent. The liquid-liquid separation yields a higher recovery than SPE but is much more labor-intensive, whereas the SPE method can be scaled easily for higher sample throughput. Both methods proved successful for purifying water-soluble sugar samples as well as digested starch (glucose) from plant leaf, wood, and root material, yielding GC amenable sugar acetates that are soluble in acetone. Sucrose octaacetate is well resolved under normal GC measurement conditions on commonly available GC columns (e.g., DB-5), without requiring instrument modification as is the case for halogenated sample compounds.

How to cite: Hugger, S., Holloway-Phillips, M., Kahmen, A., and Nelson, D. B.: A new solid phase extraction method for purifying plant sugars for compound-specific hydrogen isotope analysis, EGU General Assembly 2023, Vienna, Austria, 24–28 Apr 2023, EGU23-14146,, 2023.