Size Exclusion High Performance Liquid Chromatography: Re-Discovery of a Rapid and Versatile Method for Clean-Up and Fractionation in Chemical Ecology

Abstract

Solvent extraction of bioactive molecules from glands, tissues, or whole organisms is a common first step in chemoecological studies. Co-extraction of a surplus of high boiling materials such as triacylglycerides (TAGs) and other lipids with higher molecular weight might hamper the identification of volatile or medium-volatile semiochemicals by high resolution chromatographic and spectroscopic techniques. Therefore, effective clean-up procedures are needed to separate potential semiochemicals from the accompanying materials. Size exclusion high performance liquid chromatography (SE-HPLC), a technique often disregarded by chemoecologists, has proved to be a rapid and efficient clean-up method for complex crude extracts. We demonstrated that TAGs can be baseline separated from typical semiochemicals within less than 10 min on a porous gel stationary phase based on highly cross-linked polystyrene/divinylbenzene. We applied the method as a rapid one-step clean-up procedure for the analysis of juvenile hormone III in insect hemolymph by gas chromatography-mass spectrometry. We furthermore introduced some recent application examples on insect pheromones to demonstrate that SE-HPLC is not only an effective method for the purification of crude extracts, but can as well be used as a first fractionation step for the bioassay-guided identification of behavior modifying natural products. SE-HPLC can be well operated with low-boiling solvents such as dichloromethane, and results in fraction volumes of typically less than one ml, which decreases the danger of losing volatile analytes during subsequent concentration steps.

A hormone-related female anti-aphrodisiac signals temporary infertility and causes sexual abstinence to synchronize parental care

“…SE-HPLC settings and all further steps for quantifying JH III, including gas chromatography-mass spectrometry analysis and calibration, were performed following the protocol described in ref. 75 . We used Gaussian GLMs to test for effects of treatment and day on methyl geranate emission and JH level and a Pearson correlation to test for relationship between pheromone and hormone levels.…”

Parasitic wasps do not lack lipogenesis

“…For this purpose, we extracted pools of three Nv females of differing age and oviposition history from the 13 C feeding experiment (see above) and fractionated the resulting raw lipids by size exclusion high performance liquid chromatography (HPLC) (n=3 per treatment). This technique allows for the isolation of TAGs as a single chromatographic peak [38] (Fig. S6 in the supplementary material).…”

“…Eluting analytes were detected with an Infinity 1260 multi-wavelength UV/VIS detector at 254 nm. Under these conditions, TAGs elute as a single peak between 5.8 and 6.5 minutes [38]. To confirm this elution range, we analysed 20 μl of a dichloromethane solution (1 μg/μl) of synthetic triolein (( Z ) - 9-octadecenoic acid 1,2,3-propanetriyl ester, Sigma-Aldrich, Deisenhofen, Germany).…”

Male Sex Pheromone of the Parasitoid Wasp Urolepis rufipes Demonstrates Biosynthetic Switch Between Fatty Acid and Isoprenoid Metabolism Within the Nasonia Group

“…These raw extracts were concentrated under a gentle stream of nitrogen to a concentration of 0.5 male equivalents per µl and used for the bioassay. Additionally, we isolated DMOD from the natural extracts either by size exclusion high performance liquid chromatography (SE-HPLC) (Sperling et al, 2015) or by solid phase extraction (SPE) on silica gel. For SE-HPLC, raw extracts of 30-50 males (see section Pheromone Extraction and Quantification From Wasps) were concentrated to 20 µl and separated on a 300 × 7.5 mm PLgel SE-HPLC column (particle size 5 µm, pore size 100 Å) (Agilent Technologies, Waldbronn, Germany) with DCM as mobile phase as described previously (Sperling et al, 2015).…”