Back to Nature with Flash Chromatography

Dr Vera Thoss from Bangor University (Bangor, Wales) spoke to Bethany Degg of The Column about her work investigating the chemistry of plants and the value of flash chromatography in this area of research.

Q. Your research activities are focused on the investigation of plant secondary metabolites. How did you become interested in this field and why is this area of research important?

A: Plants can't run away so they either need to defend themselves chemically from predators or they need to attract pollinators. This results in some fascinating, mostly organic, compounds that have evolved for specific biological activities. Our work has shown that a herb Acinos suaveolens (thyme basil) produces more pulegone, which is a liver toxin, when exposed to grazing by herbivores compared to plants that evolved without herbivory pressure. This is known as the evolutionary arms race.

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I have always been interested in ecology because of the abundance of smells and colours in nature in addition to it being a joy just lying in the sun and observing animals. I keep bees and generally we do taste plants when out and about. My background in chemistry is ideal to probe whether specific compounds have ecological roles. In the future this may lead to new medicines, for example bluebells contain iminosugars that are anti-diabetic.

Q. What are your main research interests involving plants?

A: I am working on biorefining high-value chemicals from plants, and we are looking at three species particularly closely: bluebells, bracken, and an African tree Trichillea emetica. I am also working closely with industry on quality control and process monitoring as part of the WISE Network. In the WISE Network we support Welsh companies, some of which work on plant chemistry, for example in producing essential oil from lavender or assessing a specific plant-derived metabolite for its use as a nutritional supplement.

I also apply chromatography to investigate the smell of plants, particularly pines and lavender, and isolation work. Plants produce an array of metabolites, often with variation in the glycosylation pattern. In order to assess this variety, we ideally need to assess every metabolite on its own.

Q. You use flash chromatography in your work. What is flash chromatography and why do you use it in your field?

A: Flash chromatography is an instrumental technique that uses a column with varying amounts of filling material, from 12 g to 1.5 kg. The stationary phase is comparable to those used in high performance liquid chromatography (HPLC) or solid-phase extraction (SPE). The component mixture is applied either in solution or adsorbed onto silica. Elution is achieved via a pumped solvent or solvent mixtures, again similar to HPLC. In flash chromatography the eluent is monitored, our system uses 2 UV–vis wavelengths and an evaporative light scattering detector (ELSD), and when a peak occurs above a set threshold, the peak is automatically collected.

Flash chromatography is relatively cheap and can be a great workhorse in a natural product laboratory. We use flash chromatography to prepare standard materials for quantitative analysis. Working in plant chemistry often requires separating mixtures of very similar components, such as glycosides with the same aglycon, and we need to isolate these compounds to undertake structure elucidation.

In the current climate, the consumer market prefers "natural" materials. They often perceive a synthetic chemical as "bad" and seek a natural alternative. Consequently, working with plant extracts is becoming more common and there is a need to analyze the plant extract for the molecular composition and this is research we undertake in our laboratory.

Q. What are the advantages of flash chromatography versus column chromatography?

A: Traditional column chromatography requires more user attention, does not offer automated peak collection, and is less flexible in relation to stationary phases than flash chromatography. We usually use C18 cartridges in flash, which allows us to go from analytical HPLC to flash. Another important consideration is the detector. A lot of organic compounds are non UV-active, requiring aggressive spray reagents in thin layer chromatography (TLC) to make them visible, for example carbohydrates with 10% H₂SO₄. Having an ELSD detector on the flash systems allows the detection of anything, which is a big advantage. ELSD is also a universal detector and is really handy, particularly as the maintenance requirements are low.

Q. Do you have any advice to offer to analytical chemists considering using flash chromatography for plant extracts?

A: Make your own standards! Flash chromatography allows us to keep the cost lower by producing our own standards, and making isolates for biological screening. Even with flash chromatography the isolation of standards is not trivial and usually requires a number of chromatographic stages. While the thought of being able to inject a crude extract to obtain a chemically pure compound is attractive, usually we need to undertake a number of fractionation steps to achieve this. A good tip is to match your analytical column with the flash system, which means running the same stationary phase to allow for direct translation between the two systems.

Q. You have previously published work on the analysis of British bluebell (Hyacinthoides non-scripta) seed oil. What was the motivation behind this study? Can you discuss the analytical methods that you used and what were your main findings?

A: The work on bluebells is driven by a conservation interest of mine. If you have been involved in organizing conservation work, such as bracken clearing, there are generally costs involved even when working with volunteers and there is no financial incentive. I was hoping to find metabolites in bluebells that could be of commercial interest and hence potentially pay for the conservation effort.

Our work on the seed oil composition showed that bluebells are an untapped resource in Britain because they do not grow anywhere else in the densities observed in the UK. This is purely a result of the mild winter climate because bluebells grow mostly during the winter with flowering taking place in spring. It is not right that we import palm or soya oil if we have potential sources in the UK. For me this is a sustainability issue and worth pursuing further.

To analyze the oil, we undertook a method comparison using different stationary phases for gas chromatography coupled with mass spectrometry (GC–MS) of fatty acid methyl esters (FAMEs). We also used high temperature GC for the triglycerides. One of our foci was the location of the double bond in the unsaturated fatty acids; hence we included ozonolysis to assess this, in addition to nuclear magnetic resonance (NMR). So it was quite an analytical paper. As far as the results go, oils do not vary in composition from year to year.

Q. Is there anything that you would like to add?

A: Natural products chemistry is an old branch of chemistry, after all scientists have worked on this for centuries. New developments, like flash chromatography, aid in our research. In addition, we seem to have passed the high use of petroleum and are shifting back to renewable resource use. It is surprising that in the UK nearly half of native plants traditionally used for medicines are still in need of chemical exploration. I think the future for natural products chemistry, for which chromatography is essential, is bright.

Vera Thoss is a lecturer in chemistry and project manages, on behalf of the School of Chemistry, the ERDF funded pan-Wales projects WISE Network (www.wisenetwork.org) and BEACON (www.beaconwales.org). Vera's scientific interests are in natural products chemistry, chemical analysis and chemical ecology. She has a thorough understanding of analyzing materials, whether it is for elemental composition or targeting distinct compounds. With a remit for industrial liaison, Vera frequently interacts with commercial partners to identify their challenges and finds analytical approaches that yield relevant information. This is supported by access to state-of-the-art facilities either in-house or collaborating with other partners. Vera also has a strong interest in sustainability, particularly with respect to utilizing underexplored native plants, particularly bluebells.

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Website: www.bangor.ac.uk

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