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Techniques & Tools Liquid Chromatography, Gas Chromatography, Sample Preparation

Splendid Isolation

Over the past few years, several efforts have been made to obtain bioactive molecules from natural sources, but the most widespread technique for obtaining pure chemicals is organic synthesis, which plays a major role in many fields such as pharmaceutical, food, flavour and fragrances. The practice is often inefficient; in fact, sometimes kilograms, even tons of raw material are required to obtain enough pure product. And the approach certainly cannot be considered “green” because of the huge production of waste, including solvents and hazardous by-products.

Over the last century, both liquid and gas preparative chromatography appeared in analytical chemistry as a possible substitute for organic synthesis for the isolation of pure molecules. However, neither technique – and in particular, preparative gas chromatography – was seriously considered from a commercial point of view, because of the low quantities collected per run, as well as the degree of purity attained. Preparative chromatography also had other limitations: difficulty in separation of very complex samples and the low relative concentration of the components to be isolated.

Many scientific papers have been published on the subject, reporting the isolation of pure components, such as PAHs and volatiles, components from distilled spirits and small molecules from complex samples (1)(2)(3)(4). Almost all these applications exploited a huge number of repeated injections (100-500) to obtain only micrograms of pure components. The reason lies in the low injected amounts, often mandatory to preserve separation and resolution of the components of interest prior to collection.

With this in mind, during my post-doc experience in the group headed by Luigi Mondello, some colleagues and I decided to develop an all-in-one lab-made instrument – the first on-line LC-GC-GC-GC prep system capable of collecting milligrams of pure components in a very short time (5)(6). The system represented a step forward in the field of preparative chromatography because of its ability to meet the demands of private companies who, until that moment, had not considered the technique a valid tool for their needs.

The entire instrumentation may appear very complex to handle, but the results attained surely justify its use.

The instrument consists of a HPLC system equipped with a normal phase 25 cm x 4.6 mm ID LC-Silica column, connected through a special syringe-type interface to a large-volume injector (LVI) and three GC ovens. The ovens are equipped with three wide-bore columns (0.53 mm i.d.) of different selectivity and three Deans Switch devices that allow the heart cut of single fractions from each dimension to the next. Finally, the collection of the pure components is performed using two different collection devices: i) a low-cost, lab-made one, positioned in the second oven, which allows the collection of the already resolved peaks and ii) an automated commercial system positioned at the end of the third column that is capable of collecting up to 10 components in one run.

The four-dimensional preparative system described above has been exploited for the isolation of two non-commercially available sesquiterpene components from vetiver essential oil – namely alpha-amorphene and beta-vetivone – as well as the collection of seven of the most important oxygenated components belonging to sandalwood essential oil. In both cases, thanks to this system, we were able to collect milligrams of pure component in under a day.

Although the entire instrumentation may appear very complex to handle, the results attained surely justify its use in many specific applications where other techniques – such as distillation and organic synthesis – fail.

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  1. CM Reddy et al, “Radiocarbon as a tool to apportion the sources of polycyclic aromatic hydrocarbons and black carbon in environmental samples”, Environ Sci Technol 36, 1774-1782 (2002)
  2. M Mandalakis and Ö Gustafsson, "Optimization of a preparative capillary gas chromatography–mass spectrometry system for the isolation and harvesting of individual polycyclic aromatic hydrocarbons”, J Chrom A, 996, 163–172 (2003)
  3. J Ledauphin et al, “Identification of Trace Volatile Compounds in Freshly Distilled Calvados and Cognac Using Preparative Separations Coupled with Gas Chromatography−Mass Spectrometry”, J Agric Food Chem, 52, 5124-5134 (2004)
  4. G T Eyres et al, “Method for small-molecule discovery based on microscale-preparative multidimensional gas chromatography isolation with nuclear magnetic resonance spectroscopy”, Anal Chem, 80, 6293–6299 (2008)
  5. D Sciarrone et al, “Rapid isolation of high solute amounts by using a totally orthogonal on-line four-dimensional preparative system: normal phase-liquid chromatography coupled to methyl siloxane-ionic liquid-wax phase gas chromatography”, Anal Chem, 86, 4295−4301 (2014)
  6. S. Pantò et al, “Performance evaluation of a versatile multidimensional chromatographic preparative system exploiting an on-line combination of polyethylene glycol/ poly (5% diphenyl/95% dimethylsiloxane), ionic liquid and β-cyclodextrin stationary phases”, J Chrom A, 1417, 96-103 (2015)

About the Author

Sebastian Pantò

Sebastiano received his Bachelors degree in Biology in 2008 from the University of Messina, followed by a Masters degree in Biology and a PhD in “Food Chemistry and Safety” with a dissertation entitled “Development of novel multidimensional chromatography technologies for the isolation and identification of targeted and untargeted food constituents”. In the same year, he started a research grant for the project PON-Hi-Life (Health Products from the Industry of Foods) and in 2015 a second one from Chromaleont s.r.l. as a gas phase scientist. In 2012, he received an award from the Interdivisional Group of Separation Sciences (GISS) for the oral presentation at the symposium “ExTech 2012”. In 2013, his work on development of a multidimensional system for the collection of pure molecules from complex samples was recognized by “The Analytical Scientist Innovation Awards (TASIAs)”. Sebastiano says, “I love challenges in my life and I cannot stay in a steady state for any more time than is absolutely necessary!”

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