Supercritical fluid chromatography (SFC) has experienced a recent increase in popularity. Gilles Goetz, a Principal Scientist at Pfizer  in Groton, Connecticut, USA, is doing some interesting work in  non-traditional uses of SFC. He recently spoke to us about this research.

Q. Supercritical fluid chromatography (SFC) seems to be back in fashion again. Why is this?

 I think one of the main reasons supercritical fluid chromatography (SFC) has recently enjoyed a surge in popularity is because of the “green chemistry” attributes. First of all, the use of CO

 in SFC operation is carbon neutral. Compared to high performance liquid chromatography (HPLC), SFC uses much less organic solvents, reducing the cost of operation, processing (evaporating), and disposal. SFC offers a significant reduction in chemical waste, making it a more environmentally friendly alternative to HPLC. Health and safety benefits are obvious results of organic solvents reduction.

	Instrument manufacturers have also realized this as well. Recent mergers and acquisitions amongst them have brought SFC into the mainstream of analytical separation techniques. SFC now benefits from the same extensive marketing and sales networks as HPLC.

	In our purification laboratory, SFC has the upper hand over HPLC. SFC is used for over 90% of the chiral purifications and for about 50% of the achiral purifications. Because there is no diffusion occurring in SFC, resolution and speed are significantly improved.

Q. How has SFC evolved and has the technique tackled some of the problems associated with it in the past? 

 The first commercially available SFC system dates back to 1982. In the early days, capillary SFC and packed column SFC co-evolved and competed with each other. Capillary SFC lost ground and eventually faded away, mainly as a result of poor reproducibility issues. The early systems were costly and not user friendly, resulting in the technique being marginalized as too expensive and inefficient.

	The main obstacles to the adoption of SFC have been the high pressure operating conditions, the difficulty to maintain those high pressures consistently, and the handling of the depressurization and aerosolization issues post analysis.

	Current benchtop analytical systems are able to function with a simple high purity CO

 cylinder. More complex operations (that is, purification laboratories) require larger volumes of CO

, typically stored as liquid in a centralized location before being distributed, and converted into supercritical fluid in the SFC instrument.	 	Improvement in back pressure regulator technology enabled constant pressures across SFC systems even during gradient elution, when solvent compositions or flow rates change. In the realm of preparative SFC, full recovery of product upon collection is paramount. The development of effective gas/liquids separators (such as the cyclone separators) enabled CO

 to depressurize without aerosolizing and losing the dissolved product, keeping it safe to collect with the co-solvent portion of the eluent.	 	 	

Q. Are there any new trends or discoveries using “modern” SFC?

 Ultrahigh-performance SFC (UHPSFC) and the combination of a new generation of instrumentation (more robust, reliable and with improved performance) with sub-2-μm particles columns appears to be quite the hype these days. Reliability, robustness, and performances are improved with this combination; for example, core–shell 2.6 μm columns showed a 50% increase in efficiency compared to 3 μm fully porous particles.	 	Expanding the chemical space of compounds amenable to both chiral and achiral SFC has recently been the focus of significant efforts. Methods are being developed to accommodate highly polar or ionic compounds historically incompatible with SFC. (Larry Taylor at VT is working on peptide analyses using ion pairing SFC, with mobile phases consisting of CO

, 5% v/v of water in methanol containing an ion pairing agent).	
			 	 	

Q. How easy is it to implement SFC in your work?

 Because of the perception of danger associated with operating under special conditions (high pressure and high temperature) that actually change the state of the matter of the CO

 into something quite mysterious (a supercritical fluid), SFC might be perceived as intimidating and complicated to neophytes. This is a common misconception. Implementing SFC is pretty much plug and play. This was the case for this work as well. SFC was the method of choice to get us to analysis conditions with a dielectric constant mimicking the inside of a biological membrane, in my opinion, much more so than normal phase HPLC.	 	

Q. What is the appeal of SFC in pharmaceutical analysis? How can you use SFC to enable drug design?

In the pharmaceutical industry, emphasis is placed on return on investment. The many advantages of SFC in terms of analysis speed, improved resolution, and solvent consumption compensate for the original infrastructure investment needed for efficient SFC operation. As green highâthroughput pharmaceutical analysis goes, SFC is the ultimate contender, and the same is true for preparative-scale chiral purifications. Indeed, by enabling separation of enantiomers in a new chemical space previously limited by normal phase chiral HPLC, SFC reduced the need for complex and expensive asymmetric syntheses in early project development. 	 	Applications of SFC in the pharmaceutical industry are not limited to sample purity analysis, chiral enantiomer excess determinations, or fast chiral and achiral purification from milligram to multi-gram scales. With the introduction of EPSA - a novel SFC technique enabling the design of permeable cyclic peptides - we enabled SFC to enter the realm of drug design. With EPSA we are able, using a pairwise analysis, to confirm or disprove the presence of an intramolecular hydrogen bound (IMHB) in small molecules or peptides. These IMHBs are a critical factor affecting the conformation of molecules and can have a direct impact on their absorption into the human body, their permeability into target cells, and even their potency against therapeutic targets. Having a reproducible, rugged, high-throughput method able to highlight those conformation changes can be a game changer in terms of drug design.	 	

Q. You are doing some interesting work in non-traditional uses of SFC. Please can you detail the reasons behind the development of EPSA. 

 The identification of compounds likely to form intramolecular hydrogen bonds is an important drug design consideration given the correlation of intramolecular hydrogen bonding with increased membrane permeability. Having access to a fast, robust method to identify the potential for a compound to form IMHBs and as a result hide polarity that might otherwise reduce the permeability of the compound was the main idea behind EPSA. 	 	Once we successfully established EPSA as a viable polarity monitoring tool for small molecules, we went on to tackle peptides, especially cyclic peptides, which generally demonstrate insufficient permeability to be used as oral drugs. Guiding the design of such peptides without a reliable permeability monitoring method is a challenge. Since for peptides, the main obstacle to permeability is polarity, the EPSA experiment was a perfect fit. We now routinely use EPSA data to inform peptide design as a predictor of permeability and an indicator of IMHB patterns in cyclic peptides.	 	

Q. Please can you describe EPSA. Could this technique be applied to other applications, other than pharmaceuticals? 

Our method named EPSA provides, under controlled SFC conditions, an exposed/hidden polarity readout that is derived from the retention time of a compound on a specific column. The stationary phase was selected for its balance of lipophilic and polar attributes and its capacity to separate compounds with wide polarity differences. SFC, which uses inherently normal phase conditions, provides a low dielectric constant environment conducive to IMHB formation. Polar compounds are retained stronger under these conditions, and a low-slope gradient of methanol achieves elution based on increasing polarity of the mobile phase. In the cases that we have studied, using matched molecular pairs with or without IMHBs, compounds with IMHB resulted in a significant reduction in polarity and eluted earlier in the chromatogram. Results are normalized daily by the use of calibration standards generating a linear relationship between retention times and EPSA values. This method has been successfully used for small molecule medicinal chemistry projects to assess exposed polarities of their lead compounds.	 	Having spent my entire career in pharmaceutical R&D, I am probably excessively pharma-centric, so I have not yet seriously considered other applications. But I believe that any field where the polarity monitoring of chemicals has any significance can benefit from EPSA, provided that the compounds are compatible with the SFC solvent system. This means that the experiment need not be limited to the pharmaceutical industry. 	
			 	 	

Q. Where do you hope your research with EPSA will take you in the future? 

 There are still a few things that I would like to figure out about EPSA. For example, the complete recognition mechanism between the stationary phase and the polar analytes and the contribution of fully charged species on retention, notably why specific carboxylic acids behave as if they are more polar than expected.	 	

is a Principal Scientist at Pfizer in Groton, Connecticut, USA. Gilles received his B.Sc. (chemistry/biochemistry 1991) and Ph.D. (organic chemistry 1995) at the University of Strasbourg in France, studying the molecular processes of poison ivy contact dermatitis. After two post docs at the University of Hawaii (1996–97) studying marine natural products and the University of Neuchatel (CH) (1998–99) focusing on secondary metabolites from fungal and terrestrial sources, he joined Monsanto in 2000. There he worked through mergers and acquisitions for Pharmacia and Pfizer successively in the Natural Product group, the High Throughput Screening group (analytical support), and the Automated Purification Group. In 2010 he transferred to the Expert Purification Group at Pfizer Groton and in 2011 became a member of the Molecular Properties Group. He is part of the team tasked with influencing medicinal chemistry design teams through insights into molecular properties such as polarity, lipophilicity, shape, and conformation that will impact molecular behaviours such as solubility, permeability, and efflux. They develop and use mainly chromatographic techniques (EPSA) to assess those properties, and work towards predicting molecular behaviours.	 	 	

Articles

Cover Story

A Spotlight on SFC

Consumers are accustomed to all year round availability of a variety of reasonably priced fruit and vegetables in supermarkets, regardless of the season, which can only be achieved by using millions of tonnes of agricultural chemicals. Mineral fertilizers and pesticides are freely dispersed on fields and plantations, and their residues or degradation products can remain in our fruits, vegetables, and cereals. To protect consumers, fast and reliable methods for the qualification and quantification of residual chemicals, contaminants, and pesticides in food and feed are essential to ensure the safety of the food chain.

	Pesticides are used in agriculture to protect plants and crops from detrimental influences.

 There are a number of different types of pesticide classified as herbicides, insecticides, bactericides, insect repellents, animal repellents, or fungicides that are all known to be harmful and of toxicological significance. Several testing methods are available that enable the detection and monitoring of residual chemicals in food. The conventional method for sample extraction of pesticideâcontaining matrices is QuEChERS sample preparation (quick, easy, cheap, effective, rugged, and safe). QuEChERS requires a number of steps before the final extract can be analyzed by a chromatographic technique as shown in Figure 1.

			A new and efficient extraction process uses “supercritical” CO

 for extraction as well as the chromatographic separation, which can shorten analytical run times, reduce solvent consumption, and reduce the risk of losses or error of measurement during sample preparation. Furthermore, extraction selectivity can be carefully controlled by adjusting the solvating power of the CO

 through changes in pressure and temperature, making it faster, more efficient, and more versatile than liquid extractions.

 is a fluid state of carbon dioxide where it is held at, or above, its critical temperature (31.1 °C) and critical pressure (73.8 bar). In this state, the physical and thermal properties are between a liquid and a gaseous phase and the fluid density is more sensitive to temperature and pressure changes.

 The physicochemical properties of supercritical CO

 near the critical point drastically change with only small variations in temperature or pressure making supercritical conditions a nonârobust area to work with in supercritical fluid chromatography (SFC).

 In addition, solvent strength is often increased by using a polar co-solvent as CO

 is non-polar at its critical point. As soon as an organic modifier is introduced, the mobile-phase is no longer truly supercritical, but the terminology is generally used regardless. These now “subcritical” conditions imply much lower changes in fluid density with a small variation in pressure or temperature and are therefore better suited for a robust, reproducible SFC separation (see Figure 2).			 			

Case Study: SFE–SFC Analysis of a Spinach Sample

			In an on-line SFE–SFC approach, solid food samples are homogenized with an absorbent powder, and placed in the extraction vessel in the SFE unit. Supercritical CO

 is then introduced into the vessel, where extraction conditions can be adjusted. The sample is extracted under light-shielding, anaerobic conditions so that decomposition of even labile compounds can be avoided. After extraction in the SFE unit, the sample containing CO

 is introduced in the SFC flowline for chromatographic analysis by SFC.			 			The following study outlines the SFE–SFC analysis of spinach spiked with a concentration of 100 ppb of seven pesticides (sethoxydim, chlorpyrifos, piperonylbutoxide, aminocarb, chromafenozide, captan, and endosulphansulphate). 			 			

A sample of 1 g of spinach was homogenized, mixed with 1 g of absorbent, and entered into the extraction vessel. CO

 was introduced into the vessel and the sample was extracted at 100 bar and 40 °C for 2 min using static extraction, then another 2 min using dynamic extraction before introduction of the extract into the SFC flow-line for analysis.			 			SFC analysis for pesticide residues was performed on a 250 × 4.6 mm, 5-µm Inertsil ODS-EP HP column (GL Sciences) with a flow rate of 3 mL/min using CO

 and 16 mM ammonium formate in methanol as an organic modifier in a gradient run: 0 to 11% modifier (4–15 min), 11 to 30% (15–18 min) and 30 to 40% (18–21 min) at 40 °C and at a pressure of 100 bar. An LCMSâ8050 triple quadrupole mass spectrometer (Shimadzu) was used in MRM mode.						 					

					Figure 3 shows the SFC–MS chromatogram of 100 ppb pesticide residue extracted from a spiked spinach sample. Using this on-line SFE approach pesticides could be extracted, separated, and quantified in less than 20 min; using the QuEChERS method sample preparation alone takes approximately half an hour.					 					

					The on-line SFE–SFC–MS approach was used successfully to extract and analyze seven commonly used pesticides from a spiked spinach sample, in just a fraction of the time that the QuEChERS sample preparation method would take. This method offers an ideal alternative for laboratories wanting to improve effectiveness while reducing the risk of losses or error of measurement. There are also many other possible applications of SFE and SFC, such as recovery of organics from oil shale, separations of biological fluids, investigation of biomarkers, selective extraction of fragrances, oils, and impurities from agricultural and food products, pollution control, additives in polymers, drug discovery in pharmaceuticals and biopharmaceuticals, environmental controls, and cleaning validation.

	1.  http://ec.europa.eu/food/plant/protection/pesticides/index_en.htm

	2. M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, 

	5. Eric Lesellier, ICOA, UMR 7311, SFC 2014

 graduated with a diploma in chemical engineering from the Technical University, NTA in Isny, Germany, in 2004 and a Master of Science in pharmaceutical analysis from the University of Strathclyde in Glasgow, UK, in 2005. Until 2006, she worked as a consultant in HPLC method development and validation in an analytical laboratory of the FAO/IAEA in Vienna, Austria. She gained her doctorate for research in pharmaceutical sciences at the University of Strathclyde in 2010 and was employed as HPLC specialist in the R&D department at Hichrom Ltd. in Reading, UK, from 2009. Since 2013, she has worked in the analytical business unit of Shimadzu Europa in Duisburg, Germany, as a HPLC product specialist.			 						E-mail:

Features

SFE–SFC–MS for the Analysis of Pesticide Residues in Food Products

As interest in supercritical fluid chromatography (SFC) grows, practitioners require that the technique delivers reproducible results. For SFC separations to be successful in regulated quality control and release testing laboratories, the robustness of the separation system must be well understood. The industry standard mobile phase in SFC separations is compressed CO

 mixed with a co-solvent, most often methanol, chosen for cost, speed, and solubility reasons. However, the water-free nature of CO

/methanol mixtures can impose slow changes to chromatography over time as a result of interaction between the stationary and mobile phase. These changes are documented and defined in this article.

	Interest in supercritical fluid chromatography (SFC) has grown in recent years, in large part as a result of new chromatography products and stationary phases. New SFC-specific stationary phases now include bondings and ligands designed and optimized for use in CO

-based separations, which can reduce or eliminate the need for additives. One of the most compelling reasons to consider SFC over other separation modes is the green nature of CO

-based mobile phases (less organic waste and faster dry down). These mobile phases are also relatively inexpensive, offer exceptional solubility for small molecules and are particularly useful for performing preparative separations. In addition, SFC mobile phases are typically, or are nearly, water-free, which makes them ideal when separating analytes that are hygroscopic or degrade quickly in the presence of water. Also, SFC is particularly well-suited for the recovery of free bases.	 	A common misconception with stationary phases is the assumption that the retentive surface never changes and that the interaction with the mobile phase is only to affect retention. The condensation of alcohols with silanols to form silyl ethers is a wellâknown and well-understood phenomenon in silica chemistry.

 This phenomena occurs under the water-free conditions of SFC and is a result of the slow reaction of the alcohol co-solvent with silanols to create silyl ethers on the particle surface. We have recently shown that the changes in surface polarity arising from silyl ether formation (SEF) contributes to changes in retention and selectivity in SFC separation.

 In this article, we describe the observable characteristics of SEF - in particular, changes in chromatographic results over time. While not every change in retention can be or should be attributed to SEF, our investigations reveal that this can be a significant contributor to variability. This type of slow, steady change in retention of SFC stationary phases has been documented.

	 	Found on the surface of silica-based materials, silanols react with alcohols (methanol, ethanol, etc.) in the mobile phase to create surface-bound silyl ethers. As shown in the equilibrium depicted in Figure 1, a silanol can be converted to a methyl-silyl ether to produce a water molecule. In simplistic terms, the surface is being bonded with ethers. Under typical SFC conditions, the equilibrium is driven towards the formation of silyl ethers. SEF should not be confused with physical changes to a packed bed structure, for example, voids or bed shifting, resulting in significant changes in peak profiles (loss of efficiency, increase of peak tailing). The change is chemical in nature and will affect the chromatography. Furthermore, the reaction equilibrium is greatly influenced by the presence of water, even in small amounts. Because typical SFC separations use water-free, or nearly water-free, mobile phases and silanols are commonly used to retain analytes, the effects of SEF are detectable. SFC is mostly operated in normal phase mode, which functions by using polar-polar interactions between analytes and the stationary phase. The polar surface of silica-based particles affects retention and selectivity under those conditions. Decreasing the polarity of the particle surface results in an obvious loss of retention and change in selectivity. In the past, normal-phase liquid chromatography (LC) had similar equilibration issues, often as a result of the presence of residual water. Practitioners worked around the problem by either using very dry solvents or saturating the mobile phase with water (that is, vigorous shaking of hexane with water before use). Some of the reluctance to use normal-phase LC, in favour of reversedâphase LC, can be attributed to these issues. For preparativeâscale separations, compound collection is often done in a mass spectrometry-directed fashion, so the change in retention or selectivity may be ignored.	
			 	 	

	The SEF reaction requires two basic parameters: 1) that the liquid phase be nearly water-free, and 2) that the liquid phase must contain an alcohol. While SEF is prevalent under SFC conditions, the chromatographic response will not be observed in all modes of chromatography. For example, size-exclusion chromatography (SEC), reversed-phase LC, and hydrophilic interaction liquid chromatography (HILIC) separations make detection of SEF unlikely because of the nature of the mobile phases and the separation mechanisms used. When performing non-retentive separations (for example, SEC) there is little or no interaction between the analyte and stationary phase surface (well chosen SEC phases will prevent adsorption of analytes to the particle surface). For a silanol and alcohol to condense, the subunits must be in proximity and the reaction must be favourable. Other nucleophiles are expected to react in a similar fashion with silanols, but alcohols will form the defined silyl ethers. Without a catalyst, the reaction is quite slow, and can occur continually for several days. We have successfully catalyzed SEF reactions on a chromatographic column by mixing a tertiary amine into methanol and observed an acceleration in the retention drift effect.

 In the case where a reversedâphase LC material or, more generally, an endcapped stationary phase is used in SEF favorable conditions, we have observed an increase in retention over time. The retention increase is not a result of SEF, but can be explained as a gradual loss of bonded phase, exposing silanols, which increases retention of hydrophilic analytes. We speculate that it would be possible to chromatographically detect SEF under HILIC conditions, where the weak eluent is acetonitrile and the strong eluent is methanol. However, confirmational testing has not been attempted and those conditions are rarely used. When there is a significant amount of water (>1% by volume) in the mobile phase, the equilibrium will shift towards the free silanol form, which is nearly always the case for reversed-phase LC and HILIC separations.	 	Detecting SEF on chiral stationary phases used under SFC conditions is more difficult. The most popular chiral phases are made of wide-pore, low surface area (about 10 times less surface area than comparable smaller pore materials) silica particles that have been thickly coated or functionalized with polysaccharides. The chiral separation mechanism takes place in the chiral environment afforded by the high order structure of the polysaccharides - very different from the interactions of small-ligand bonded phases. It has been well-documented that these phases are susceptible to changes when exposed to strong acids or bases,

 which is related to changing the chemical nature of carbamateâlinked selectors, but not the silica surface. SEF is not expected to play a significant role or be detected in polysaccharide-coated or functionalized phases because of limited access to silanols. In the case of bonded chiral selectors (brushâtype), if the phase has accessible silanols (not endcapped), SEF is expected to occur and retention changes would be detectable. If a brush-type chiral phase is endcapped, increases in retention over time could be observed as endcaps hydrolyze from the surface. 	
			 	 	

Explanation of Evidence and Mitigation Routes 

	We have documented both chromatographic and nuclear magnetic resonance (NMR) evidence of the formation of silyl ethers on both bonded (2-ethylpyridine) and unbonded silica (and inorganic/organic hybrid silica

 Our chromatographic findings allowed us to learn more about the SEF process, such as the relative reaction speeds on silica and hybrid silica particles; the latter being about two times slower because of lower silanol activity and not as a result of the number of silanols (stoichiometrically there are only ~10% fewer silanols). Figure 2 is an example of the retention change difference between a traditional SFC stationary phase and a modern phase that uses a high-density bonding to mitigate the effects of SEF. This is accomplished through limiting access of analytes to silanols on the particle surface. Even though SEF is still believed to occur at the particle surface, retention over time is unaffected because of stronger interaction of the analytes with the polar ligand. 	 	

	 	Our experiments show that a column exhibiting lower retention as a result of SEF can be returned near to its initial retention state by flushing with many volumes of water. Furthermore, we successfully baseâcatalyzed SEF and hydroxylation using hybrid silica particles, as monitored by the change in selectivity of a pair of well-chosen analytes. We were also able to form silyl ethers with different alcohols, ethanol and n-propanol. We found the reaction rates to be many, many times slower than that of methanol, but the chromatographic response was similar. These results all point to a characterizable chemical reaction taking place on the particle surface, which has a detectable effect on the chromatography. In our investigations, we have not been able to attribute significant changes in peak shape to SEF. None of our evidence suggests that the acidity or reactivity of CO

/methanol mobile phases, because we have performed the SEF reaction in pure methanol mobile phases; only the exposure time and frequency or concentration of methanol had an effect. As a final note, SEF can be mitigated by doping mobile phases with water; however, the controllability and reproducibility is questionable.	 	

	We have identified changes in surface chemistry resulting from SEF as a significant source of retention drift in SFC when using alcohol modifiers under non-aqueous conditions. This observation is important to users of SFC because the gradual decrease in retention times can result in chromatographic irreproducibility for a given method. 	 	

	2) J.N. Fairchild, D.W. Brousmiche, J.F. Hill, M.F. Morris, C.B. Boissel, and K.D. Wyndham, 

	5) K.D. Wyndham, J.E. O’Gara, T.H. Walter, K.H. Glose, N.L. Lawrence, B.A. Alden, G.S. Izzo, C.J. Hudalla, and P.C. Iraneta, 

is a senior research scientist at Waters Corporation in the Consumables Business Unit (CBU). After receiving his PhD from the University of Tennessee in 2010, he has been researching and developing novel SFC stationary phases. The research team at Waters has a goal to understand selectivity in CO

-based mobile phases and improve the relative ruggedness of columns. 

 is a principal scientist in the CBU Synthesis Group at Waters Corporation. He is involved in the development of new stationary phases, SPE materials, and tagging reagents. 

Explaining Silyl Ether Formation (SEF) in Supercritical Fluid Chromatography (SFC)

The 5th International Network of Environmental Forensics (INEF) 2015 conference will be held from 3–6 August 2015 at Victoria College at the University of Toronto, Ontario, Canada. 

	The International Network of Environmental Forensics (INEF) conference series began in 2008 to provide a forum for scientists, environmental consultants, regulators, and lawyers to share information regarding the use of environmental forensics. 

	The theme of this year’s conference is “Emerging Trends and New Technologies”. A combination of plenary presentations, short courses, and oral and poster presentation technical sessions will be hosted alongside a lively and varied social programme to encourage networking. There will also be exhibitor stands showcasing the latest instrument and software products available to help solve complex environmental forensics problems.	 	The short courses will be held on 3 August, focusing on specific areas of environmental forensics and will be given by internationally recognized experts in their respective fields including: statistical fingerprinting as a line of evidence in a forensic evaluation by Mike Bock, application of environmental forensic techniques to track oil spills and “fake” oils by Ioana Petrisor, and applications of GC×GC by Jack Cochran and Frank Dorman. 	 	The programme will also have a number of plenary lectures including “Experts in Environmental Litigation” by Marc McAgree, Law Society of Upper Canada Certified Specialist in Environmental Law, “Where Does All the Oil Go?” by Stephen Mudge, Exponent, and “The Use of Nuclear Magnetic Resonance for Determining Molecular Structure” by Andre Simpson, University of Toronto.	 	The full scientific programme is listed on the conference website and includes over 100 presentations in a wide variety of topics listed in the following sessions: multidimensional chromatography; microbial source tracking; passive sampling; wastewater; human exposure; monitoring freshwater environments; petroleum hydrocarbons; legal case studies; emerging contaminants of concern 1; emerging contaminants of concern 2; atmospheric source tracking; inorganic forensics; persistent organic pollutants; statistics; advances in analytical assessment; and compound specific isotope analysis.	 	As well as the series of high-quality lectures there will also be breakout periods with poster presentation sessions and exhibitor booths for delegates to browse with complimentary refreshments provided. There will also be talks from students currently studying environmental forensics. To boost student participation INEF are offering several travel grants to prospective students to help them attend the conference. Students are also encouraged to enter the student competition for the best presentation when submitting their abstracts. The top three students will all receive a generous monitory prize. 	 	To supplement the scientific programme and refuel those tired minds, a reception is planned on the Tuesday and will be held in Dinosaur Hall at the Royal Ontario Museum (a short 2 minute walk from Victoria College). Delegates will have the opportunity to browse special exhibits in the museum collection.	 	 The conference will be held in the prestigious Victoria College at the University of Toronto, which is located in the heart of Toronto with excellent local transport facilities. Conference delegates may wish to extend their stay in Toronto to experience the Panam and Parapanam games that are happening either side of the conference. There is also plenty to see and do in and around Toronto, with a large choice of shopping opportunities, plus vibrant cafes, bars, and restaurants to enjoy. Delegates with a head for heights can visit and abseil off the famous CN Tower. Those looking for a more peaceful time can visit the local Islands, or take a couple of hours drive to explore Algonquin Park by foot or canoe or experience the impressive Niagara Falls. 	 			E-mail: 

The 5th International Network of Environmental Forensics (INEF) 2015 conference will be held from 3–6 August 2015 at Victoria College at the University of Toronto, Ontario, Canada.

The 5th International Network of Environmental Forensics 2015 (INEF 2015) Conference

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Determining aroma compounds and off-flavours in edible oils by gas chromatography coupled to mass spectrometry (GC–MS) is routine in many food-producing companies; however, the oily matrix needs to be kept out of the analytical instrument to avoid impacting analytical stability. The following article describes a simple, yet efficient, way of eliminating the matrix while determining the flavour compounds in a sensitive and selective manner.  

	Products that taste good will be well liked and will sell well, but only as long as consumers are satisfied with the quality. This is nothing new, but it can be challenging for producers to ensure the uniform taste of products when raw products come from different sources, and different suppliers are used over the course of the year in different production locations worldwide.

	Even though variations in flavour and taste rarely affect consumer safety, the offâflavours and aroma profile of a product can make a big difference in its acceptability. It is well known that consumers remember negative experiences more vividly than positive experiences - and that they tend to share a negative experience more readily with others. Therefore even the occasional negative eating experiences have a wide ranging and long lasting impact on future buying decisions. As a consequence of this producers are interested in frequent quality controls.

	Sensory testing is traditionally performed to confirm an even product quality. This is very clearly a fully valid approach and we should not attempt to replace this with instrumental chemical analysis. However, the strength of instrumental analysis is that it can explain sensory findings and determine the reasons for flavour deviations. Analytical instruments also never get tired, are available at any time, and results are traceable to a calibrated balance.

	In order to take advantage of the benefits of aroma analysis as a complementary tool to sensory testing, aroma specifications need to be established. These specifications require a reliable instrumental technique, which is robust and delivers correct results reproducibly. As aroma compounds very often are trace compounds hidden amongst a multitude of volatile matrix compounds, the analytical method of choice also needs to be quite sensitive. The selectivity of a GC in combination with a mass spectrometer is sufficient for most applications.

	Determination of aroma compounds in edible oils (such as olive oil, sunflower oil, fish oil) is important for both manufacturers and vendors. Off-flavours derived from unsaturated fatty acid degradation are especially important (for example, hexanal, 2-[E]-nonenal and 2,4-[E,E]-decadienal), because these compounds can compromise the taste and therefore quality of a product even in the ng/g concentration range.1,2,3,4 Sensitive and fast analysis methods, ideally combined with simple sample preparation, are needed. 

	A sensitive analysis method based on dynamic headspace sampling was previously developed in 2008.5 At the time, direct thermal desorption from standard microvials placed in thermal desorption tubes was also tested, but although the technique performed well for volatile analytes, the resulting sensitivity for less volatile analytes was not satisfactory. In order to have good reproducibility, it is important to achieve high recovery of volatile aroma compounds such that the quality of the analysis results is not critically dependent on minor day-to-day variations in the analytical system. For very volatile and chemically stable compounds this is relatively easy to achieve. The difficulties arise with less volatile compounds. Therefore the focus in the development of any instrumental technique needs to be on these compounds. Generally speaking, it is desirable to minimize the distances in the system that the analytes must travel, minimize surface areas, and eliminate cool zones in the analyte flow path where these high boiling compounds may be lost through adsorption or condensation. In the case of this work, the oil samples were placed in microvials inside a thermal desorption unit for thermal extraction close to the site where the aroma compounds were subsequently trapped before being transferred to a GC system for analysis. In addition, the position of the slit in the microvials used to purge the analytes through to the trap was optimized for best recovery, that is, for most efficient purging of the analytes, minimizing the length of the flow path. In this project we returned to the microvial approach and evaluated different microvial designs to improve the transfer of high boiling compounds while maintaining the excellent performance for the volatile fraction.

	Materials: Standard empty TDU tubes with a single notch were used for thermal desorption. Oil samples were placed in different types of microvials, in which purge slits were placed at different distances from the bottom of the microvial or which were cut to different lengths. Analyte recovery depends significantly on the distance between the surface of the oil sample and the purge slit. With increasing distance, the volume between the two is increasingly unswept by the purge gas. Analyte recovery is therefore reduced since it relies mainly on diffusion from the surface of the oil sample to the area near the purge slit.

 Thermal desorption of oil samples in microvials was conducted in a Thermal Desorption Unit (TDU) and analytes were refocused in a Cooled Injection System (CIS 4), PTV-type inlet (Gerstel) at a low temperature before being transferred to the GC column. A 7890/5975 GC–MS system (Agilent Technologies) was used for separation and determination of the analytes of interest. Thermal desorption tubes containing the samples were delivered to the TDU automatically using a MultiPurpose Sampler (MPS) (Gerstel). 

Edible oil was spiked with the analytes listed in Table 1 in the concentration range from 10 to 1000 ng/g. A set of 30 mg samples of the oil were weighed into individual microvials and placed in thermal desorption tubes. Each tube was capped with a transport adapter and placed in the autosampler tray for later desorption. The presented case focuses on edible oils, in this case of potato chip production quality. A range of aroma compounds was chosen, which cover a few relevant aroma attributes: methional (potato), pyrazines (roasted), alkenes (fatty/rancid, cardboard), 3-methylbutanal (malty), dimethyltrisulphide (vegetable, cabbage), and gamma-decalactone (creamy, coconut). For calibration purposes three internal standard compounds were added during sample preparation: one more volatile (4-Heptanone), one less volatile (2,3-Dimethoxytoluene), and finally 2-methylpentanoic acid as internal standard for volatile acids. These compounds do not occur naturally in food material and have excellent chromatographic properties making them well suited as internal standard compounds.

TDU: solvent venting; temperature programme: 30 °C; 200 °C/min; 90 °C (15 min); PTV: glassbead liner; 0.2 min solvent vent (30 mL/min); split 3:1; temperature programme: -70 °C; 12 °C/s; 280 °C (30 min); column: 15 m × 0.25 mm, 0.25âmm ZB-FFAP (Phenomenex); pneumatics: He, constant flow = 1.3 mL/min; oven temperature programme: 35 °C (1 min); 4 °C/min; 120 °C (5 min); 50 °C/min; 250 °C (8 min); MSD: SIM, cf. Table 1.

Ten oil samples of 30 mg each were prepared in microvials of the selected design and individually desorbed in the TDU to determine the repeatability.

	Thermal extraction of aroma compounds from edible oils using microvials can prevent contamination of the analysis system by highâboiling matrix compounds while allowing effective transfer of semi-volatile organic compound (SVOC) analytes onto the analytical column. Thermal desorption of the aroma from the oil sample in the microvial should be performed at relatively mild temperature conditions to avoid both analyte degradation and the formation of additional aroma compounds by thermal degradation of the sample. On the other hand, the recovery of aroma compounds during thermal desorption increases dramatically with increasing temperature, especially for less volatile compounds. Depending on the nature of the sample, a good compromise has to be found. After analysis the microvial can be disposed of and the desorption tube is ready to take up the next sample. 

	In our work, we determined that microvials with a purge slit placed at a height of 1 cm from the bottom were the most effective for analyte transfer, especially for high boiling compounds (gamma-decalactone), as shown in Figure 1. Analyte recovery expressed as relative peak area for the different types of microvials used are shown in Figure 2.

	It is reasonable to assume that the vapour phase directly above the sample surface would be more efficiently purged when using the slit design than when using a standard microvial, which mainly relies on analyte diffusion through a stagnant vapour phase volume because of the extended distance between the sample surface and the purge flow path. In other words, a microvial with a slit enables faster and more efficient analyte transfer from the sample to the analysis system. A different effect seems to be responsible for the improved transfer of 2-methylpentanoic acid when using the microvial cut to a 1 cm length. The reduced glass surface area in the analyte transfer path could be beneficial for transfer of active compounds such as acids.

	Relative standard deviations for 10 repeat measurements with the 1 cm slitted microvial were between 7.2% and 16.8% with a median of 9.7% (Figure 3). This is highly acceptable considering the complex matrix, the low concentrations, and the straightforward sample preparation. A longer desorption time would likely improve the relative standard deviations further. 

		Aroma compounds can be determined in edible oil down to the ng/g concentration range by direct thermal desorption from a slitted microvial.

		Introduction of liquid samples to the microvials is easily performed using a pipette and is the only required sample preparation step.

		The microvial design with the slit at 1 cm distance from the bottom was found to deliver the best performance. 

		Slitted microvials can be beneficial for the determination of volatile and semi-volatile compounds in other liquid, paste, or solid materials.

Journal of Agricultural and Food Chemistry

Comprehensive Reviews in Food Science and Food Safety

Journal of the American Oil Chemists Society

	5. O. Lerch and C. Gil, Gerstel App Note 03/2008.

, Ph.D., is a specialist in flavour analysis and flavour technology. He has worked for nearly 20 years in various positions in flavour research at Kraft Foods, Germany. In 2011 he and Claudia Geyer founded FlavoLogic GmbH, a flavour consultancy in Munich, Germany. Together with his team, he provides solutions for off-flavour issues, aroma analysis, sensory testing, innovative flavour technology, and customized flavours and flavour recipes. 

, Ph.D., works as a scientist in the Analytical Services department at Gerstel. After receiving his Ph.D. from the Ruhr University Bochum, Germany, in 2003, he worked as a postdoctoral fellow at the Max-Planck-Institute of Molecular Plant Physiology in Potsdam, Germany, before joining Gerstel in 2005.

GC–MS Analysis of Aroma Compounds in Edible Oils by Direct Thermal Desorption