Rapid, High Capacity Evaporation Solutions for Sophisticated Fractionation Processes
IntroductionIn the quest to find novel flavor ingredients from nature, powerful equipment that can produce and concentrate fractions derived from natural extracts is essential. Conventionally, many steps of fractionation and evaluation are necessary until a compound is obtained at the purity required for structural analysis.
International Flavors & Fragrances Inc (IFF) has developed a state of the art fractionation process using the most modern equipment for this purpose. Key components of this system are the Sepbox® instrument made by Sepiatec GmbH (Germany) and the Genevac® HT-24 (Genevac Ltd., UK). The Sepbox allows for two dimensional, preparative scale fractionation of natural products using high performance liquid chromatography – solid phase extraction (HPLC-SPE). In sequential HPLC-SPE / HPLC-SPE steps individual components are isolated from complex mixtures. The Sepbox automates all of these stages.
How it all startsThe increasing demand for “natural” products makes natural product research a potentially lucrative point of research emphasis. The vast variety of plant and animal sources that have been traditionally consumed by mankind makes selecting staring materials crucial for a time and resource efficient approach.
In order to narrow down possible sources for extraction, IFF conducted wide ranging consultation both internally and externally. IFF employs research chefs and flavourists, which were very helpful in directing our sample sourcing. The IFF chefs provided ideas on materials but also prepared several foods as a starting point for extraction. Besides internal recommendations we also researched traditionally used foodstuffs from all around the globe and consulted many other food professionals.
Little is known about the properties a compound must exhibit in order to be taste-active, we believe an exhaustive extraction over a wide range of polar compounds to be the most promising approach. A series of three successive extraction techniques were used, cold and hot organic solvent as well as water are employed. It is quite a challenge for a researcher to look at a flask full of dark, crude “sludge” and liquid as a great deal of work is necessary to isolate just one particular molecule from all of the other materials present. The predicament is that we do not want to loose any potentially successful compounds, but we also don’t want excessive equipment downtime because our samples deteriorate system performance. Several techniques are described in order to remove unwanted components such as chlorophylls and fats  which prove useful.
The fractionation process
Figure 1: Design of the Sepbox
The Drying ChallengeEach separation run yields 320 sample vials total, each with a volume of approximately 45ml. The Genevac HT-24 (figure 2) was chosen to dry the samples, because it has two chambers with a large individual vial loading capacity. The vials used to collect the fractions aren’t a standard size, therefore
Genevac designed a custom sample holder to fit. The system can evaporate 192 vials at a time (96 per chamber). Within two days all fractions are dried and ready for further liquid handling and testing. 285 vials contain 45ml of ternary organic solvent mix, with approximately 5% water. Another 35 vials come from a water acetonitrile gradient, from 0 to 100% of each solvent. Conditions for evaporation must be gentle since degradation or even evaporation of possible target compounds would be a great loss. In the
evaporator the evaporation conditions are carefully controlled to provide fast and safe evaporation. The Dri-Pure® system built into the evaporator prevents bumping and cross contamination, and the software in the system controls and monitors the pressures and temperatures of the samples. Temperature measurement is via a thermocouple placed in the sample holder, an additional thermocouple is placed into a sample to provide further feedback on the evaporation process.
ConclusionsNatural product derived discovery of novel flavor molecules is a highly complex process. Natural product sources yield complex extracts containing many components which may not lend themselves to processing and analysis via traditional laboratory equipment. Based on wide consultation and literature search scientists at IFF have developed a well optimized process to extract, separate, process and evaluate natural product derived sources of flavor molecules.
Every stage of the process must be fully evaluated to ensure that by introducing one element of automation a bottleneck or other problem is not created down stream.
The authorsStephen Pickrahn is a senior scientist working at International Flavors & Fragrances R&D facility in Union Beach, New Jersey, USA. (Phone: 732-335-2587, e-mail: email@example.com).
Rob Darrington is Product Manager for Genevac, based at their Ipswich, UK head office.
 Hostettmann et al., Preparative Chromatography Techniques, Springer 1998.
Sepbox® is a registered trade mark of Sepiatec GmbH, Berlin, Germany.
Genevac® & Dri-Pure® are registered trade marks of Genevac Ltd, Ipswich, UK.
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