Oven extraction is another very simple extraction technique in which the sample is placed in a suitable container or even the sample itself along with a solvent and then place in an oven for a period of time.  It can either be placed in a static environment or far more commonly in a dynamic way through shaking/agitation [1].  It is this approach of elevated temperatures and agitation that the BioPhorum published in their study plan for single-use systems (40ºC and 50 rpm on an orbital shaker)[2]

Oven extraction has a particular advantage in the size and scale of the extraction, it can go from a few microlitres to litres (the automotive industry has ovens that are used to look for volatile organic compounds of complete cars [3].

The complexity of an oven extraction can be increased by increasing the level of automation.  This can start at simultaneous extraction and analysis to a whole range of additional activities e.g. centrifugation.  Two systems exist that can carry out simultaneous extraction and analysis for volatile and semi-volatile/non-volatile species. This simultaneous extraction and analysis can be a tremendous time-saver and hence can also maximise equipment utilisation and minimise analyst involvement. As with all extraction techniques, the principle is reliant on the use of appropriate temperature, time and solvent conditions.  Direct analysis of the very volatile species after heating e.g. by headspace gas chromatography will not be discussed in this article.

For the volatile species, the automated extraction and analysis process is as follows. A vial is loaded with an appropriate amount of material (weight) and the vial capped and crimped. The vial is then loaded onto the equipment. The remainder of the process can be fully automated, as shown in the following example. A known volume of solvent is added by the instrument, containing an appropriate internal standard. The vial is automatically transferred to the heating agitator, where it is shaken at elevated temperatures for a set time.  See Figure 2 for the apparatus. After the set time, a sample is taken automatically and injected directly into the GC with mass spectrometry (MS) detection and/or flame ionisation detection.  The apparatus can also add a range of solvents during the analysis, allowing for prolonged unattended operation. This standard analysis can allow for the usual identification and quantification to take place. The time and the temperature in the agitator can be varied to produce asymptotic extraction levels. The asymptotic levels can be readily plotted in graphs.

The equipment shown in Figure 2 is Gerstel MPS 2 dual-rail multipurpose autosampler fitted on top of an Agilent 7890GC,5975B MSD. The solution was supplied and configured by Anatune Limited. One rail is equipped with a 1 mL syringe to address liquid handling for the sample preparation whilst on the other rail a 10-µl syringe allows injection of the freshly prepared sample onto the GC-MS. The automated sample preparation was performed using the following objects: Solvent Filling Station, Agitator, wash station and 10mL/20mL vial trays. The solution offers a vial capacity of up to 240 vials for long unattended experiments. The system is controlled with Gerstel Maestro software with full integration into an Agilent MSD Chemstation producing a single sequence table. For a more simplified system, a single rail can be used but, in this case, the solvent containing an internal standard would have to be added manually.  Since this work was done there have been advances with the hardware allowing an increased range of activities in sample preparation which is outside the scope of this article.

Figure 1 Shaking apparatus Agilent 7890GC, 5975B MSD and Gerstel MPS 2 dual-rail system.

Example data generated using the shaking apparatus is shown in Figure 2. This was generated using the prescribed extraction duration with the agitator set to 10 °C below the boiling point of the solvent. It shows that DCM is the most effective solvent at extracting compounds and that asymptotic levels are reached after ~30 min. In this case, the optimum extraction time for DCM is very close to the GC analysis time. This allows maximum analysis to be undertaken in the minimum time if the extraction is carried out while the analysis of the previous samples is carried out.

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Figure 2 Asymptotic plot showing the levels of a given species with each solvent against time (presented previously [i])

The shaking system described above could also be connected to a HPLC inlet system. For such a system, careful considerations must be made when deciding whether an extraction solvent could be used with a reverse-phase (aqueous-based) chromatographic setup. For example, hexane is not water-soluble. In addition, if the system were to be used for HPLC sample preparation, then it would take up a considerable amount of laboratory space.

For the semi-volatile-to-non-volatile species, a similar mechanical extraction system exists: the I‑Chem explorer An example of the Agilent set up is shown in Figure 3. But can also be used with Waters and Shimadzu [4].

Figure 3 The I-Chem explorer

The sample, with solvent, is heated together with agitation for appropriate times, similar to the previous system. By sampling at prescribed intervals, asymptotic extraction conditions can be determined. Sample agitation is achieved by the use of a magnetic flea. The vial can be heated up to 150 ºC and with stirring up to 1500 rpm. After heating, the sample is automatically analysed by liquid chromatography with suitable detectors, such as ultraviolet and/or MS. This technique does suffer from several drawbacks. As discussed previously, solvents such as DCM and hexane cannot be easily used with this system because they are not miscible with typical HPLC mobile phases. The solvent also has to be added before the analysis/heating being started, so accurate timings of asymptotic levels may not be feasible. However, as long as these can be produced reproducibly, this should not be an issue.

[1] A Feilden., Update on Undertaking Extractable and Leachable testing ISBN 978-1-84735-455-6

[2] BioPhorum best practise guide https://www.biophorum.com/wp-content/uploads/Best-practices-guide-for-extractables-testing-April-2020.pdf accessed August 2020

[3] ISO 12219-1:2012(E) Interior air in Road Vehicles-part 1: The Whole Vehicle Testing Environmental Chamber-specification and Method for the determination of volatile Organic Compounds inside the Vehicle

[4] https://reactionanalytics.com/products/ accessed August 2020