## 2 Isolation Techniques for Volatiles in Honeys
SDE extracts were rich in terpen and esters while the other two techniques embraced the formation of artifacts due to heating. To avoid the generation of artifacts (furan and pyran derivatives) and also the consumption of expensive and toxic organic solvents, some new methods have been put forth recently. These methods include solid phase extraction (SPE), headspace (HS) and solid phase micro-extraction (SPME) methods. The main advantage of the HS analysis is that it is carried out on an untreated sample [28] and the profile of the isolated volatiles is closely associated with sensory perception [20]. Dynamic SPE is a technique that eliminates the use of solvent entirely and removes interfering substances such as sugars and acids by merely washing with water [29], but requires extensive modification of the gas chromatographic injector or the addition of a desorption module. Consequently, SPME resolves the handicap associated with the former technique. SPME is a method that shows lucid advantages like; it eliminates the use of (toxic) organic solvents, allows the quantification of a large number of molecules, little or no manipulation/preparation of samples, substantially shortens the time of analysis and moreover, it is simple and covers a wide range of sampling techniques including _in situ_ and air sampling [26]. Moreover, it can be easily coupled to various analytical instruments, e.g., GC, GC-MS, HPLC, LC-MS and GC-O (GC-olfactometry) [23]. However, the efficiency of SPME technique is affected by the following important parameters: fiber coating, sample amount, matrix modification by water and sodium chloride addition, agitation of sample matrix, extraction temperature, extraction time and analyte desorption [30]. All these parameters could be optimized to ensure efficient extraction in order to obtain high recoveries of honey volatiles. For example, [PERSON] _et al_. [28] showed differences in volatile profiles of chestnut honey extracted by SPME using polydimethylsiloxane (PDMS)-coated fiber, polyacrylate (PAC)-coated fiber or Carboxen-coated fiber. Overall, PAC-coated fiber gave the best results while Carboxen-coated fiber showed peak distortion attributed to band broadening due to poor focalization inside the column; this could be related to slow desorption from Carboxen because of the high polarity of this phase. [PERSON] _et al_. [16] equally indicated the advantage of using fiber composed of polydimethylsiloxane/Divinylbenzene (PDMS/DVB). Most recently, [PERSON] _et al_. [6] evaluated the extraction efficiency of different types of phases of stationary fibers employed in HS-SPME analysis of VOCs in honey; divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) gave the best results in comparison to the previously mentioned fibers. SPME sampling can be performed in three basic modes: direct extraction, headspace extraction (HS) and extraction with membrane protection. HS extraction has been reported as the most appropriate sampling mode since it protects the fiber from adverse effects caused by non-volatile compounds that occurs in the sample matrix (namely, sugars) and also allows modifications (for example pH) with no effect on the fiber. Nonetheless, the drawbacks of this technique include its sensitivity against organic solvents and the limited range of stationary phase that are available commercially [26]. Several authors have used HS SPME for the extraction of VOCs from honey matrix [2, 31, 32]. Conclusively these varied isolation methods have been employed for the extraction of VOCs from honey matrix which were analyzed by GC-MS; valuable information were obtained that enabled the determination of honey's botanical origins. A novel approach for the isolation of VOCs was validated in the study by [PERSON] _et al_. [33]; we identified volatile compounds in solvent extracts of honeys produced in South Africa. Depending on our previous work on the antimicrobial activity of Selected South African honeys; we extracted our honeys with proven antibacterial activity with chloroform and n-hexane solvents.