6 February / March 2020
Figure 2: Heat-maps showing the extraction response obtained using regular and Vac-HS-SPME at 30 and 43 °C for different extraction time, namely 10, 20, 30 and 40 min. Compounds identification as reported in Table 1.
3. Results and Discussion
The heat maps reported in Figure 2 illustrate the overall change in the profile of the 12 targeted compounds when sampled using Regular and Vac-HS-SPME at both 30 and 43°C for different extraction times (10, 20, 30, and 40 min). The two heatmaps are normalised separately to emphasise the change in the response within a single temperature tested.
It can be observed as the Vac-HS-SPME sampling increased the general profile (colour turning toward red). However, an important distinction needs to be made between the compounds with the highest and lowest volatilities. For the former, generally, the same performance can be observed between regular and Vac-HS- SPME at 30 and 43°C, with even a slightly lower extraction yield at 43°C. The kinetics of these compounds is usually rapid; thus the effect of using reduced pressure conditions is limited or even non existent as these analytes have reached ‘equilibrium’. The slight reduction of the signal at 43°C using Vac-HS-SPME indicates that the effect of temperature for the earlier eluted compounds is comparable to the gain in extraction yield obtained using vacuum. This behaviour can be related to both an acceleration of the extraction kinetic (not assessable in the range of extraction time tested) and competition effect. It is interesting to notice that for the rest of the compounds the effect of vacuum and temperature is instead synergic, significantly improving the extraction efficiencies at an earlier sampling time, in fact for the latest
Figure 3: Total ion chromatogram obtained using regular (yellow, lower chromatogram) and Vac-HS- SPME (blue, upper chromatogram). Compound identification as for Table 1.
compounds (V6-V12) almost the same intensity of response is obtained at 43°C after 20 min using Vac-HS-SPME, meaning that the equilibrium is almost reached; while under regular conditions, the response is much lower compared to Vac-HS-SPME and in a clear ascending trend moving from 10 to 40 min.
Noteworthy is the improvement obtained for α-farnesene (V12), reported as an important marker for discriminating the geographical origin of extra-virgin olive oil [21,22]. An almost 10-fold increment was observed when Vac-HS-SPME sampling and higher temperatures are applied. Figure 3 shows the comparison of the chromatographic traces between regular- and Vac-HS-SPME when sampling at 43°C for 10 min.
It is important to highlight that the effect of increasing the temperature in Vac-HS- SPME of water-based solutions was not always successful especially for absorbent type SPME fibres [11]. In fact, the increased humidity in the headspace increased the pressure in the vial, thus reducing its benefits. In edible oil samples, like olive oil, water is not present (or is in trace amounts); therefore, heating can be exploited with beneficial effect, although care must be paid to avoid artifact formation and thermal degradation products. Moreover, in high viscous samples like olive oil, the increase in temperature decreases the viscosity of the fluid. The high viscosity value of olive oil (49 mPa at 30°C, 60-times larger than water) increased the liquid-phase resistance, ‘delaying’ the analyte diffusion through the liquid boundary layer of the
olive oil matrix. This phenomenom is of high importance when targeting analytes with a small affinity for the headspace, regardless of the pressure conditions used [16]. In fact, for these analytes, a multi-stage process occurs: analyte molecules are transferred from the liquid sample to the gas phase every time the headspace concentrations fall below equilibrium levels [14,16]. This ‘replenishment’ process depends on the resistance in the liquid phase (related to the viscosity). It was shown here, that heating the sample from 30°C to 43°C led to a 40% decrease in viscosity of the sample (i.e., ~30 mPa s) improving the liquid-phase diffusivity and thus the overall extraction yield. Since Vac-HS-SPME sampling impact significantly on the kinetics of extraction, this process of ‘replenishment’ becomes even more limiting than in regular HS-SPME. However, the use of still milder temperature (43°C) in combination with reduced pressure provide a synergic improvement on the overall extraction yield..
Two-variable CCD: a study of the response surfaces
To better characterise the gain in performance obtained using Vac-HS- SPME over regular, a full factorial central composite design (CCD) was used to optimise at the same time and temperature ranges both the regular and Vac-HS-SPME. Based on previous findings that highlighted that time and temperature were the main significant variables [19,20], a two-variable (k=2; temperature and time) CCD was applied. The extraction temperature
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