Two reference paraffin samples and two reference stearic acid samples were purchased. For both reference types, one sample was purchased online and the other from Kahlwax (NL) company, a leading specialist in the production of natural wax and several other products. The two paraffin samples were checked by IR spectroscopy and their corresponding spectra were almost identical. The same observation was also made for the stearic acid samples.
The calibration set was prepared by mixing three different non-adulterated beeswax samples and subsequently spiking them with paraffin and stearic acid in increasing amounts. Τhe absence of adulteration in the three beeswax samples was verified by PCA, where it was observed that these samples were projected in a big cluster away from the adulterants and together with honeycomb sidewalls that are considered pure, since the latter would only contain background levels of the two adulterants. For paraffin calibration samples, beeswax was spiked with paraffin at: 0, 5, 10, 15, 20, 25, 50 and 100% (w/w) denoted as CP1, CP2, CP3, CP4, CP5, CP6, CP7, CP8 and for stearic acid calibration samples, beeswax was spiked with stearic acid at: 0, 0.5, 1, 5, 15, 25, 50 and 100% (w/w) denoted as CA1, CA2, CA3, CA4, CA5, CA6, CA7, CA8. For each calibration sample, the solid mixture was homogenized by melting at 85 °C for 1 h and re-solidified by cooling the mixture at room temperature.
Note that different concentrations were considered for the two calibration sets, particularly for spiking levels below 15%. This was primarily carried out to account for the different LOD values reported for stearic acid (1%) and paraffin (5%), respectively. For this reason, spiking with stearic acid started at very low concentrations (<1%), whereas in the case of paraffin, spiking at such low levels would not allow differentiation from pure beeswax. Τhus, the first paraffin spiking level was established at ~5%. Above 15%, approximately the same concentrations were considered for both adulterants, covering the entire range needed for calibrations. The calibration samples and their nominal concentrations are shown in detail inTable S1 .
The calibration set was prepared by mixing three different non-adulterated beeswax samples and subsequently spiking them with paraffin and stearic acid in increasing amounts. Τhe absence of adulteration in the three beeswax samples was verified by PCA, where it was observed that these samples were projected in a big cluster away from the adulterants and together with honeycomb sidewalls that are considered pure, since the latter would only contain background levels of the two adulterants. For paraffin calibration samples, beeswax was spiked with paraffin at: 0, 5, 10, 15, 20, 25, 50 and 100% (w/w) denoted as CP1, CP2, CP3, CP4, CP5, CP6, CP7, CP8 and for stearic acid calibration samples, beeswax was spiked with stearic acid at: 0, 0.5, 1, 5, 15, 25, 50 and 100% (w/w) denoted as CA1, CA2, CA3, CA4, CA5, CA6, CA7, CA8. For each calibration sample, the solid mixture was homogenized by melting at 85 °C for 1 h and re-solidified by cooling the mixture at room temperature.
Note that different concentrations were considered for the two calibration sets, particularly for spiking levels below 15%. This was primarily carried out to account for the different LOD values reported for stearic acid (1%) and paraffin (5%), respectively. For this reason, spiking with stearic acid started at very low concentrations (<1%), whereas in the case of paraffin, spiking at such low levels would not allow differentiation from pure beeswax. Τhus, the first paraffin spiking level was established at ~5%. Above 15%, approximately the same concentrations were considered for both adulterants, covering the entire range needed for calibrations. The calibration samples and their nominal concentrations are shown in detail in
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