**1. Introduction**

Cuticular waxes are compounds ubiquitously present on the surface of all kinds of vegetable matter. They cover leaves, flowers, seeds and other vegetable structures, exerting the main functions of (i) controlling the perspiration, (ii) insulating the plant from external water and (iii) protecting it from pathogens [1,2], biotic and abiotic stresses and plant-insect interaction [3–5]. A cuticular wax is a complex mixture of long-chain alkanes, alkenes, alcohols, aldehydes, alkyl esters, fatty acids and other compound families [2,4,5]; although the large majority is represented by long-chain hydrocarbons [2]. Depending on the plant species, the total amount and composition of cuticular waxes can vary widely [4,6]: i.e., every vegetable species (and even organs from the same vegetable) can exhibit a unique composition. Cuticular waxes are not only interesting from an analytical point of view; they can have industrial applications in the field of cosmetic formulations and healthcare products [7,8], since they show a very large affinity with human skin thanks to the prevalence of odd long-chain hydrocarbons with respect to the analogous products coming from fossil feedstocks [8,9].

The current methods for extracting natural waxes from vegetable matter use large quantities of toxic organic solvents [10]. Guo and Jetter [11] studied cuticular waxes coming from potato leaves and other potato organs, after extraction using chloroform; the samples were extracted twice for 30 s. The same procedure was adopted by Jetter et al. [12] to process *Prunus laurocerasus* L. leaves. Cheng et al. [1] extracted cuticular waxes from rose petals and leaves using chloroform as the extraction solvent, in which the samples were immersed three times for 30 s. Trivedi et al. [2] used the same organic solvent to extract cuticular waxes from bilberry fruits; the immersion was 1 min long. Pimentel et al. [13]

**Citation:** Scognamiglio, M.; Baldino, L.; Reverchon, E. Fractional Separation and Characterization of Cuticular Waxes Extracted from Vegetable Matter Using Supercritical CO2. *Separations* **2022**, *9*, 80. https:// doi.org/10.3390/separations9030080

Academic Editor: Hailei Zhao

Received: 15 February 2022 Accepted: 16 March 2022 Published: 20 March 2022

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processed *Croton* leaves using three consecutive immersions of 30, 20 and 10 s duration in dichloromethane. They systematically identified C19 to C33 alkanes and C18 to C34 alcohols.

Therefore, the extraction of cuticular waxes is carried out, as a rule, by liquid solvent extraction and chloroform is the most frequently used solvent. Moreover, the process is performed in a very fast manner to minimize the co-extraction of other undesired compounds [14]. However, when other extraction techniques are used, as in the case of Soxhlet method that can last some hours, other compounds and intracuticular waxes can be also extracted and the authors generally do not give indications about these co-extracts. In particular, cuticular waxes represent interfering compounds that are extracted together with the desired ones, since the target compounds generally have a biological/industrial interest, such as essential oil, coloring matter, antioxidants and active principles for pharmaceutical applications [8]. However, they are systematically co-extracted during solvent extraction, as previously discussed [15], and during alternative processes [16], like ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE). Therefore, they have to be eliminated by post-processing procedures, such as the so-called winterization [17], in which the extract, dissolved in the organic solvent, is cooled at very low temperatures (e.g., from −10 ◦C to −40 ◦C) for several hours to precipitate cuticular waxes that are subsequently separated by filtration [18].

CO2 at supercritical conditions (SC-CO2) is largely used to extract the compounds of interest from vegetable matter. In particular, above its critical point (Pc = 73.8 bar and Tc = 31 ◦C), CO2 shows a liquid-like density and a gas-like diffusivity that favor the extraction of chemically affine compounds from solid matrices. Several studies [19–24] reported in the scientific literature describe the main advantages of using this green technique instead of the traditional ones, such as lower operative temperatures and higher selectivity. Moreover, post-processing steps, adopted to purify the extracts from cuticular waxes, are not required when winterization is performed in series to the extraction process in the same operative plant. In particular, Reverchon and co-workers demonstrated in several papers [25–27] that, by using SC-CO2 extraction coupled with high-pressure fractional separation, it is possible to separate cuticular waxes during the extraction process by the selective precipitation from the overall extract [28]. Specifically speaking, the compounds of interest for extraction are generally located well inside the vegetable structure; whereas cuticular waxes are located on the surface of the vegetable material and show a non-negligible solubility in SC-CO2 [28]. For this reason, they are inevitably co-extracted at all SC-CO2 processing conditions due to the overlap of mass transfer limitations and thermodynamic (solubility) limits [28]. However, since they are generally considered as an interfering matter that reduces the quality (purity) of the extracts, a procedure has been developed that allows the selective separation of cuticular waxes from the extract by cooling the mixture CO2 plus overall extract at the exit of the extractor down to temperatures lower than 0 ◦C [25–27,29,30]. Operating at these process conditions, the solubility of cuticular waxes reduces to near zero in SC-CO2 and, therefore, they can be precipitated in a separator before the final collection of the extract of interest.

Therefore, according to the previous discussion of the literature, the scope of this work is to attempt, for the first time, a systematic analysis of cuticular waxes extracted by SC-CO2 plus fractional separation from several vegetables. After performing the specific SC-CO2 extraction processes, several high-resolution gas chromatography-mass spectroscopy (GC-MS) identifications are carried out on cuticular waxes obtained from more than ten different vegetable species, to analyze their composition and dependence on the vegetable tested, and to show that their composition can be specific for the different vegetable species and tissue analyzed.

#### **2. Materials and Methods**

#### *2.1. Materials*

Basil leaves (*Ocimum basilicum* L.), cannabis inflorescence (*Cannabis sativa* L.), chamomile flower heads (*Chamomilla recutita* L. Rausch.), clove buds (*Eugenia caryophyllata* Thun.), ginger rhizomes (*Zingiber officinale* Roscoe), lavender inflorescence, marjoram leaf (*Origanum Majorana* L.), rosemary leaf (*Rosmarinus officinalis* L.), tangerine peels and tobacco leaves were supplied by Planta Medica srl (Pistrino di Citerna (PG), Italy). Jasmine concrete (*Jasminum grandiflorum* L.) was supplied by Chauvet (Seillans, France). Vegetable materials (except for jasmine concrete) were dried and ground using an electric stainless-steel grinder (KYG, mod. 304, China); mean particle size was determined by mechanical sieving. Carbon dioxide (CO2, 99.9% purity, Morlando Group srl, Naples, Italy) was used to carry out SFE processing.

#### *2.2. SFE Plant Description*

SC-CO2 extraction experiments were carried out in a homemade laboratory apparatus equipped with a 490 cm3 internal volume extractor. One hundred grams of vegetable matter, with a mean particle size of 600 μm, were used in all the experiments. In the case of jasmine concrete, since it was a semi-solid material and can produce undesired caking/channeling phenomena during extraction, it was mixed with glass beads (3 mm diameter) to create an inert core surrounded by a thin shell of jasmine concrete. Extracts were recovered using two separation vessels with an internal volume of 200 cm3 each, operated in series. The first separator was cooled down to −10 ◦C using a thermostated bath (Julabo, mod. F38-EH, Milan, Italy); the second one allowed the continuous discharge of the extract using a valve located at the bottom of the vessel. It was operated at 25 bar and 15 ◦C. A high-pressure pump (Lewa, mod. LDB1 M210S, Leonberg, Germany) pumped liquid CO2 at the desired flow rate. CO2 was then heated to the extraction temperature in a thermostated bath (Julabo, mod. CORIO C-B27, Milan, Italy). CO2 flow rate was monitored by a calibrated rotameter (ASA, mod. d6, Sesto San Giovanni (MI), Italy), located after the last separator, coupled with a volumetric meter (Sacofgas 1927 SpA, mod. G.4, Milan, Italy). Temperature and pressure along the plant were measured by thermocouples and test gauges, respectively. More details about the apparatus and the experimental procedure are published elsewhere [25,27,30,31].

The operative conditions selected for the experiments carried out in this work were 90 bar and 50 ◦C (ρCO2 ≈ 0.280 g/cm3) in the extractor, 90 bar and −<sup>10</sup> ◦C in the first separator and 25 bar and 15 ◦C in the second one. CO2 flow rate was fixed at 0.8 kg/h for all the experiments. The first separator, used for cuticular waxes precipitation, was operated at the same extraction pressure and at a temperature lower than 0 ◦C since, operating in this way, the solubility of cuticular waxes in CO2 drastically reduced [25,27,28,30,31].

#### *2.3. Characterization of Cuticular Waxes*

Gas chromatography-mass spectroscopy (GC-MS) analysis was carried out using a Varian 3900 apparatus (Varian, Inc., San Fernando, CA, USA), equipped with a fused-silica capillary column (mod. DB-5, J & W, Folsom, CA, USA) of 30 m length, 0.25 mm internal diameter and 0.25 μm film thickness, and connected to a Varian Saturn Detector 2100T (Varian, Inc., San Fernando, CA, USA). Helium was used as the carrier gas, at a flow rate of 1 mL/min. Column temperature was set at 120 ◦C and held for 5 min; then, it was ramped up to 320 ◦C, at 2 ◦C/min, where it was held for 10 min. An injection step was performed using 1 μL of a 1:10 *n*-hexane solution in split mode; the injector temperature was set at 320 ◦C. The mass spectrometer operated at an ionization voltage of 70 eV in the 40–650 a.m.u. range, at a scanning speed of 5 scans/s. The retention indices (RI) were determined considering the retention time (Rt) values of homologous series of *n*-alkanes (C21-C40) obtained at the same operating conditions. The various components were also identified by a comparison of their RI with published data in the scientific literature. Further identifications were performed, by comparison, of the mass spectra with those stored in the

NIST 02 (National Institute of Standards and Technology, Gaithersburg, MD, USA) library. The relative amounts of the components were evaluated as a percentage of normalized peak area.
