3.2.1. Solid Phase Selection
Regarding the solid phase, those that contain silica as a carrier to which various groups, such as octyl, octadecyl, and phenyl groups are attached, are most often used. Recently, an increasing number of studies indicated the high efficiency of the application of phases composed of phenyl silica in the separation of different groups of phenols. The separation of analytes possessing aromatic rings with phenyl groups of the solid phase is based on the formation of π-π interactions and dispersive hydrophobic forces, and therefore, such phases are more efficient materials for separating phenolic compounds than conventional phases used in a reverse phase system, such as octadecyl silica or octa silica [
26,
27]. In addition to the above materials, various polymeric materials composed of polystyrene-divinylbenzene are very often used for phenol separation. Such a group also includes various Amberlite XAD resins. According to a European Commission regulation, the use of these solid phases is allowed in the food, pharmaceutical, and cosmetic industries [
28,
29,
30]. The big disadvantage of solid phases with bound phases and polymer phases is their high price. Sea sand is a very inexpensive material that has a wide range of applications in destroying plant specimens. Although it is considered not to act as an analyte solvent during the mixing process, due to its structure in the form of very sharp edges and a rough surface, it allows for shearing during the mechanical mixing of samples and the solid phase, making it a very effective means of destroying solid sample cells. Some studies showed that sea sand is a more efficient solid phase for phenol extraction compared with various C18 materials [
31,
32,
33]. Based on the above findings, Sepra Phenyl, Amberlite XAD-2, and sea sand were used as solid phases in optimizing the MSPD process.
3.2.2. Optimizing the MSPD Method Using the BBD
During the optimization process of the MSPD method, the type of solid phase, type and volume content of organic phase in the elution solvent, volume of elution solvent, and sorbent-to-sample ratio with the levels given in
Table 4 were used as extraction factors, with a total of 90 experiments. Statistical processing of the obtained content of anthocyanins, flavonols, and flavan-3-ol showed that they can best be described using a quadratic polynomial equation, whose coefficients, together with other parameters determined using an analysis of variance, are shown in
Table 7.
The most significant extraction factor that influenced the content of all tested groups of compounds was the applied solid phase. The most effective solid phase was phenyl silica, which had a dual role during application in the MSPD process. It served as an abrasive to destroy the cell wall structure of grape skins, but it also acted as a kind of solvent for flavonoids. Bound-phase phenyl and flavonoids possess aromatic rings in their structures and, in addition to the usual hydrophobic interactions, can also form strong π-π interactions, which ultimately result in high efficiency during the application of the MSPD process. The content of anthocyanins, flavonols, and flavan-3-ol obtained by using sea sand as the solid phase was slightly lower compared with those obtained by using phenyl silica. Due to its structure, the sand was very effective at destroying the cell wall structures of grape skins. Comprehensive destruction of cell walls results in easier access of the elution solvent to the cell components, and thus, an increase in extraction. The content of individual groups of flavonoids obtained using XAD-2 as a solid phase was significantly lower compared with those obtained using phenyl silica and sand. The largest differences were observed in the case of anthocyanins, while the least was observed regarding the content of flavonols. Such a result is in line with previous studies that found that this type of solid phase has the highest affinity for flavonols and an extremely low affinity for anthocyanins, while the affinity for flavan-3-oils was moderate [
34,
35].
The choice of elution solvent plays an important role during the MSPD process. It has the role of a common mobile phase in the chromatographic system during the MSPD, but it also has a role as a solvent for the desired analytes. The type of organic phase contained in the elution solvent is an important extraction factor that significantly affects the content of individual groups of flavonoids. Anthocyanins are the most abundant group of flavonoids in grape skins, and ethanol must be used for their most efficient extraction using MSPD. Methanol, in contrast to acetonitrile, is thought to enhance the π-π interactions between the anthocyanin and the phenyl-bound phase, resulting in better separation. Since ethanol and methanol have very similar physicochemical properties, this property of methanol can probably be applied to ethanol as well [
36]. To achieve the maximum content of flavonol and flavan-3-ol, it is necessary to use an elution solvent containing acetonitrile.
The volume of the elution solvent was an important factor that influenced the extraction efficiency of anthocyanin, flavonol, and flavan-3-ol. The volume of the elution solvent depended on the weight of the sorbent and the content of the analyte. The use of larger volumes of elution solvent ensured complete elution of the desired analytes.
The content of anthocyanin and flavan-3-ol was a function of the sorbent-to-solid ratio. Increasing this ratio resulted in a significant increase in the content of anthocyanins, while decreasing this ratio had a positive effect on flavan-3-ol extraction. Such an observation may have been due to the content of these groups of compounds. Anthocyanins were present in significantly higher contents; therefore, their dissolution in the bound phase required a higher weight because the application of smaller weights led to supersaturation, and thus, the solvent could not further dissolve these analytes. Flavan-3-ols were contained in much smaller contents; therefore, the application of a larger weight in the solid phase could have a negative effect on their extraction because they could lead to their stretching in the solid phase, and thus, to loss in the final extract.
The effect of interactions between individual extraction factors on the content of anthocyanins, flavonols, and flavan-3-ol was determined from contour graphs (
Figure 4,
Figure 5 and
Figure 6).
The content of anthocyanin, regardless of the applied solid phase and the elution solvent, was a function of the interaction between the sorbent-to-sample ratio and the proportion of the organic phase or the volume of the elution solvent. Increasing the proportion of the organic phase with the application of a smaller sorbent-to-sample ratio led to a significant increase in the content of this group of compounds. The highest content of anthocyanins was observed in the extracts obtained by applying a small sorbent-to-sample ratio and a large elution volume. The elution solvent acted as a solvent that competed with the bound phase for the analytes, and therefore, a larger volume of solvent compensated for the small weight of the bound phase (
Figure 5).
The content of flavonols, regardless of the elution solvent used and the type of solid phase was influenced by the interactions between the organic phase content in the elution solvent and the sorbent-to-sample ratio or volume. The applied volume of solvent did not significantly depend on the applied solid phase, and its increase led to an increase in the content of flavonols (
Figure 6).
As in the case of anthocyanins, the content of flavan-3-ol depended on the interaction between the sorbent-to-sample ratio and the volume of the elution solvent or its content. The proportion of the organic phase in the extraction solvent, as well as its volume, depended on the organic solvent used (
Figure 7).
The optimization of the MSPD procedure was performed using the Derringer function. The obtained optimal conditions for the extraction of all the individual flavonoids are listed in
Table 8. Since the use of sea sand as a solid phase and ethanol as an organic solvent is allowed in the food, pharmaceutical, and cosmetic industries, optimal conditions were determined in the case of the use of these extraction conditions.
By conducting experiments under the obtained optimal conditions and comparing the values predicted by the model with those obtained experimentally, the suitability of the obtained equations for individual models was determined. The differences between the experimentally obtained values and the values predicted by the model were very small, thus confirming that the obtained models were reliable and accurate. The optimal conditions obtained for anthocyanins determined using HPLC and the spectrophotometric method were the same, as well as those obtained using the Folin-Ciocaulteu method because the anthocyanins represented a great majority of the analyzed flavonoids.
The SLE technique is the most applied technique for the extraction of polyphenolic compounds from grape skins. Methanol, EtOH, acetone, ethyl acetate, and their aqueous solutions are the most frequently applied extraction solvents for the recovery of polyphenolics from grapes. The extraction time can be in the range from 8 min to 48 h, while the phase ratio can be in the range from 1:1.5 up to 1:125 g mL−1. The application of these newly optimized methods, namely, MAE and MSPD, enables the use of smaller volumes of solvent compared with SLE, as well as a shorter duration of the extraction procedure.