2.1.1. Stoichiometry of Ascorbic Acid Binary Complexes
Although various studies confirm that AA forms inclusion complexes with α- and β-cyclodextrin [22
], inclusion complexes between AA and γCD are yet to be verified. The phase solubility technique is well known for drug/CD inclusion complex confirmation. However, in the case of water-soluble drugs such as ascorbic acid, the major disadvantage of this technique is the preparation of saturated AA in γCD solutions. To perform phase solubility studies, saturated AA in various concentrations of γCD should be prepared; hence, a large amount of AA is required. The prepared solutions should also be placed on a horizontal shaker at a constant speed for at least three days to verify that that those solutions are saturated. Because AA is a labile compound that is easily degraded, a preparation of saturated AA in γCD solutions may not be a suitable option. Therefore, the continuous variation technique was proposed. Furthermore, Karim and coworkers [30
] demonstrated that PVA can encapsulate AA into its structure so that the formation of AA/PVA complexes could be verified. Considering the maximum of the curve on Job’s plots (Figure 1
), the AA/γCD and AA/PVA complexes were confirmed and demonstrated a symmetrical shape with a maximum at r = 0.5 that represented the formation of an equimolar complex (i.e., 1:1, 2:2 or higher n:n, within the investigated concentration range).
2.1.2. Binary and Ternary Complexes in Solid State
Fourier Transform Infrared (FTIR) spectroscopy was used to determine the molecular interaction between two or three molecules, which were binary or ternary complexes, considering the change of intensity and peak in the spectra upon complexation. The low-intensity and very broad band (3000–3600 cm−1
) with a maximum at 3260.53 cm−1
was assigned to the stretching vibration of the O-H groups in the glucose rings of γCD. The C=O and C=C stretching vibration at 1750.89 and 1651.54 cm−1
, respectively, were assigned to the characteristic bands of the lactone ring of AA [31
]. The C-O stretching vibration of PVA was assigned to the moderate-intensity band with a maximum at 1078.72 cm−1
] (Table 1
). The spectra with their characteristic bands are shown in Figures S1–S4
and Table 2
, respectively. The similarities between spectra of inclusion complexes and their physical mixtures at a mole ratio of 1:1 and the shifts of the maxima of the characteristic bands were studied.
In the spectrum of the AA/γCD inclusion complex, an increase of intensity and shifting of characteristic bands was observed. The shifts of characteristic groups corresponding to the hydroxyl groups of glucose rings in γCD (45.52 cm−1
) and lactone ring in AA (10.83 and 37.48 cm−1
for carbonyl and alkene groups, respectively) were assumed to be due to the interaction of the lactone ring with hydroxyl groups inside the γCD toroid. The increment of intensity compared to the 1:1 physical mixture indicated that the AA molecule was encapsulated inside the cavity of γCD, resulting in the AA/γCD inclusion complex. In the case of the AA/PVA complex, the disappearance of O-H and C-H alkyl stretching bands (3293.30 and 2914.85 cm−1
, respectively) shown in Figure S2
, along with the shift of C-O stretching vibration band of PVA (7.90 cm−1
), represented the interaction of the PVA molecule with the carbonyl group in the lactone ring of AA. Furthermore, in the γCD/PVA spectrum, the shift of characteristic bands corresponding to the hydroxyl group of γCD and C-O groups of PVA (60.97 and 1.40 cm−1
, respectively) and increasing of their intensity indicated the formation of the γCD/PVA inclusion complex. According to the characteristic bands in the spectrum of the AA/γCD/PVA complexes, the dominant shift of the hydroxyl group (46.15 cm−1
) indicated that the formation of a ternary complex was preferred. In the lactone ring of the AA molecule, the shift of the C=C stretching vibration band was approximately 6.79 times higher than in the binary complexes, whereas the shift of C=O stretching vibration bands was almost identical.
2.1.3. Binary and Ternary Complexes in Aqueous State
H NMR spectra of binary and ternary mixtures (Figures S5–S8
) were used to support the inclusion phenomena in aqueous solutions. A chemical shift (δ) of protons belonging to AA, γCD, and PVA (Figure 2
) was implied in the complexation process. The inclusion complex of γCD (i.e., the host) with AA and PVA (i.e., the guest molecules) was confirmed by the resonance signals of the H3
protons located inside the cavity change, which was their chemical shift (Δδ). The magnitude and ratio of ΔδH3
provided information regarding the stability and depth of the inclusion complex. The change of chemical shifts induced on the protons of the AA, γCD, and PVA as a result of the interactions in the binary and ternary complexes are summarized in Table 2
. To thoroughly understand those complexes, two-dimensional rotational Overhauser enhancement experiment (ROESY) NMR experiments were performed. The inspection of a ROESY map established a spatial proximity between protons of two and three molecules.
In the AA/γCD spectrum, the interaction between AA and γCD induced upfield shifts of protons located inside of the CD toroid indicated that inclusion complexes of AA/γCD were formed, and this corresponded to the result of the FTIR spectrum. The high value of ΔδH3
ratio (1.11) suggested that AA penetrated into the macrocyclic cavity of γCD from the wider rim. The minor upfield of the H3
signals showed that weak inclusion complexes were formed, agreeing with the inclusion complexes of AA with αCD and βCD from previous works [22
]. According to the bidimensional spectrum of the AA/γCD complex (Figure S9
), a very weak dipolar correlation supported the formation of a weak complex. In the presence of γCD, the protons of AA, particularly Hx
upfield shifts, suggested that the lactone ring of the AA molecule was entrapped in the hydrophobic cavity of γCD.
In the 1
H NMR and 2D ROESY spectra of AA/PVA (Figure S10
), the interaction between AA and PVA was confirmed by the absence of HCH
signals and intermolecular cross-peak between HA,B
protons of AA and HCH2
protons of PVA, respectively. A pronounced upfield shift of Hx
(−0.0084) indicated that the cyclic lactone structure of the AA molecule, which is rich in π electrons, was associated with the alkyl and hydroxyl groups of PVA. This finding corresponded to the absence of O-H and the notable shift of C-O stretching vibration of PVA in the FTIR spectrum. Furthermore, the inclusion complex of γCD/PVA was observed through the upfield shifts of H3
protons and the disappearance of HCH
signals (3.9512 and 3.7716 ppm, respectively) in the 1
H NMR spectrum. The high value of the ΔδH3
ratio (1.21) suggested that PVA penetrated deeply into the toroidal shape of the γCD cavity. The bidimensional spectrum of γCD/PVA (Figure S11
) exhibited several intermolecular cross-peaks between protons of γCD (i.e., H3
) and HCH2
protons of PVA demonstrated the inclusion complex of PVA with γCD. Additional dipolar correlations were found between HCH2
protons of PVA and other protons in the exterior structure of γCD confirmed that a γCD/PVA non-inclusion complex had formed. Because PVA is a linear chain polymer, it was possible that the long chain of PVA threaded through the γCD hydrophobic cavity at the same time as the polymer swathed around the external surface.
According to the 1
H NMR spectrum, the formation of AA/γCD/PVA ternary inclusion complexes was confirmed by the deviation of chemical shifts assigned to AA and γCD molecules. A pronounce upfield shift of HX
compared to HM
indicated that the cyclic lactone part of AA preferably interacted with the other two molecules. The absence of HCH
signals also confirmed that the PVA molecule associated with AA and γCD. The high ratio of ΔδH3
(1.22) suggested that AA and/or PVA penetrated through the γCD cavity. Several intermolecular cross-peaks, shown in Figure S12
, between HCH2
protons of PVA and assigned protons in AA and γCD (i.e., HA,B
, and H4
), exhibited the formation of AA/γCD/PVA ternary complexes.
The results from 1H NMR and 2D ROESY spectra agreed with the FTIR spectra and confirmed the formation of binary and ternary inclusion complexes. It is worth noting that the conformation of the ternary complex might be different from those of binary complexes. The conformation of AA/γCD/PVA ternary complexes should be verified by molecular modeling stimulation in future research. Moreover, even though the chemical shifts from proton NMR can be used to confirm complexes, in this case, the carbon-13 NMR spectroscopy can be performed for more information on the intermolecular interaction.