Values of surface tension (
Host‐guest inclusion complex (IC) of vitamin C with β‐cyclodextrin (β‐CD) in aqueous medium has been explored by spectroscopic, physicochemical and calorimetric methods as stabilizer, carrier and regulatory releaser. Job plot has been drawn by UV‐visible spectroscopy to confirm the 1:1 stoichiometry of the host‐guest assembly. Stereo‐chemical nature of the inclusion complex has been explained by two‐dimensional (2D) NMR spectroscopy. Surface tension and conductivity studies further support the inclusion process. Association constants for the vitamin C‐β‐CD inclusion complex have been calculated by UV‐visible spectroscopy using both Benesi‐Hildebrand method and non‐linear programme, while the thermodynamic parameters have been estimated with the help of van’t Hoff equation. Isothermal titration calorimetric study has been performed to determine the stoichiometry, association constant and thermodynamic parameters with high accuracy.
- vitamin C
- inclusion complex
β‐CD has been widely employed as not only excellent receptors for molecular recognition but also excellent building blocks to construct functional materials, where they could be applied to construct stimuli‐responsive supramolecular materials . Series of external stimuli, e.g. enzyme activation, light, temperature, changes in pH or redox and competitive binding may be employed to operate the release of guest molecules from the inclusion composites [10, 11]. Recently cyclodextrin‐modified nanoparticles are of great interest as these supramolecular macrocycles significantly combine and enhance the characteristics of the entities, such as the electronic, conductance, thermal, fluorescence and catalytic properties expanding their potential applications as nanosensors, drug‐delivery vehicles and recycling extraction agents . Different sophisticated probes based on semiconductor nanocrystals and other nanoparticles have been designed for this purpose because of their potential applications in the fabrication of molecular switches, molecular machines, supramolecular polymers, chemosensors, transmembrane channels, molecule‐based logic gates and other interesting host‐guest systems [13–15].
In this article vitamin C (Figure 1), is an essential human nutrient with many important functions in biological systems. Scurvy, fatigue, depression and connective tissue defects are the common syndromes caused by deficiency of vitamin C [16, 17]. Thus to protect this important bio‐molecule from external effects (e.g. oxidation, structural modification, etc.) and for its regulatory release, it is crucial to investigate whether this molecule can be encapsulated into the β‐CD molecule and to explore the thermodynamic aspect of the process. In this present chapter, the formation of host‐guest inclusion complex (IC) of the vitamin C with β‐CD (the cavity dimension of which is more appropriate than other CDs to encapsulate a great variety of molecules) has been explored particularly towards its formation, stabilization, carrying and controlled release without chemical modification by different dependable methods like two‐dimensional rotating‐frame nuclear overhauser effect spectroscopy (2D ROESY) NMR, UV‐Vis spectroscopy, surface tension (
2. Result and discussion
2.1. Job plot reveals the stoichiometry of the host‐guest inclusion complex
One of the best methods used to recognize the stoichiometry of the host‐guest inclusion complexes is the Job’s method, known as the continuous variation method, which has been applied here by using UV‐visible spectroscopy . A set of solutions for the vitamin and β‐CD was prepared varying the mole fraction of the guest in the range 0–1. Job plot was generated by plotting Δ
2.2. 2D NMR spectra analysis
Two‐dimensional (2D) NMR spectroscopy gives most powerful evidence about the spatial proximity between the host and the guest atoms by observations of the intermolecular dipolar cross‐correlations [21, 22]. Any two protons that are located within 0.4 nm in space can produce a nuclear overhauser effect (NOE) cross‐correlation in NOE spectroscopy (NOESY) or rotating‐frame NOE spectroscopy (ROESY) [23, 24]. In the structure of β‐CD the H3 and H5 protons are situated inside the conical cavity, particularly, the H3 are placed near the wider rim while H5 are placed near the narrower rim, the other H1, H2 and H4 protons are located at the exterior of the β‐CD molecule (Figure 3) [25, 26]. Thus the inclusion phenomenon within the cyclodextrin cavity may be confirmed by the appearance of NOE cross‐peaks between the H3 or H5 protons of the host and the protons of the guest identifying their spatial contacts [27, 28]. For this purpose, 2D ROESY has been obtained of the 1:1 molar mixture of vitamin C with β‐CD. The ROESY spectra in D2O shows significant correlations between the H‐3, H‐5 protons of β‐CD and the C
2.3. Surface tension study elucidates the inclusion as well as stoichiometric ratio of the host and guest
Surface tension (
|Concentration of β‐CD (mM)||Concentration of vitamin (mM)|
2.4. Conductivity study demonstrates inclusion process and its stoichiometric ratio
|Concentration of β‐CD (mM)||Concentration of vitamin (mM)|
2.5. Ultraviolet spectroscopy: association constants and thermodynamic parameters
Association constants (
The thermodynamic parameters can easily be derived basing upon the association constants found at various temperatures by the above method with the help of van’t Hoff equation Eq. (2).
Association constants (
The association constant (
Here, [IC], [
The values of Δ
2.6. Isothermal titration calorimetry: characterization of the complexation
Isothermal titration calorimetry (ITC) is the most sensitive and accurate analytical technique for determination of binding constant and various thermodynamic parameters in host‐guest complexation with precise accuracy . It has become an efficient method for direct determination of the thermodynamic parameters rather than using the earlier van’t Hoff equation technique . Top of Figure 9 shows the data obtained from the ITC titration of vitamin C with β‐CD in water at 298 K, which describes production of exothermic heat after each injection and the magnitude of the released heat decreases progressively with each injection until complete complexation is achieved. Bottom of Figure 9 shows the experimental data and the calculated best fit binding curve of vitamin C with β‐CD, that provides the stoichiometry (
(J mol−1 K−1)
|0.99 ± 0.0111||3.655 ± 0.335||−22.28 ± 1.06||−5.21|
The stoichiometry (
Formation of the host‐guest IC is the dimensional suitability between the two species, which is favoured by the unique cyclodextrin molecule that provides an appropriate condition by encapsulating the apolar part of the guest molecule inside the cavity, as well as stabilizing the polar part by the polar rims . The other driving force for the formation of IC is the release of the water molecules from the hydrophobic cavity into the bulk thereby increasing the entropy of the system [1, 51]. The inclusion of the guest molecule is likely from the wider rim of the β‐CD molecule to make maximum contact with the cavity (Figure 5), which is also supported by ROESY spectrum. The polar —OH group of the vitamin can also make H‐bonds with the —OH groups at both the rims of the β‐CD molecule, thereby stabilizing the IC.
Vitamin C and β‐cyclodextrin of puriss grade were bought from Sigma‐Aldrich, Germany and used as purchased. The mass fraction purity of vitamin C and β‐cyclodextrin was ≥0.99 and ≥0.98, respectively.
3.2. Apparatus and procedure
Prior to the start of the experimental work, solubility of β‐cyclodextrin and the vitamin has been precisely checked in triply distilled and degassed water (with a specific conductance of 1 × 10−6 S cm−1) and observed that the selected vitamin was freely soluble in all proportion of aqueous β‐cyclodextrin. All the stock solutions of the vitamin were prepared by mass (weighed by Mettler Toledo AG‐285 with uncertainty 0.0003 g), and then the working solutions were obtained by mass dilution at 298.15 K. Adequate precautions were made to reduce evaporation loss during mixing.
UV‐visible spectra were recorded by JASCO V‐530 UV/VIS Spectrophotometer, with an uncertainty of wavelength resolution of ±2 nm. The measuring temperature was held constant by an automated digital thermostat.
Two‐dimensional (2D) ROESY spectra were recorded in D2O at 300 MHz using Bruker Avance 300 MHz instrument at 298 K.
The surface tension experiments were done by platinum ring detachment method using a Tensiometer (K9, KRU″SS; Germany) at the experimental temperature. The accuracy of the measurement was within ±0.1 mN m−1. Temperature of the system has been maintained by circulating auto‐thermostat water through a double‐wall glass vessel containing the solution.
Specific conductance values of the experimental solutions were measured by Mettler Toledo Seven Multi conductivity meter with uncertainty of ±1.0 µS m−1. The measurements were made in an auto‐thermostated water bath maintaining the temperature at 298.15 K and using the HPLC grade water with specific conductance of 6.0 µS m−1. The cell was calibrated using a 0.01 M aqueous KCl solution. The uncertainty in temperature was ±0.01 K.
Isothermal titration calorimetry was used to obtain association constant at 298 K using a MicroCal VP‐ITC (MicroCal, Inc., Northampton, MA, USA). The thermal equilibration step at 298 K was followed by an initial 120 s delay step and the subsequent 25 injections of each vitamin to β‐CD (injection duration of 10 s and spacing of 180 s). Each injection generated a heat‐burst curve between micro cal s−1 versus time (min). The saturation curve between kcal/mol of injectant versus molar ratio was determined by integration, using Origin 7.0 software (Microcal, Inc.) to give the measure of the heat associated with the injection. The binding affinity and thermodynamic parameters of the binding process were obtained by fitting the integrated heats of binding the isotherm to the one site binding model to give the association constant (
The present study explains that vitamin C forms IC with β‐CD in aqueous medium, which can be used as regulatory releaser of the vitamin. Two‐dimensional (2D) ROESY NMR study confirms the inclusion phenomenon and its mechanism. Surface tension and conductivity studies also show that the ICs have been formed, the stoichiometry of which were confirmed as 1:1 by Job plots. The association constants and thermodynamic parameters have been estimated for both the ICs by reliable spectroscopic and calorimetric techniques with high accuracy. Thus, this work communicates both qualitative and quantitative idea about the formation of IC of β‐CD with vitamin C suggesting its potential applications in pharmaceutical industries and medical sciences.