Exploring Existence of Host-Guest Inclusion Complex of β-Cyclodextrin of a Biologically Active Compound with the Manifestation of Diverse Interactions

The host–guest interaction of p-nitro benzaldehyde as guest β-Cyclodextrins have been investigated which have significant applications in the field of medicine such as controlled drug delivery. The 1H NMR study confirms the formation of inclusion complex while surface tension and conductivity studies support the formation inclusion complex with 1:1 stoichiometry. The stoichiometry of the inclusion complex was also supported with Job’s plot method by UV-Visible spectroscopy. FT-IR spectra and SEM study also support the inclusion process. Association constants of the inclusion complexes have been calculated using the Benesi–Hildebrand method, while the thermodynamic parameters have been estimated with the help of van’t Hoff equation.


2-1-Source and Purity of Samples
P-nitro benzaldehyde and β-cyclodextrin of puriss grade were bought from Sigma Aldrich, Germany and used as purchased.The mass fraction purity of PNB and β-cyclodextrin are ≥ 0.99 and ≥ 0.98 respectively.

2-2-Apparatus and Procedure
The guest molecule PNB and the β-CDs are freely soluble in triply distilled, deionized and degassed water.The stock solutions of PNB and aqueous CD were prepared by mass at 298.15 K. Mettler Toledo AG-285(uncertainty 0.0001 g) was used for weighing.
UV-visible spectra were taken by JASCO V-530 UV/VIS Spectrophotometer, with an uncertainty in wavelength as ±2 nm.The temperature during experiment is kept constant using a digital thermostat.
The surface tension study was performed with platinum ring detachment technique using a Tensiometer (K9, KRSS; Germany).The temperature is maintained at 298.15 K by circulation of thermostated water through a double wall glass vessel containing the solution.The accuracy of the instrument is about ±0.1mN m -1 .
Conductivities of the solutions are measured using a Mettler Toledo Seven Multi conductivity meter having uncertainty 1.0 µSm -1 having uncertainty ±0.01 K. Thermostated water bath kept the temperature constant at 298.15 throughout the experiment.HPLC grade water was used with specific conductance 6.0l Sm -1 .The conductivity cell was calibrated using 0.01 M aqueous KCl solution.
Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer FT-IR spectrometer according to the KBr disk technique.Samples were prepared as KBr disks with 1 mg complex and 100 mg KBr.The FTIR measurements were performed in the scanning range of 4000−400 cm −1 at room temperature.
The solid inclusion complex [β-CD+PNB] has been prepared taking 1:1 molar ratio of both the PNB and CD.In this case 1.0 mmol CD was dissolved in 20 mL water and 1.0 mmol PNB was dissolved in 20 mL ethanol and stirred separately for 4 hrs.Then the ethanol solution of PNB was added drop wise to the aqueous CD solution.The mixture was then allowed to stir for 72 hrs at 50-55 o C. It was filtered at this temperature, then cooled to 5 o C and kept for 12 hrs.The resulting suspension was filtered and the white polycrystalline powder was found, which was washed with ethanol and dried in air.

3-1-Job Plot Demonstrates the Stoichiometry of the Host-Guest Inclusion Complex
The stoichiometry of the host guest inclusion complex can be predicted with the help of Job's method of continuous variation using UV-Visible spectra [6].The difference of absorbance of PNB in presence and absence of β-CD was calculated and designated as ∆A.Then a plot is constructed taking ∆A × R against R where R = [PNB]/([PNB]+[CD]).(Figure 3)Job plots are constructed with variation of mole fraction of PNB in the range of 0-1 [7,8].Absorbance values were measured at 293 nm at 298K for a series of solutions.(Table 1) The stoichiometry of the inclusion complex can be found with the value of R at the point of maximum deviation.(R=0.5 for 1:1 complexes; R=0.33 for 1:2complexes; R=0.66 for 2: 1 complexes) [9,10].In this study the plot show maxima at R=0.5 which means that the inclusion complex was formed with 1:1 molar ratio of PNB and β-CD.

3-2-1H-NMR Study
1H-NMR study gives us valuable information about the formation of inclusion complex between PNB and β-CD [11].When the guest PNB molecule inserts into the hydrophobic cavity of β-CD molecule the protons of both PNB and β-CD show considerable chemical shift.The positions of different protons in the CD molecule are shown in scheme.The H3 and H5 protons are placed in the cavity while H1, H2 and H4 are located outside the cavity.(Figure 4) H3 is situated near the wider rim and H5 is close to the narrower rim [12,13].The respective δ values of PNB, β-CD and inclusion complex are mentioned in Table 2.The protons of PNB and β-CD show considerable upfield shift in the spectra of inclusion complex.(Figure 5) From NMR data it can be concluded that the aromatic protons of PNB interacts more with the H3 protons of β-CD in comparison to H5 perhaps due to the reason that the PNB molecule enters in the hydrophobic cavity from the wider rim.All the protons of both host and guest show considerable chemical shift due to

3-3-Surface Tension Study Confirms the Stoichiometry of the Inclusion Complex
Cyclodextrin is freely soluble in water.The aqueous solution of β-CD does not show any notable change with increasing concentration [14].The guest molecule p-nitro benzaldehyde shows surfactant like behavior due to its structure.This means that the presence of PNB in water reduces the surface tension value (γ) of water.In this experiment the γ values of PNB has been measured with increasing concentration of host β-CD at 298.15 K. (Table 3) The aqueous solution of PNB shows a regular increase with increasing concentration of host β-CD.Perhaps this occurs due to the removal of surface active PNB molecules from the solution into the hydrophobic cavity of β-CD after forming the inclusion complex.The surface tension plot also contains a sharp break point which is indication of the formation of inclusion complex with 1:1 stoichiometry (Figure 6) [15,16].Appearance of more number of break points in the surface tension curves reveal more complex stoichiometry such as 1:2, 2:1, 2:2 etc. the value of γ at the break point with corresponding concentration of β-CD is listed in table.

3-4-Conductivity Study Again Proves the Stoichiometry of the Inclusion Complex
The formation of inclusion complex is again confirmed with the help of conductivity study [17,18].The guest molecule PNB exists in ionic form in aqueous solution having a measureable value of κ.In this study we have added aqueous CD solution to the aqueous solution of PNB; it has been found that κ value shows a regular declining trend.It is again probably due to insertion of PNB molecules from the solution to the hydrophobic cavity of CD.At certain concentration of both the host and guest molecules a single break point appeared in the conductivity plot, this confirms the formation of inclusion complex.(Figure 7) The value of κ with corresponding concentration of β-CD is listed in Table 3 [19].The appearance of single breakpoint again suggests the stoichiometry of the inclusion complex 1:1.

3-5-Association Constants and Thermodynamic Parameters
The association constant Ka of the inclusion complex of PNB with β-CD can be calculated from UV visible spectra.When the PNB molecule enters into the cavity of β-CD the environment of the guest changes, it results in the change of molar extinction coefficient ε [20].The value of binding constant has been determined from the double reciprocal plots on the basis of Benesi-Hildebrand method which can be stated as: The equation is valid in case of 1:1 host-guest IC only [21,22].We get the value of binding constant by dividing the intercept by the slope from double-reciprocal plots.The Ka value is calculated at three different temperatures.The thermodynamic parameters ∆G ∘ , ∆H ∘ and ∆S ∘ for the formation of IC is measured from the famous Van't Hoff equation with the help of association constant Ka [23,24].
lnKa varies linearly with 1/T.The ∆H ∘ value depends upon the equilibrium constant at different temperatures.(Table 4) The ∆G ∘ value for the formation of inclusion complex is negative which indicates the process to be thermodynamically favourable.The negative value of ∆H ∘ shows that the process is exothermic.The entropy value ∆S ∘ is also negative.The randomness of the molecules decrease due to insertion in the cavity of CD, as a result the ∆S ∘ value is negative.The higher negative value of ∆G ∘ overcomes the effect and the whole process is spontaneous.

3-6-FT-IR Study
The encapsulation of PNB by β-CD is confirmed by FT-IR study [25,26].There are many changes in the position of peaks in the solid inclusion complexes.The various frequencies of PNB, β-CD and PNB+β-CD are reported in Table 5.All the spectra are shown in the Figure 9.The spectra of PNB is identified by various characteristic peaks of -CHO, -NO2, aromatic protons at 1707, 1535, 3424 etc. Broad characteristic peaks of -OH at about 3349 cm −1 is present in the spectrum of β-CD.In the spectra of solid inclusion complexes many peaks of the PNB are either absent or shifted due to change of environment after insertion.The -O-H frequency of β-CD are shifted to lower region probably due to involvement of the -O-H groups of the host molecules in hydrogen bonding with the PNB molecule.No additional peaks are recognized in the solid inclusion complex which means no chemical reaction occurred between the molecule PNB and CD [27].

Figure 2 .
Figure 2. Two dimensional and three dimensional structure of PNB

Figure 7 .
Figure 7. Variation of conductivity of aqueous PNB solution with increasing concentration β-CD at 298.15K.

Figure 8 .
Figure 8. Plausible Schematic representation of mechanism for the formation of 1:1 inclusion complex of PNB with βCyclodextrin.