INTRODUCTION:

In cyclodextrin complexation jobs plot is used for determination of the Stoichiometry for preparation of binary/ternary complex. The theoretical aspect of cyclodextrin complexation contributes in understanding the molecular recognition, molecular interactions and the role of different structural factors of the guest molecule.

The stoichiometry plays a vital role in complexation. Without stoichiometric ratio of guest and host; it would be difficult to prepare complex. According to literatures and datas, it is stated and considered that both on experimental grounds and molecular dynamics calculations the efficient stoichiometry for complexation is 1:2 guest: host, providing best effect. Stoichiometry can be estimated by spectral method. Through stoichiometry and or phase solubility study following parameters relevant to complexation can be determined –

a. Stability constant

b. Complexation efficiency

c. Coefficient of regression

d. Intrinsic solubility of guest

e. Solubilizing efficiency

The stoichiometry/stoichiometric ratio

The stoichiometry of the inclusion complex is given by the number of guest [G] and host [H] molecules present in the supramolecular complex/inclusion system. The general notation being GnHm; The most common stoichiometry is 1:1 (G:H), implying the inclusion of a single guest molecule. However, several other stoichiometries like G1H2, G2H1, G2H2, G1H3, G3H1, etc., can also be encountered. Besides 1:1 stiochiometry, the formation of the G1H2 complex may be due to two successive equilibriums, the simultaneous presence of 1:1 and 1:2 complexes is also frequently mentioned.

JOBs Method

The first method used for determination of the stoichiometry of inclusion complexes was Job’s method given by Mr. Job in year 1928. It is also known as the continuous variation method. In this method, the stock solutions of equimolecular concentrations of Host and Guest are used for the study. The samples are prepared by mixing different volumes of these two solutions in such a way that the total concentration [H]+[G] remains constant and the molar fraction of the guest, XG varies in the range 0 –1. Graph is plotted between the variation of the experimental property measured ΔP, in presence of the host in respect with the value for the free guest and XG or XH.

The value of XG for which the plot presents the maximum deviation gives the value of stoichiometry of supramolecular system/the inclusion complex. In general; XG = 0.5 for 1:1 or 2:2 G:H complexes; XH = 0.33 for 1:2 G:H complexes). In most of the cases, in a Job plot ΔP represents the change in absorbance (by UV) of the guest during addition of the host, ΔA. Also, other properties correlated with the concentration of the complex can be the change in chemical shifts (Δδ) (by NMR) or the enthalpy changes (ΔH).

Job’s method was described by Landy for the determination of the stoichiometry of CD inclusion complexes and named it as Competitive Continuous Variation Plot. This approach represented a coupling of Job’s method with the competitive experiments, spectral displacements, well known in the study of biopolymer– ligand interactions. The basic aim was to monitor the changes of a given experimental property and to build a Job plot when a competitor ligand, for which the features of the inclusion complex were previously determined, is introduced in the system. This method is recommended or suitable for those cases in which either the low solubility prevents the usual experimental determinations or the spectral properties of the guest are not in the experimental accessible range. The linear and non-linear mathematical models and equations for stoichiometry of inclusion complexes are discussed in table 1.

Table 1 – Equation based on stoichiometry of inclusion complex

Stoichiometric ratio	Equation
1:1	G + H	GH
1:2	G + 2H	GH₂
2:1	G + H	GH, GH + G	G₂H
2:2	G + H	GH, GH+ GH G₂H₂	G₂H₂
1:1 + 1:2	G + H	GH, GH + H	GH₂

In certain cases, where complexes with different stoichiometry, are present in the system, the necessity to introduce several fitting parameters reduces the reliability of the fits. In that case, it is better to work with sets of data read at different wavelengths and to perform a multivariable analysis of the whole set of data, imposing the condition that the association constants are the same, independent on the wavelength. An ideal jobs plot is shown in figure 1.

Binary cyclodextrin complex system

When the inclusion complex is prepared by mixing only two components i.e. drug and cyclodextrin, the system is called as the binary inclusion system.

Ternary cyclodextrin complex system

This type of complexation is also known as multicomponent complexation (MCC). When the inclusion complex is prepared by mixing more than two components i.e. the complexation involves use of third component called excipient eg: surfactant, emulsifier, polymer etc along with the drug and cyclodextrin, the system is called as the ternary inclusion system.

Figure 1 - A typical jobs plot

Earlier, jobs plot was widely used for its applications in pharmacology, biomedicine, pharmaceutics, microbiology, forensic science and allied fields. Some of the contributions are discussed as ---

Iacovina et. al; in year 2012 prepared jobs plot for investigating the complexation between Hydroxymethyl ferrocene and β-Cyclodextrin. In solid state XRD and FT IR were used for the study and for solution state - solubility studies, NMR and UV-Vis spectroscopy were used. The 1:1 stoichiometry of the complex is confirmed by the continuous variation method with R = 0.5.

Similarly, Yanez et al, in 2012 improved the physicochemical property of herbicide bentazon through complexation with cyclodextrin. The stoichiometry was 1:1 with jobs plot and phase solubility study. Pipemidic acid, a therapeutic agent used in urinary tract infection was complexed with cyclodextrin for the enhancement of solubility, bioavailability and pharmacological activity. The 1:1 stoichiometry was established by a Job plot and the inclusion mechanism was clarified using docking experiments (Iacovino, 2013). Mostly herbals and phytoconstituents were studied through jobs plot.

Recently, the use of jobs plot is replaced by phase solubility study. Now-a-days very less emphasis is focused on jobs plot for determination of stoichiometry.

Phase solubility study

The method resembles the jobs plot or continuous variation method. Through phase solubility study, type of solubility curve indicates the relationship between host and guest.

Phase solubility/equilibrium solubility study is the analysis or investigation of dependence of guest solubility on the concentration of host [CD], and may reveal both the stoichiometry of the complex formation as well as the stability constant of the complex. The solubility of guest molecules is often determined by UV spectrophotometry, as CDs do not absorb in the UV spectrum. Phase-solubility of drug/CD complexes comprises two processes. The experimental techniques for phase-solubility analysis involve multiple vials of equal masses of substrate (compound of interest) and solvent, with increasing amounts of the ligand (complexing agent), mixing a constant temperature and pressure. This is maintained until equilibrium in reached. As complexation lowers the thermodynamic activity of the dissolved drug, more drugs dissolves until the activity of the free drug, which is in chemical equilibrium with the complex, becomes equal to the thermodynamic activity of the pure solid drug. The interpretation of the resultant equilibrium plots according to the phase rule results in the determination of the stability constant of the complex.

Phase-solubility diagrams are generated by plotting the total molar concentration of the substrate against the molar concentration of the ligand. The diagrams obtained fall into two main categories, Type A and Type B. Type A diagrams are characterized by a continual increase in substrate concentration [S] with increasing ligand concentration [L], presumably due to the formation of soluble complexes between the substrate and the ligand. A strict linear rise in substrate concentration with increasing ligand concentration, designated AL, occurs when complexes are of the first order in Ligand (shown in figure 2). The linear relationship between solubility and CD concentration has a number of advantages, one of which is the lack of precipitation of the formulation upon dilution.

Positive curvature, type AP, arises when the complexes contain the ligand at higher orders than 1. Negative curvature, type AN, may arise when the presence of the ligand at high concentrations changes the stability constant of the complex or induces self-association of the ligand. This behavior is rare. The plateau may also represent saturation of the solution with ligand, owing to its own solubility limits.

Type B phase-solubility diagrams arise when complexes of limited solubility are formed between the substrate and the Ligand. The limited solubility of the natural CDs often gives rise to type B phase diagrams when complexed with other compounds with limited aqueous solubility. Type BS diagrams indicate some solubility of the complex.

Figure 2 – Phase solubility diagram

Through this phase solubility study –

Stoichiometry can be determined or estimated

Stability constant [K]

Complexation efficiency

Solubilizing Efficiency can be determined as the Ratio between drug solubility in aqueous solution of CD and in water.

Cyclodextrin complexation is mainly suitable for drugs, molecules, compounds of BCS class II and class IV. Not all the drugs and molecules can be complexed with cyclodextrin. The minimum criteria for complexation are –

Ø More than five atoms (C, P, S, N) form the skeleton of the drug molecule.

Ø Melting point temperature of the substance is below 250 ⁰C.

Ø Solubility in water is less than 10 mg/mL.

Ø The guest molecule consists of less than five condensed rings.

Ø Molecular weight between 100 and 400.

CONCLUSION:

In this article an attempt has been made to discuss and explore the concept of jobs plot or continuous variation method for determination of the Stoichiometry of cyclodextrin inclusion complexes. This can be done mainly by using spectral methods like UV, FTIR, NMR, circular diochrism etc. Both continuous variation and phase solubilty are used to estimate the stoichiometry. This is applicable for drugs with poor biopharmaceutical and physic-chemical properties.

REFERENCES:

1. Tablet C. The Determination of the Stoichiometry of Cyclodextrin Inclusion complexes by Spectral Methods: Possibilities and Limitations. Stoichiometry and Research – The Importance of Quantity in Biomedicine. www.intechopen.com

2. Ficarra, R., Ficarra, P., Di Bella, M.R., Raneri, D., Tommasini, S., Calabro, M.L., Gamberini, M.C. and Rustichelli, C. Study of β-blockers:-cyclodextrins inclusion complex by NMR, DSC, X-ray and SEM investigation. Journal of Pharmaceutical and Biomedical Analysis. 2000; 23 (1): 33-40.

3. Li, J. and Loh, X.J. Cyclodextrin-based supramolecular architectures: Syntheses, structures, and applications for drug and gene delivery. Advanced Drug Delivery Reviews. 2008; 60 (9): 1000-1017.

4. Martin Del Valle, E.M. Cyclodextrins and their uses: a review. Process Biochemistry. 2004; 39 (9): 1033-1046.

5. Connors, K. A. Binding Constants: The Measurement of Molecular Complex Stability; John Wiley and Sons, Inc.: New York, 1987.

6. Iacovino R. β-Cyclodextrin Inclusion Complex to Improve Physicochemical Properties of Pipemidic Acid: Characterization and Bioactivity Evaluation. Int. J. Mol. Sci. 2013; 14: 13022-13041.

7. Yanez C. Cyclodextrin Inclusion Complex to Improve Physicochemical Properties of Herbicide Bentazon: Exploring Better Formulations. PLOs One. 2012; 7 (8): e41072.

8. Iacovino R. Physicochemical Characterization and Cytotoxic Activity Evaluation of Hydroxymethylferrocene: β-Cyclodextrin Inclusion Complex. Molecules. 2012; 17: 6056-6070.

9. Challa R, Ahuja A, Ali J, Khar R. Cyclodextrins in drug delivery: an updated review. AAPS Pharm Sci Tech. 2005; 6: 329–357.

10. Connors KA. The stability of cyclodextrin complexes in solution. Chemical Reviews. 1997; 97: 1325–57.

11. Harada P. Preparation and structures of supramolecules between cyclodextrins and polymers. Coord. Chem. Reviews. 1996; 148: 115—133.

12. Higuchi T, Connors KA. Phase-solubility techniques. In: Reilly CN, editor. Advances in analytical chemistry and instrumentation. pp. 117–212. Wiley, New York (1965).

Received on 18.05.2016 Modified on 02.06.2016

Research J. Pharm. and Tech. 2016; 9(7):1013-1016.

DOI: 10.5958/0974-360X.2016.00191.8