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Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) is one of the most powerful spectroscopic techniques in analytical chemistry. It is an important tool for the studies of host-guest complexes, for elucidating the structures of the various complexes existing in the form of aggregates, ion pair or encapsulated systems.As the name suggests, NMR identifies the different nuclei in the molecules (most commonly, proton), by measuring their chemical shift. The binding activity of two molecules causes a considerable change in their electronic environments. This leads to a shift in the signals in the NMR spectrum, and this basic principle is made use of to study the phenomena of host-guest chemistry. The driving forces for host-guest binding are the various secondary interactions between molecules, such as hydrogen bonding and pi-pi interaction. Thus, NMR also serves as an important technique to establish the presence of these interactions in a host-guest complex.

Previous NMR studies have given useful information about the binding of different guest to hosts. Fox et al. calculated the hydrogen-bond interactions between pyridine molecules and poly(amido amine (PAMAM) dendrimer; on the basis of the chemical shift of the amine and the amide groups. In a similar study, Xu et al. titrated carboxylate based G4 PAMAM dendrimer (the host) with various amine based drugs (the guests) and monitored the chemical shifts of the dendrimer. In conjunction with the 2D-NOESY NMR techniques, they were able to precisely locate the position of the drugs on the dendrimers and the effect of functionality on the binding affinity of the drugs. They found conclusive evidence to show that the cationic drug molecules attach on the surface of anionic dendrimers by electrostatic interactions, whereas an anionic drug localizes both in the core and the surface of the dendrimers, and that the strength of these interactions are dependent on the pKa values of the molecules.

In a different study, Sun et al. studied the host-guest chemistry of ruthenium trisbipyridyl-viologen molecules with cucurbituril. Whilst monitoring the change in the chemical shifts of the pyridine protons on viologen, they found that the binding modes for the 1:1 complexes are completely different for different cucurbituril molecules. In cucurbit[7]uril, only four aromatic protons per single bipyridyl ligand take part in the binding process, whilst all the aromatic protons of the ligand form a part of the complex when cucurbit[8]uril is used.

However, an important factor that has to be kept in mind while analyzing binding between the host and the guest is the time taken for data acquisition compared to the time for the binding event. In a lot of cases, the binding events are much faster than the time-scale of data acquisition, in which case the output is an averaged signal for the individual molecules and the complex. The NMR timescale is of the order of milliseconds, which in certain cases when the binding reaction is fast, limits the accuracy of the technique.

Raman spectroscopy
Raman spectroscopy is a spectroscopic technique used in the study of molecules which exhibit a Raman scattering effect when monochromatic light is incident on it. The basic requirement to get a Raman signal is that the incident light brings about an electronic transition in the chemical species from its ground state to a virtual energy state, which will emit a photon on returning to the ground state. The difference in energy between the absorbed and the emitted photon is unique for each chemical species depending on its electronic environment. Hence, the technique serves as an important tool for study of various binding events, as binding between molecules almost always results in a change in their electronic environment. However, what makes Raman spectroscopy a unique technique is that only transitions which are accompanied by a change in the polarization of the molecule are Raman active. The structural information derived from Raman spectra gives very specific information about the electronic configuration of the complex as compared to the individual host and guest molecules.

Solution phase Raman spectroscopy often results in a weak scattering cross-section. Therefore, recent advancements have been made to enhance the Raman signals, such as surface enhanced Raman spectroscopy, and Resonance Raman spectroscopy. Such techniques serve an additional purpose of quantifying the analyte-receptor binding events, giving a more detailed picture of the host-guest complexation phenomena where they actually take place; in solutions. In a recent breakthrough, Flood et al. determined the binding strength of tetrathiafulvalene (TTF) and cyclobis(paraquat-p-phenylene) using Raman spectroscopy. Prior work in this field was aimed at providing information on the bonding and the structure of the resulting complex, rather than quantitative measurements of the association strengths. The researchers had to use Resonance Raman spectroscopy in order to be able to get detectable signals from solutions with concentrations as low as 1 mM. In particular they correlated the intensity of the Raman bands with the geometry of the complex in the photo-excited state. Similar to UV-vis spectroscopy based titration; they calculated the binding constant by “Raman titration” and fitted the binding curves to 1:1 models, giving a $$\Delta G$$ of -5.7±0.6 kcal/mol. The study is now providing a basis for similar studies involving charge transfer complexes in solutions.

Isothermal titration calorimetry
Spectroscopic techniques give information about the binding constant $$K_a$$ and Gibbs free energy, $$\Delta G$$. To get the complete set of thermodynamic parameters such as $$\Delta H$$ and $$\Delta S$$, a Van’t Hoff analysis using the Van't Hoff equation would be required. However, recent advents in calorimetric techniques allows for the measurement of $$K_a$$ and $$\Delta H$$ in a single experiment, thus enabling determination of all the thermodynamic parameters using the equation:$$\Delta G$$
 * $$\Delta G = \Delta H - T \Delta S \,$$,

provided that the experiment is carried out under isothermal conditions; hence the name isothermal calorimetry. The procedure is similar to a conventional titration procedure wherein the host is added sequentially to the guest and the heat absorbed or evolved is measured, compared to a blank solution. The total heat released, Q corresponds to the association constant, $$K_a$$ and $$\Delta H_0$$ by the equation: $$Q = {V\Delta H_0[H.G]}$$

Which can be simplified as

$$Q = \frac { V\Delta H_0 K_a[H_0][G]}{1+K_a[G]}$$

Where
 * $$[H_0]$$ = Initial molar concentration of the host
 * $$[G]$$ = Molar concentration of the guest
 * $$V$$ = volume of the vessel

The above equation can be stored by non-linear regression analysis to obtain the value of $$K_a$$ and $$\Delta G$$ and subsequently $$\Delta H$$ and $$\Delta S$$ for that particular reaction. The advantages of isothermal titration calorimetry over the other commonly used techniques, apart from giving the entire set of thermodynamic parameters, are that it is more general and suited for a wide range of molecules. It is not necessary to have compounds with chromophores or UV-visible functional groups in order to monitor the binding process as the heat signal is a universal property of binding reactions. At the same time, the signal to noise ratio is pretty favorable which allows for more accurate determination of the binding constants, even under very dilute conditions. A recent example of the use of this technique was for studying the binding affinity of the protein membrane surrounding Eschericia coli to lipophilic cations used in drugs in various membrane mimetic environments. The motivation for the above study was that these membranes render the bacteria resistant to most quaternary ammonium cation based compounds which have the anti-bacterial effects. Thus an understanding of the binding phenomena would enable design of effective antibiotics for E. coli. The researchers maintained a large excess of the ligand over the protein to allowing the binding reaction to go to completion. Using the above equations the researchers proceeded to calculate $$K_a$$, $$\Delta G$$, $$\Delta H$$ and $$\Delta S$$ for each drug in different environments. The data indicated that the binding stoichiometry of the drug with the membrane was 1:1 with a micromolar value of $$K_a$$. The negative values of $$\Delta G$$, $$\Delta H$$ and $$\Delta S$$ indicated that the process was enthalpy driven with a value of 8-12 kcal/mol for each drug.

UV-vis spectroscopy
UV-vis spectroscopy is one of the oldest and quickest methods of studying the binding activity of various molecules. The absorption of UV-light takes place at a time-scale of picoseconds, hence the individual signals from the species can be observed. At the same time, the intensity of absorption directly correlates with the concentration of the species, which enables easy calculation of the association constant. Most commonly, either the host or the guest is transparent to UV-light, whilst the other molecule is UV-sensitive. The change in the concentration of the UV-sensitive molecules is thus monitored and fitted onto the Benesi-Hilderbrand plot, from which the association constant can be directly calculated. Additional information about the stoichiometry of the complexes is also obtained, as the Benesi-Hilderbrand method assumes a 1:1 stoichiometry between the host and the guest. The data will yield a straight line only if the complex formation also follows a similar 1:1 stoichiometry. A recent example of a similar calculation was done by Sun et al., wherein they titrated ruthenium trisbipyridyl-viologen molecules with cucurbit[7]urils and plotted the relative absorbance of the cucurbit molecules as a function of its total concentration at a specific wavelength. The data nicely fitted a 1:1 binding model with a binding constant of $$1.2 * 10^5 M^{-1}$$. As an extension, one can fit the data to different stoichiometries to understand the kinetics of the binding events between the host and the guest. Zhu et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2005, 62, 886-895 made use of this corollary to slightly modify the conventional Benesi-Hilderbrand plot to get the order of the complexation reaction between barium-containing crown ether bridged chiral heterotrinuclear salen Zn(II) complex $$BaZn_2L(ClO_4)_2$$ (host) with various guests imidazoles and amino acid methyl esters, along with the other parameters. They titrated a fixed concentration of the zinc complex with varying amounts of the imidazoles and methyl esters whilst monitoring the changes in the absorbance of the pi to pi* transition band at 368 nm. The data fit a model in which the ratio of guest to host of 2 in the complex. They further carried these experiments at various temperatures which enabled them to calculate the various thermodynamic parameters using the Van’t Hoff equation.