The long-wavelength emission is attributed to the formation of a TICT state facilitated by H-bonding of the solvent to the pyridine nitrogen of DMAPIP-b. The double emission observed in all protic solvents and the equilibrium between the locally excited state and the TICT state is not established even close to the boiling point of the solvents.
Structures of the N-alkylated products
In the last part of the chapter, the effect of adding a long alkyl chain on the photophysics of DMAPIP-b is discussed. The interaction of DMAPIP-b and its alkylated products (1 and 2) with BSA is presented in the last part of this chapter.
List of Abbreviation
List of Tables
143 Table 4.4.1: Absorption band maxima (λmaxab, nm), fluorescence band maxima (λmaxfl), fluorescence quantum yield (φ) and lifetime (τ, ns) of DMAPIP-b and its alkylated products in the absence and presence of BSA. Fluorescence characteristics (maximum fluorescence band, λmaxfl, nm and fluorescence lifetime, τ, ns) of BSA in the absence and presence of ligands.
Contents
Materials, methods and instrumentation 31 Chapter 3: Effect of homogeneous media on the dual fluorescence of
Chapter 4: Effect of microheterogeneous media on the dual fluorescence of
Introduction
- Molecular fluorescence
- Intramolecular charge transfer
- Organized media
- Scope of the present work
The molecules are thus more active in their excited state than the ground state. In the case of 2-naphthol, the fluorescence emission is not a mirror image of the absorption spectrum.
Materials, Methods and Instruments
Introduction
This chapter contains details of the chemicals and solvents used for the work and the procedures for the synthesis of the fluorophores. The methods used for the analysis, calculations, preparation of the samples are also expanded in the chapter.
Materials
- Other Chemicals
DMAPIP-b was synthesized by heating an equimolar mixture of 2,3-diaminopyridine and 4-dimethylaminobenzoic acid in polyphosphoric acid at 230 °C for 4 h as reported for similar compounds for synthesis [193,194]. The compound was extracted with dichloromethane and further purified by column chromatography followed by recrystallization from a mixture of methanol and ethanol.
Preparation of Samples
Surfactant solutions were prepared by dissolving the appropriate surfactant (SDS, CTAB, or TX-100) in Millipore water. TX-100/benzene/hexane/water reverse micelles were prepared by mixing TX-100 and the desired amount of water in a constant volume ratio of hexane and benzene (7:3).
Methods
The pKa or 'acid dissociation constant' is a measure of the strength of an acid or a base. The best-known Hammetts equation, which is used for the determination of ionization constant (pKa) of the dissociation reaction of an acid in aqueous medium is given below.
Instruments
Fluorescence spectra must be corrected for distortion due to the wavelength dependence of several instrument components. The excitation spectrum is distorted by variations in the intensity of the excitation light. These changes are due to the dependence of the lamp intensity on the wavelength and the transmission efficiency of the excitation monochromator.
Because the quantum counter bypasses the wavelength dependence of the sensitivity of the reference photomultiplier, the ratio of the fluorescence signal from the sample to that from the quantum counter or photodiode, as a function of the excitation wavelength, in principle yields corrected excitation spectra. The relationship between the measured excitation spectrum for this reference compound – as described above using the quantum counter – and the absorption spectrum provides the correction factors that can be stored in the computer.
Introduction
Effect of solvents and pH
Dual emission is observed only in protic solvents, and the ratio of shorter wavelength to longer wavelength emission increases with increasing H-donating capacity of the solvent (Figure 3.1.1), except in water. This shows that the longer wavelength emission is strongly influenced by the position of the pyridine nitrogen. The lifetime of the shorter wavelength emission is weakened in protic solvents compared to aprotic solvents.
Nevertheless, we speculate that H-bonding between the protic solvents and the unbonded electron pair at the pyridine nitrogen plays an important role in the formation of the TICT in DMAPIP-b. The intensity of the longer wavelength emission of DMAPIP-b increases with increasing H-bond donating capacity of the solvent, but decreases in water.
Effect of temperature
The relative population obtained from the Boltzmann distribution (not shown) showed that the population of isomer II in all solvents was less than 1% even at the boiling point of the solvents. 1-propanol and ethanol have a clear iso-emission point on the blue side of the band maximum. In propanol, an additional quasi-isoemission point is observed at 460 nm at the rear of the band maximum.
The lifetime is shortened with protic solvents and the lifetime weakening increases with the hydrogen bonding capacity of the solvent. The lifetime of the locally excited states decreases in 1-propanol and ethanol, but increases in methanol (Figure 3.2.4a).
LE k 1 TICT k 2
Since the orbitals involved in the radiative transition from the TICT state to the corresponding Franck-Condon state are located on different parts of the molecular system (donor and acceptor) with minimal overlap, the decay is expected to have a small transition moment due to its forbidden nature. After coupling to the appropriate vibration, the TICT emission is predicted to decrease in intensity from the allowed higher excited states and is responsible for its significant quantum yield. Due to this vibronic coupling, the radiative decay of the TICT emission is temperature dependent and a larger fraction of the TICT emission arises from less forbidden higher vibronic levels than hot fluorescence.
However, it is shown that both solvent parameters viscosity and polarity, and solute parameters such as spin angle of ground state, rotational volumes, molecular shape and electronic nature are found to determine the rate for this forward [13]. The kinetics of TICT emission can be explained by the general scheme (Scheme 3.2.1) for the TICT process as shown below [13].
Effect of metal ions
The absorption maximum of DMAPIP-b remains unchanged, only a small hypochromic shift was observed upon addition of Li+ (Figure 3.3.1) or Na+ (Figure 3.3.2). This is consistent with addition of Ni2+ and Cd2+ at the imidazole nitrogen of DMAPIP-b to form metal complexes (Diagram 3.3.2). The fluorescence band maximum of DMAPIP-b is unaffected by the addition of alkali metals (not shown).
These changes are consistent with Zn 2+ -induced enhancement of the TICT emission of DMAPIP-b by complexation. Zn+2 binds specifically to the pyridine nitrogen of DMAPIP-b and produces a significant change in the absorption and emission spectra.
Effect of long alkyl chain
The fluorescence spectrum of DMAPIP-b in methanol is compared with that of the alkylated molecules in Figure 3.4.4. DMAPIP-b exhibits TICT emission in the protic solvents due to hydrogen bonding of the solvent molecule with the pyridine nitrogen. In water, the relative amplitude of the TICT emission increases (Table 3.4.1) and the fluorescence spectrum also has a long tail (Figure 3.4.5a).
Unlike DMAPIP-b and 1, in 2 the imidazopyridine acceptor ring and the benzene ring are out of plane by 41.4° in. b.
Introduction
Effect of -cyclodextrin
The TICT fluorescence of DMAPIP-b is induced by hydrogen bonding of the solvent with pyrion nitrogen. As seen previously (section 3.1), the intensity ratio of the TICT to normal emission increases with an increase in the hydrogen bonding capacity of the solvent, but in water the TICT emission is almost completely quenched (lifetime data indicate only 1% for decay measurements below 420°C). nm). This indicates that limiting the increased viscosity in the CD cavity does not affect the formation of the TICT state in DMAPIP-b.
This is consistent with the observation of TICT emission in the monocation formed by the protonation of the pyridine nitrogen in DMAPIP-c [182]. The study of DMAPIP-b in -CD also reveals that H-bonding of the solvent with the donor does not play a role in the formation of the TICT state in DMAPIP-b.
Effect of normal micelles
DMAPIP-b forms an inclusion complex with -CD, and the host-guest ratio of the DMAPIP-b/-CD complex is 1:1. Reduced polarity within the hydrophobic -CD cavity is more important in controlling the emissive nature of DMAPIP-b in the complex than limiting molecular motion. Of the three possible monocations, only two types of monocations are found in the -CD solution, MC1, which is formed by protonation of the pyridine nitrogen, and MC2, which is formed by protonation of the imidazole nitrogen.
In other words, micelles can uniquely affect the TICT emission of DMAPIP-b. Thus, we investigated the effects of TX-100 (nonionic), CTAB (cationic), and SDS (anionic) micelles on the photophysics of DMAPIP-b.
Na SDS
Attention has been directed to the micellar activities on the nature and characteristics of various photophysical and photochemical processes. When bound, a molecule will experience a different environment inside the micelle's microheterogeneous structure than bulk solvents for e.g. polarity, viscosity and diffusion of water molecules change as they move towards the micelle core. The studies result in an interesting observation that the TICT emission is only induced in SDS and TX-100, but not in
NMe 3 Br CTAB
- Effect of reverse micelles
- Effect of Bovine serum albumin (BSA)
The orientation also mediates the hydrogen bonding of the water with pyridine nitrogen of DMAPIP-b and such hydrogen bonding is necessary to induce the TICT emission in DMAPIP-b. It is well known that TICT emission is highly dependent on the polarity of the environment. It is reported that the fluorescence quantum yield decreases sharply upon the initial addition of water when the molecule is present in the water pool of the reverse micelle [320].
Only such an orientation ensures the hydrogen bond between the pyridine nitrogen of the probe and the water molecule, which is crucial for inducing the TICT emission of DMAPIP-b. The observation of TICT emission inside BSA suggests that the protein did not resist the formation of TICT.
Time/ns
The prolongation of the fluorescence lifetime of 2 with increasing BSA concentration can be explained by a change in the non-radiative decay pathway of the emitting species due to their interaction with the BSA protein. With an increase in BSA concentration, there is a decrease in the nonradiative decay of 2 due to the formation of an inclusion complex due to the restriction of internal motions. The quenching of tryptophan fluorescence by BSA can be described by the Stern-Volmer equation.
0 is the lifetime of the protein in the absence of a quencher, and [Q] is the quencher concentration. A progressive quenching of the fluorescence intensity of BSA is observed as the concentrations of the ligands increase (Figure 4.4.5).
Molecule 1], µµµµM
However, predictions for the distance of ligands from tryptophan using resonance energies. The fluorescence spectral shifts of the ligands in BSA suggest the hydrophobic nature of the binding environments. With increasing ligand concentrations, the fluorescence spectrum of BSA is progressively red shifted from 345 in the native form to 378 nm in the presence of 40 μM.
The intrinsic fluorescence decay of BSA is quite complex due to the presence of tryptophan in different environments. However, the fluorescence of 2 only increases due to the reduction of the internal motion of the molecule in BSA.
![Figure 4.4.7: The plot of I 0 /(I 0 -I) versus 1/[Molecule 1].](https://thumb-ap.123doks.com/thumbv2/azpdfnet/10449006.0/188.918.150.786.364.989/figure-4-plot-i-i-i-versus-molecule.webp)
