**1. Introduction**

184 Hydrodynamics – Advanced Topics

Wena, Z., Liua, B., Zhenga, Z., Youa, X., Pua, Y., Li, Q. (2010). Preparation of liposomes

Winters, M.A., Knutson, B.L., Debenedetti, P.G., Sparks, H.G., Przybycien, T.M., Stevenson,

Yeo, S.-D., Lim, G.-B., Debenedetti, P.G., Bernstein, H. (1993). Formation of microparticulate

technique, *Chem. Eng. Res. Des.*, 88, pp. 1102–1107.

*J. Pharm. Sci*., 85, pp. 586-594.

341-346.

entrapping essential oil from Atractylodes macrocephala Koidz by modified RESS

C.L., Prestrelski, S.J. (1996). Precipitation of proteins in supercritical carbon dioxide,

protein powders using a supercritical fluid antisolvent, *Biotechnol. Bioeng*., 41, pp.

The absorption of photons by a molecule leads to its excitation. An electronically excited molecule can lose its energy by emission of ultraviolet, visible, infrared radiation or by collision with the surrounding matter. Luminescence is thus the emission of photons from excited electronic energy levels of molecules. The energy difference between the initial and the final electronic states is emitted as fluorescence or phosphorescence (Lakowicz, 2006). Fluorescence is a spin-allowed radiative transition between two states of the same multiplicity (e.g., S1 → S0) whereas; phosphorescence is a spin-forbidden radiative transition between two states of different multiplicity (e.g., T1 →S0).

The mechanisms by which electronically excited molecules relax to ground state are given by the Jablonski diagram as shown in Fig. 1. The absorption of a photon takes a molecule from ground state (singlet state, S0) to either first excited state (singlet state, S1) or second

Fig. 1. Jablonski diagram of transitions among various electronic energy levels

Rotational Dynamics of Nonpolar and Dipolar

**1.2 Fluorescence anisotropy** 

**1.1 Steady-state and time resolved fluorescence** 

transition).

2006)

Molecules in Polar and Binary Solvent Mixtures 187

emission spectrum is generally a mirror image of the absorption spectrum (S0 to S1

Fluorescence measurements can be broadly classified into two types of measurements: steady-state and time-resolved. Steady-state measurements, the most common type, are those performed with constant illumination and observation. The sample is illuminated with a continuous beam of light, and the intensity or emission spectrum is recorded as function of wavelength. When the sample is first exposed to light steady state is reached almost immediately. Because of the ns timescale of fluorescence, most measurements employ steady-state method. The second type of measurement is time-resolved method which is used for measuring intensity decays or anisotropy decays. For these measurements the sample is exposed to a pulse of light, where the pulse width is typically shorter than the decay time of the sample. The intensity decay is recorded with a high-speed detection

system that permits the intensity or anisotropy to be measured on the ns timescale.

The photoselection of fluorescent probe by polarized light offers the opportunity to study some relevant processes occurring at molecular level in heterogeneous systems. The fluorescence, emitted from the samples excited with polarized light, is also polarized. This polarization is due to the photoselection of the fluorophores according to their orientation relative to the direction of the polarized excitation. This photoselection is proportional to the square of the cosine of the angle between the absorption dipole of the fluorophore and the axis of polarization of the excitation light. The orientational anisotropic distribution of the excited fluorophore population relaxes by rotational diffusion of the fluorophores and excitation energy transfer to the surrounding acceptor molecule. The polarized fluorescence emission becomes depolarized by such processes. The fluorescence anisotropy measurements reveal the average angular displacement of the fluorophore, which occurs between absorption and subsequent emission of a photon. The degree of polarization, *P*, and steady state fluorescence anisotropy *r*, are thus respectively given by equations (Lakowicz,

> || || *I I*


*I I* ⊥ ⊥

where || *I* and *I*⊥ represent the fluorescence intensities when the orientation of the emission polarizer is parallel and perpendicular to the orientation of the excitation polarizer, respectively. The fluorescence anisotropy (*r*) is a measure of the average depolarization during the lifetime of the excited fluorophore under steady-state conditions. A steady-state observation is simply an average of the time-resolved phenomena over the intensity decay of the sample. But the time resolved measurements of fluorescence anisotropy using ultrafast polarized excitation source (laser) give an insight into the time dependent

*I I* ⊥ ⊥

<sup>−</sup> <sup>=</sup> + (2)

<sup>−</sup> <sup>=</sup> + (3)

*P*

*r*

depolarization. The time dependent fluorescence anisotropy decay, *r(t)*, is defined as

excited state (S2). The excited molecule then relaxes to the lowest vibronic level of the first excited state through internal conversion (IC), which generally occurs within 10-12 s or less. Since fluorescence lifetimes are typically near 10-8 s, IC is generally complete prior to emission. Now it can relax from the singlet excited state to the ground state via three mechanisms. First by emitting a photon (radiative process), second without emitting photon (nonradiative mechanism) and third it goes to a triplet state (T1) by intersystem crossing (ISC) which also is a nonradiative process. The transition from triplet (T1) to ground singlet state is forbidden and hence is a very slow process relative to fluorescence. Emission from T1 is called phosphorescence and generally is shifted to longer wavelength relative to the fluorescence.

In fluorescence spectroscopy the observed spectral intensity is a function of two variables: the excitation wavelength (*λex*) and the emission wavelength (*λem*). The fluorescence property of a compound is conventionally studied by examining both the excitation spectrum and the emission spectrum. The intensity vs. wavelength plot of the fluorescence spectrum obtained is characteristic of a fluorophore and sensitive to its local surrounding environment. It is consequentially used to probe structure of the local environment. Generally, the wavelength of maximum fluorescence intensity is shifted to longer wavelength relative to the wavelength of its absorption maximum. The difference between these two wavelengths, known as Stokes' shift, arises because of the relaxation from the initially excited state to the 'ground' vibronic level of S1 which involves a loss of energy. Further loss of energy is due to the transitions from S1 to higher vibrational levels of the ground state S0. The Stokes' shift further increases because of general solvent effects. The energy difference between the absorption maximum (ν*a*) and the emission maximum (ν*<sup>f</sup>*) is given by Lippert equation (Birks, 1970) in which the energy difference (ν*a*ν*<sup>f</sup>*) of a fluorophore as a function of the refractive index (*n*) and dielectric constant (ε) of the solvent is related as

$$\nu\_a - \nu\_f = \frac{2}{hc} \left[ \frac{\varepsilon - 1}{2\varepsilon + 1} - \frac{n^2 - 1}{2n^2 + 1} \right] \frac{(\mu^\* - \mu)^2}{a^3} + const \tag{1}$$

where *h* is the Planck's constant, *c* the velocity of light and *a* is the radius of the cavity in which the fluorophore resides. Also, μ and μ*\** are the ground and excited state dipole moments, respectively.

Fluorescence emission is generally independent of excitation wavelength. This is because of the rapid relaxation to the lowest vibrational level of S1 prior to emission, irrespective of excitation to any higher electronic and vibrational levels. Excitation on the extreme red edge of the absorption spectrum frequently results in a red-shifted emission. The red-shift occurs because red-edge excitation selects those fluorophores which are more strongly interacting with the solvent (solvation dynamics) (Demchenko, 2002). The red-edge effect can also be thought as ground state heterogeneity, which is common in most complex systems like a probe distribution in microheterogeneous media. In the case of ground state heterogeneity or the presence of multiple species in the ground state, the fluorescence emission spectrum is dependent on the excitation wavelength and the fluorescence excitation spectrum is dependent on the emission wavelength. Also fluorescence excitation spectrum observed for a given emission wavelength differs from that of the absorption spectrum for heterogeneous system. The large spectral width of the emission spectrum compared to absorption spectral width is also due to the presence of multiple species in the excited state. Fluorescence

excited state (S2). The excited molecule then relaxes to the lowest vibronic level of the first excited state through internal conversion (IC), which generally occurs within 10-12 s or less. Since fluorescence lifetimes are typically near 10-8 s, IC is generally complete prior to emission. Now it can relax from the singlet excited state to the ground state via three mechanisms. First by emitting a photon (radiative process), second without emitting photon (nonradiative mechanism) and third it goes to a triplet state (T1) by intersystem crossing (ISC) which also is a nonradiative process. The transition from triplet (T1) to ground singlet state is forbidden and hence is a very slow process relative to fluorescence. Emission from T1 is called phosphorescence and generally is shifted to longer wavelength relative to the

In fluorescence spectroscopy the observed spectral intensity is a function of two variables: the excitation wavelength (*λex*) and the emission wavelength (*λem*). The fluorescence property of a compound is conventionally studied by examining both the excitation spectrum and the emission spectrum. The intensity vs. wavelength plot of the fluorescence spectrum obtained is characteristic of a fluorophore and sensitive to its local surrounding environment. It is consequentially used to probe structure of the local environment. Generally, the wavelength of maximum fluorescence intensity is shifted to longer wavelength relative to the wavelength of its absorption maximum. The difference between these two wavelengths, known as Stokes' shift, arises because of the relaxation from the initially excited state to the 'ground' vibronic level of S1 which involves a loss of energy. Further loss of energy is due to the transitions from S1 to higher vibrational levels of the ground state S0. The Stokes' shift further increases because of general solvent effects. The energy difference between the

*a*) and the emission maximum (

2 1 2 1 *a f*

ε

μ and μ

ε

ε

ν*a*ν

2 1 1( )

where *h* is the Planck's constant, *c* the velocity of light and *a* is the radius of the cavity in

Fluorescence emission is generally independent of excitation wavelength. This is because of the rapid relaxation to the lowest vibrational level of S1 prior to emission, irrespective of excitation to any higher electronic and vibrational levels. Excitation on the extreme red edge of the absorption spectrum frequently results in a red-shifted emission. The red-shift occurs because red-edge excitation selects those fluorophores which are more strongly interacting with the solvent (solvation dynamics) (Demchenko, 2002). The red-edge effect can also be thought as ground state heterogeneity, which is common in most complex systems like a probe distribution in microheterogeneous media. In the case of ground state heterogeneity or the presence of multiple species in the ground state, the fluorescence emission spectrum is dependent on the excitation wavelength and the fluorescence excitation spectrum is dependent on the emission wavelength. Also fluorescence excitation spectrum observed for a given emission wavelength differs from that of the absorption spectrum for heterogeneous system. The large spectral width of the emission spectrum compared to absorption spectral width is also due to the presence of multiple species in the excited state. Fluorescence

*hc n a*

 − −− −≈ − <sup>+</sup> + +

ν

) of the solvent is related as

μ

*<sup>n</sup> const*

2 \*2 2 3

 μ *<sup>f</sup>*) is given by Lippert equation

(1)

*<sup>f</sup>*) of a fluorophore as a function of the

*\** are the ground and excited state dipole

fluorescence.

absorption maximum (

moments, respectively.

ν

(Birks, 1970) in which the energy difference (

ν ν

refractive index (*n*) and dielectric constant (

which the fluorophore resides. Also,

emission spectrum is generally a mirror image of the absorption spectrum (S0 to S1 transition).
