Question

Consider recording an emission spectrum of a solution of quinine in 0.05 M H2SO4, using a spectrofluorimeter. As a preliminary step you record a spectrum of the blank solution. With the excitation monochromator set at 395 nm and scanning the emission monochromator from 404 nm to 800 nm, you observe three sharp peaks.

A. At what wavelength would Rayleigh scattering appear in the emission spectrum as a result of spectral overlap from the second order of the grating?

B. At what wavelength would the Raman scattering from the O-H stretching vibration (at 3600 cm-1) for water appear in the emission spectrum?

C. At what wavelength would Raman scattering from the O-H stretching vibration (at 3600 cm-1) for water appear in the emission spectrum due to spectral overlap from the second order of the grating?

Thanks!

Answer 1

Quinine Fluorescence

Fluorescence spectroscopy can be used to quantify the

concentration of quinine in tonic water, providing the instrument

conditions remain constant and the quinine is in a dilute acid

solution. The chloride ion is the only normal interfering species as it

quenches quinine fluorescence. Although this has been found to be

negligible, providing the concentration of the chloride ion in tonic

water is below 0.4 mM, which it usually is. In dilute sulfuric acid,

quinine has two analytically useful excitation wavelengths; 250 and

350 nm. However, the wavelength of maximum fluorescence

(emission wavelength) is always 450 nm, regardless of the excitation

wavelength used. The fluorescence intensity will vary depending on

the relative strength of absorption.6,7

Excitation and Emission peaks observed for quinine analysis:

• First excitation peak at 250 nm, which corresponds to

an S0 →S2

* transition

• Second excitation peak at 350 nm, which corresponds

to an S0 → S1

* transition

• Single emission peak at 450 nm, which corresponds to

the S1 → S0

transition

Only one emission peak is observed because, following light

absorption and subsequent excitation to higher energy levels,

quinine undergoes both fluorescence and internal conversion.

Fluorescence is a radiative process which occurs following excitation

and thermal degradation (relaxation) to the ground vibrational state

of the excited electronic state. The radiation emitted on return to

the ground electronic state has a longer wavelength and, thus,

lower energy than that of the absorbed radiation. Internal

conversion, on the other hand, is a non-radiative and very efficient

process in which energy is dissipated if vibrational energy levels of

different electronic energy states with the same multiplicity strongly

overlap (e.g. S1

* and S2

*). Internal conversion is very efficient

between the two electronically excited states of quinine and,

therefore, only one emission at 450 nm is observed  
The types of transitions associated with fluorescence include π*-to-π and π*-to-n; σ*-to-
σ transitions are seldom observed because fluorescence spectra from absorptions at wavelengths
shorter than 250 nm are not likely to occur – the high energy causes deactivation of excited states
by predissociation or dissociation. At 200 nm (equal to 140 kcal/mol) bond ruptures often take
place. The molar absorptivity for a π-to-π* transition (excitation) is 100 to 1000 times that for a
n-to-π* transition. The time involved for the former is of the order of 10-7 to 10-9 s; that for the
latter is 10-5 to 10-7 s.
Under usual conditions and at a concentration of about 2 µg/mL quinine, the two
excitation peaks (250 and 350 nm) and one emission peak (450 nm) will be seen. However, for
carefully controlled conditions other peaks appear due to grating monochromator peculiarities
and Rayleigh, Tyndall, and Raman scattering. Rayleigh scattering refers to radiation scattered in
all directions by elastic collisions (impinging and dispersed radiation are the same wavelength
and are radiated in a random manner). In Raman scattering, the collisions are nonelastic, due to
the mixing of the electromagnetic energy with the rotational and vibrational energy of the
colliding molecule, and the emerging radiation will be at a different wavelength.