Title: MOLECULAR SPECTROSCOPY

section 4: Rayleigh and Raman scattering at low fluorescence

intensity

section 5: Effect on quantum yield of fluorescein

Full name: NAUMAN MITHANI

Student no.: 301016320

Sections: LA02: group C

Date of expt.: Jan. 31, 2008

ABSTRACT:

MS 4: {Rayleigh and Raman scattering at low fluorescence, with quinine sulphate}

The lesser intensity of Raman Stokes and anti-Stokes relative to the strongest

signals of Rayleigh and the fluorescence signals has been shown. The Rayleigh peak

occurred at 352 nm in the excitation spectrum (450 nm); the fluorescence peaks

occurred at 456 nm, 458nm and 476 nm in the emission spectra (350 nm, 350 nm and

370 nm respectively), reasonably verified with literature values.

MS 5: {Effect on quantum yield of fluorescein}

The effect of environment, specifically pH, were studied and explained;

changing conditions altered the chemical species and thus the detected emission

spectrum. The de-protonation of fluorescein began at pH of 8 and by 10, virtually all

of it was in its anionic form, which is when the maximum intensity was observed.
! "!

INTRODUCTION:

MS 4: Incident radiation is, for the most part, scattered elastically by the

molecules/atoms of the liquid or gas subjected to it. In other words, the molecule

returns to a stable/ground/de-excited state by emitting photons bearing the same

frequency as the incident photons; thus, there is no net gain or loss in energy. This is

known as Rayleigh scattering; the opposite is Raman or inelastic scattering. In this

form of scattering, the emitted photons are of a lower frequency and energy (Stokes)

or of a higher frequency and energy (anti-Stokes). This experiment saw the

identification of the Rayleigh and Raman peaks in the absorption and fluorescence

spectrum of a quinine sulphate solution of a low 0.01 ppm concentration.

MS 5: Intensities of excitation and fluorescence spectra of molecules are

susceptible to changes in the environment e.g. pH; this is defined in terms of quantum

"(molecules that fluoresce)

yield/efficiency, . The experiment conducted, measured
"(excited molecules)

average fluorescence intensities as a function of pH, ranging from 7 to 13. The

!
phenomena of molecular absorption spectroscopy is such that every chemical specie

(a molecule or its ions) has its own unique emission profile, which may be used for

identification.
! #!

EXPERIMENTAL:

MS 4:

The experiment was commenced with the preparation of a 50 mL solution of

100 ppm (100 mg/L) quinine sulphate (aq). 5 mg of quinine sulphate solid were

weighed out and dissolved in 0.05 mol/L H2SO4 (aq) in a 50 mL volumetric flask. The

flask was filled to the mark with the acid. 5 mL of this solution were diluted to the

mark with the acid in a new volumetric flask, the quinine sulphate concentration now

being 10 ppm. Then, 5 mL of this solution were diluted to the mark with the acid in a

new volumetric flask, the quinine sulphate concentration now being 1 ppm. Such

dilutions were carried out until a quinine sulphate solution of 0.01 ppm was obtained.

Next, an excitation scan of the quinine sulphate solution was recorded. A clear

quartz cuvette (thickness / path length of 1 cm) was rinsed then filled with the 0.01

ppm quinine sulphate solution, placed in the spectrofluorometer and its spectrum

recorded. The excitation scan was measured at 450 nm (quinine sulphate’s fluorescence

emission wavelength as determined in the preceding set of experiments), the parameters were

wavelengths of 200 to 600 nm, step size of 2 nm. Two emission scans of the solution

followed, both measured at 350 nm and same scan parameters (quinine sulphate’s

absorption wavelength as determined in the preceding set of experiments); a third emission scan

followed, this was measured at 370 nm (20 nm higher) with the scan parameters

unchanged.

The cuvette was cleaned, rinsed then filled with the 0.05 mol/L H2SO4 (aq).

Then four scans were repeated with this substance as the analyte.

MS 5:

1.881 mg of fluorescein was dissolved in 1 litre of water (in a 1 litre

volumetric flask) to produce a 5 µmol/L solution of the substance.

! $!
Starting with a solution of pH of 13 (0.1 mol/L NaOH supplied by the

laboratory), 100 mL solutions of pH 12 to 7 inclusive were prepared next. This was

done by diluting 10 mL of the immediately higher pH solution to the mark in a new

100 mL volumetric flask (de-ionised water was used for the dilutions).

5 mL of the 5 µmol/L fluorescein (aq) were added to seven new 50 mL

volumetric flasks each, which were then diluted to the mark with a particular pH

solution, one flask for dilution with each pH. A clear quartz cuvette (thickness / path

length of 1 cm) was rinsed then filled with the fluorescein (aq), placed in the

spectrofluorometer and its time-based fluorescence intensity spectrum recorded. The

scan’s parameters were set at a duration of 40 seconds (1 data point/s), and excitation

and emission wavelengths of 490 and 518 nm (literature values) respectively. A scan

of each fluorescein (aq) – pH solution was recorded.

! %!

DATA and RESULTS:

MS 4: -

!
! &!

!
! '!

!
! (!

!
! )!

! graphs based on borrowed data: -

! *+!
! **!
! *"!
 ! *#!
MS 5: -

5 µmol/L fluorescein (sodium salt):

g
5 "10#6 mol • 376.2 = 1.881 mg , which was added to 1 litre of water.
mol

DISCUSSION:

! Reason for borrowing data: -

The results based on the data obtained from the performed experiments were

deemed, in light of comparison with the literature, absolutely flawed.

The emission scans of 0.01 ppm quinine sulphate, dissolved in 0.05 mol/L

H2SO4, are identical to that of 0.05 mol/L H2SO4 (aq) only. Secondly, the spectra bear

no Rayleigh peak at ~350 / ~370 nm (wavelengths of the incident radiation), the

maximum emission (fluorescence) peaks are shifted far from the literature value of

450 nm and that these peaks are exceedingly broad.

Possible reasons for such untenable results: error(s) in calibration of the

instrument; a glitch/bug in the software. Possible reason of contaminating the quinine

sulphate (aq) solutions is dismissed since the preparation of quinine sulphate (aq)

solutions was a simple procedure involving the dissolution of solid quinine sulphate in

0.05 mol/L H2SO4 (aq), both substances provided by the laboratory. Another possible

reason of compromising the transparency of the cuvette with fingerprints is also

dismissed since all members of the group were wearing latex gloves at all times.
! *$!

! MS 4: -

! Q1:

A Rayleigh peak corresponds to an elastic exchange of energy by the

molecules of the substance subjected to incident radiation. Detected photons (as they

are emitted by the substance as it de-excites) are of the same energy as those incident

on the substance and the peak in the spectrum is observed at the wavelength of

incident radiation.

Raman Stokes peaks correspond to a net gain in energy by the substance in

question. The substance emits photons of lesser energy than of those absorbed; the

Stokes peak appears at a higher wavelength than that of the incident radiation. The

opposite holds for the anti-Stokes peak. Stokes and anti-Stokes peaks appear an equal

distance apart from the Rayleigh peak either side of it.

! Q2:

Raman peaks may appear at any frequency, unlike fluorescence (resonance)

peaks, which appear at a particular frequency for a substance. This is seen in the

emission spectra on pg 9 and the table below. Fluorescence peaks are also of greater

intensity than Raman peaks.

! *%!
! Q3:

quinine sulphate (aq) 0.05 mol/L H2SO4 (aq)

! abs./emis. ! abs./emis. classification
(nm) intensity (nm) intensity

352 78135 352 90105 Rayleigh

excitation (450 nm)
322 5220 322 5496 Stokes

456 386571 458 398882 fluorescence

emission #1 (350 nm)
482 13325 484 13841 anti-Stokes

458 200294 458 398518 fluorescence

emission #2 (350 nm)
484 13443 484 13758 anti-Stokes

476 798590 476 798597 fluorescence

emission #3 (370 nm)
508 16591 508 16461 anti-Stokes
! *&!

! MS 5: -

! Q1: -
! *'!
! Q2, Q3: -

O De-protonated forms of this molecule are its anion

C OH
and di-anion; which in turn can form multiple resonance

species, giving rise to changes in emission.

HO O O

fluorescein
{str. 1}

The acid dissociation constant, pKa, of a molecule in

O

C O- its ground state differs from that of its excited state, at

times 5 orders of magnitude or more.

HO O O From pH = 7 onwards, as more base is added, more

fluorescein anion
{str. 2} and more fluorescein is de-protonated to its anion; at pH

! 10.5, virtually, all the excited fluorescein molecules

O
exists as its anion {str. 2}.
C O-

pH = 12 is considered an anomaly since it is deemed

unlikely that such a radical change in fluorescein could

-O O O
occur in a pH difference of 1, and for that particular
fluorescein dianion
{str. 3}
value only, when the prevalent trend shows otherwise.

As the pH is further raised, the fluorescein anion starts

to become further de-protonated to its di-anion form,

with its own emission profile.

! *(!

CONCLUSION:

MS 4: {spectroscopy with quinine sulphate}

It was observed that the resonance peaks of Rayleigh and fluorescence were

the strongest, the Raman Stokes and anti-Stokes were significantly less intense and

that there exists a greater probability of anti-Stokes than Stokes. The Rayleigh peak in

the excitation spectrum (450 nm) was observed at 352 nm; fluorescence peaks were

observed at 456, 458 and 476 nm for the 350, 350 and 370 nm emission spectra, fairly

in synchrony with the literature value of 450 nm.

MS 5: {spectroscopy with fluorescein}

The effect of environment, specifically pH, were studied and explained;

changing conditions altered the chemical species and thus the detected emission

spectrum. The highest emission signal was observed at pH of ~10.5 when ~all the

fluorescein had been de-protonated to its anionic form.