MS 4 and 5 report
-
Upload
nauman-mithani -
Category
Documents
-
view
473 -
download
1
description
Transcript of MS 4 and 5 report
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
yield/efficiency,
!
" molecules that fluoresce( )" excited molecules( )
. The experiment conducted, measured
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):
!
5 "10#6mol • 376.2
g
mol=1.881mg , which was added to 1 litre of water.
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)
!
(nm)
abs./emis.
intensity
!
(nm)
abs./emis.
intensity
classification
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: -
HO O O
C
O
OH
fluorescein {str. 1}
HO O O
C
O
O-
fluorescein anion {str. 2}
-O O O
C
O
O-
fluorescein dianion {str. 3}
De-protonated forms of this molecule are its anion
and di-anion; which in turn can form multiple resonance
species, giving rise to changes in emission.
The acid dissociation constant, pKa, of a molecule in
its ground state differs from that of its excited state, at
times 5 orders of magnitude or more.
From pH = 7 onwards, as more base is added, more
and more fluorescein is de-protonated to its anion; at pH
! 10.5, virtually, all the excited fluorescein molecules
exists as its anion {str. 2}.
pH = 12 is considered an anomaly since it is deemed
unlikely that such a radical change in fluorescein could
occur in a pH difference of 1, and for that particular
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.