Title

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MOLECULAR SPECTROSCOPY
section 4: Rayleigh and Raman scattering at low fluorescence intensity section 5: Effect on quantum yield of fluorescein

Full name: Student no.: Sections:

NAUMAN MITHANI
301016320 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.

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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) . 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.

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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

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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.

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DATA and RESULTS:
MS 4: -

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)! graphs based on borrowed data: -

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! MS 5: 5 µmol/L fluorescein (sodium salt):

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5 "10#6 mol • 376.2

g = 1.881 mg , which was added to 1 litre of water. mol

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DISCUSSION:
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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.

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*$! MS 4: Q1: A Rayleigh peak corresponds to an elastic exchange of energy by the

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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.
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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.

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*%! Q3: quinine sulphate (aq) ! abs./emis. (nm) intensity excitation (450 nm) 352 322 456 482 458 484 476 508 78135 5220 386571 13325 200294 13443 798590 16591 0.05 mol/L H2SO4 (aq) ! abs./emis. (nm) intensity 352 322 458 484 458 484 476 508 90105 5496 398882 13841 398518 13758 798597 16461

classification

Rayleigh Stokes fluorescence anti-Stokes fluorescence anti-Stokes fluorescence anti-Stokes

emission #1 (350 nm)

emission #2 (350 nm)

emission #3 (370 nm)

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*&! MS 5: Q1: -

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*'! Q2, Q3: De-protonated forms of this molecule are its anion
OH

O C

and di-anion; which in turn can form multiple resonance species, giving rise to changes in emission.

HO

O fluorescein {str. 1}

O

O C O-

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

HO

O fluorescein anion {str. 2}

O

O C O-

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

-O

O fluorescein dianion {str. 3}

O

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.

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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.

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