section 1: section 2: Absorption and fluorescence spectra Determination of quinine sulphate in tonic water using fluorescence spectroscopy section 3: Quenching interference in fluorescence spectroscopy

Full name: Student no.: Sections:

301016320 LA02: group C

Date of expt.: Jan. 24, 2008

A series of three molecular absorption-emission spectroscopy experiments were carried out with quinine sulphate as the primary object/analyte. MS 1: The objective was to verify quinine sulphate’s molar absorptivities using Beer’s law with the molecular absorption spectroscopy technique, calculated to be 51871, 12812, 7248 and 9280 L mol-1 cm-1 for wavelengths of 210, 250, 316 and 346 nm respectively. MS 2: The objective was to determine the concentration of quinine sulphate in a sample of tonic water, it was determined to be 3.371!10-7 mol L-1; the technique employed was fluorescence spectroscopy. MS 3: The effect of sodium halides as quenching agents on fluorescence spectroscopy was observed.



The experiment was comprised of three sub-experiments on molecular absorption- fluorescence spectroscopy. It involves irradiating a substance/analyte with a beam of radiation and measuring the particular magnitudes of energies (corresponding wavelengths) absorbed and/or emitted as the substance returns to a deexcited state. MS 1: The first section explored the application of Beer’s law of molecular absorption spectroscopy, it states A=!bc A ! ! ! ! absorption molar absorptivity (L mol-1 cm-1) path length in medium (cm-1) concentration of medium (mol L-1)

b c

Quinine sulphate of varying concentrations was used as the analyte, and the absorbance measurements were conducted with a UV-visible spectrophotometer. MS 2: Fluorescence spectroscopy of quinine sulphate-containing liquid was conducted using a spectrofluorometer. A sample of tonic water was analysed in order to determine the concentration of quinine sulphate in it. MS 3: Interference of quenching agents on the fluorescence spectra of quinine sulphate was investigated using varying concentrations of aqueous sodium halides. The data was processed as per the Stern-Volmer equation (adapted):

Ii = 1+ K sv [Q] If

! Ii If Ksv [Q] ! ! ! ! intensity/rate of fluorescence without quencher intensity/rate of fluorescence with quencher Stern-Volmer coefficient concentration of quencher


MS 1: The sub-experiment was commenced by weighing out (5.1 ± 0.1) mg of quinine sulphate and diluting it to mark in a volumetric flask of 50 mL with 0.05 mol L-1 H2SO4 (aq). This resulted in a quinine sulphate solution of 100 ppm concentration (1.277 ! 10-4 mol/L). (5 ± 0.02) mL of the solution, and subsequent solutions, were diluted to the 50 mL mark with the H2SO4 (aq) in new volumetric flasks resulting in quinine sulphate solutions of 10, 1, 0.1, 0.01, 0.001 and 0.0001 ppm concentrations respectively. The next step was the commencement of the molecular absorption spectroscopy, for which the HP-8453 UV-visible spectrophotometer was employed in conjunction with the UV-visible HP-8453 software program. A quartz cuvette (frosted opposite faces) of 1 cm thickness (path length) was filled with the H2SO4 (aq) and placed in the spectrophotometer for calibration (setting the baseline / zero mark) and determination of the limit of detection. Six measurements (spectra) were taken, the last five were deemed a consistent detector response and so the experiment moved ahead. The cuvette was removed from the spectrophotometer, rinsed with the lowest concentration quinine sulphate solution of 0.0001 ppm, filled with it, placed in the

! spectrophotometer and its absorption spectra taken. Absorption spectra of the other quinine sulphate solutions were taken in increasing order of concentration.


Next, quinine sulphate solutions were subjected to fluorescence spectroscopy. A clear quartz cuvette (thickness / path length of 1 cm) was rinsed then filled with the 0.1 ppm quinine sulphate solution, placed in the spectrofluorometer and its spectra were recorded. The emission scan parameters were 200 to 800 nm with the optimal wavelength set at 350 nm and a step size of 2 nm. An excitation scan of the same solution was conducted in the spectrofluorometer and its spectrum recorded; the parameters were the same as before except the optimal wavelength, which was set at 450 nm. MS 2: The spectrofluorometer’s parameters were changed so as to perform time-based scans, the scan length was 40 seconds, the data acquisition rate was 1 point/second. The excitation and emission wavelengths were set at 350 and 450 nm respectively. The first time-based scan was performed on the 0.05 mol/L H2SO4 (aq) then the quinine sulphate (aq) in increasing concentration. A clear quartz cuvette was used for this sub-experiment; it was first rinsed with the solution to be analysed then filled with before being inserted in the spectrofluorometer. A 1 mL sample of tonic water (containing an unknown concentration of quinine sulphate (aq)) was diluted to the mark with the 0.05 mol/L H2SO4 (aq) in a 100 mL volumetric flask. A sample of this solution was placed in the spectrofluorometer and its spectrum, fluorescence intensity were recorded. MS 3: Fifteen 0.5 mL extractions from the 100 ppm solution of quinine sulphate were added to new 50 mL volumetric flasks. 0.5, 1, 2, 3 and 4 mL of NaCl (aq), NaBr (aq) and NaF (aq) of each were added to the new volumetric flasks and diluted to the mark with the 0.05 mol/L H2SO4 (aq). This resulted in 1 ppm quinine sulphate solutions with halide concentrations of 0.005, 0.01, 0.02, 0.03 and 0.04

! mol/L (corresponding to 0.5, 1, 2, 3 and 4 mL respectively); (3 halides ! 5 concentrations each = 15 solutions). NOTE: plastic volumetric flasks were used for


quinine sulphate (aq) containing NaF (aq). Fluorescence intensity spectrum of each of the solutions was recorded with the spectrofluorometer; the parameters were timebased scan with the scan length of 30 seconds and data collection at 1 point/second.



MS 1: --------------------------------------------------------------------------------!


0.05 mol/L H2SO4 (blank) absorption measurements: blank no. 2 3 4 5 6 avg. absorption -0.00002519 0.0004038 -0.000813 -0.0004194 -0.0004122

" = 0.000460955


limit of detection = 3#" = 0.001382864


MS 1 Q1: !"#$%&' Concentrations (mol/L)
1.28E-10 (0.0001 ppm) 1.28E-09 (0.001 ppm) 1.28E-08 (0.01 ppm) 1.28E-07 (0.1 ppm) 1.28E-06 (1 ppm) 1.28E-05 (10 ppm)








Wavelengths (nm)
























A = (!b)c ! Y = (m)X ! if the path length is 1 cm then ! = 51,871 L mol-1 cm-1.


A = (!b)c ! Y = (m)X ! if the path length is 1 cm then ! = 12,812 L mol-1 cm-1.



A = (!b)c ! Y = (m)X ! if the path length is 1 cm then ! = 7,248 L mol-1 cm-1.

A = (!b)c ! Y = (m)X ! if the path length is 1 cm then ! = 9,280 L mol-1 cm-1.


)! MS 1 Q5: 210 nm: 250 nm: 316 nm: 346 nm: " = 0.2664 " = 0.4289 " = 0.03741 " = 0.04767 ! ! ! ! limit of detection = 3" = 0.7992 limit of detection = 3" = 1.286 limit of detection = 3" = 0.1122 limit of detection = 3" = 0.1430 {calculations based on data in Table 1.}




MS 2: -----------------------------------------------------------------------------


MS 2 Q1: concentration (mol/L) 1.2773 ! 10-10 1.2773 ! 10-09 1.2773 ! 10-08 1.2773 ! 10-07 1.2773 ! 10-06 1.2773 ! 10-05 intensity (counts/s) 7869 9251 25835 198065 1.79E+06 3.77E+06

concentration (ppm) 0.0001 0.001 0.01 0.1 1 10


**! MS 2 Q5: [quinine sulphate] in tonic water: intensity of fluorescence of tonic water: 347,845
347,845 = 1"1012 x + 10,709 #quinine sulphate& 347845 )10709 x =% (= 1"1012 $ in tonic water ' #quinine sulphate& )7 )1 x =% ( = 3.371"10 mol L $ in tonic water '





MS 3: NaX as (Q)uenchers----------------------------------------------------


MS 3 Q1:


*#! MS 3 Q4:


Ii = 1+ K sv [Q] If Y = c + (m) X where : m = K sv ; X = [Q]



*$! MS 3 Q5:


If [Q] = 0 then Y = 190.2(0) + 0.952 Y= Ii = 191.5 If Ii 1.785 "10 6 = = 9321 counts /s 191.5 191.5

If =





MS 1: ----------------------------------------------------------------------------------------------All molecular absorption spectroscopy experiment trials on quinine sulphate were to be conducted using a quartz cuvette where as all spectroscopy trials (future) on fluoroscein with a plastic cuvette. This was so that the cuvette’s material’s absorption region did not overlap with that of the analyte, and so we could record and study the molecular absorption spectra of the desired analyte only. The absorption spectra were recorded in increasing order of concentration as mentioned earlier. Reason: lingering droplets of a higher concentration solution in the cuvette would have added significantly to the overall concentration, and thus changed it, of the analyte solution. Beer’s law on molecular absorption spectra seems to have been fairly obeyed in this sub-experiment. Errors in the preparation of the quinine sulphate solutions resulting in inaccurate concentrations could account for discrepancies, apart from erroneous spectrophotometer settings. Secondly, the spectrophotometer compartment where the analyte is placed and irradiated was not completely covered from outside light, though it was, and judging from the results, deemed sufficient. MS 1 Q3: Reason: the slit widths (and thus the corresponding bandwidth)

are sufficient such that, for this concentration, the resultant spectrum is of a higher resolution. Since, the two peaks are the most prominent ones, they (the corresponding wavelengths) ought to be selected in further experiments.

! MS 1 Q4: The absorption and the fluorescence spectra are similar in


the sense that there is a minimal response signal but for the particular wavelengths at which the substance is excited or at which it de-excites. (There are of course the inherent differences between the two types of molecular absorption of emission and absorption.)

! MS 3: --------------------------------------------------------------------------------------------


Unlike the NaCl (aq) and NaBr (aq) quenching agents, which were prepared in glass volumetric flasks, NaF (aq) solutions were prepared in plastic volumetric flasks. Once again, its so that the container material’s range of wavelengths of absorption/emission do not overlap with those of the analyte. Secondary reason: fluoride’s reactivity with glass.

MS 3 Q2 and Q3:

Fluorescence intensity generally decreases with increasing concentration of either NaCl (aq) or NaBr (aq) quenching agent; the opposite is true for NaF (aq). Reason: NaCl (aq) and NaBr (aq) obey/cause dynamic quenching whereas NaF (aq) obeys/causes static quenching.

! MS 3 Q6: As in the fluorescence experiment conducted with known concentrations of quinine sulphate and one experiment run with tonic water containing an unknown concentration of the quinine sulphate, an identical experiment could be setup with known concentrations of NaCl and a sample of sea-water with and unknown concentration of NaCl. The data could be processed in a similar manner shown previously.




MS 1: But for the rare, off measurement, Beer’s law was successfully applied in determining the molar absorptivities of varying concentrations of quinine sulphate solutions; calculated to be 51871; 12812, 7248 and 9280 L mol-1 cm-1 for wavelengths of 210, 250, 316 and 346 nm respectively. MS 2: Fluorescence spectroscopy was satisfactorily employed as an analytical tool; the concentration of quinine sulphate in a sample of tonic water was calculated to be 3.371!10-7 mol L-1. MS 3: The effect of sodium halide quenchers was studied, it was determined that NaCl, NaBr (aq) follow dynamic quenching and the opposite for NaF (aq).

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