ms 1 - 3 report

Title: MOLECULAR SPECTROSCOPY

section 1: Absorption and fluorescence spectra

section 2: Determination of quinine sulphate in tonic water using

fluorescence spectroscopy

section 3: Quenching interference in fluorescence spectroscopy

Full name: NAUMAN MITHANI

Student no.: 301016320

Sections: LA02: group C

Date of expt.: Jan. 24, 2008

ABSTRACT:

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.

Nauman Mithani

! "!

INTRODUCTION:

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 de-

excited state.

MS 1: The first section explored the application of Beer’s law of molecular

absorption spectroscopy, it states

A = ! b c

A ! absorption

! ! molar absorptivity (L mol-1

cm-1

)

b ! path length in medium (cm-1

)

c ! concentration of medium (mol L-1

)

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

I f=1+Ksv Q[ ]

! #!

Ii ! intensity/rate of fluorescence without quencher

If ! intensity/rate of fluorescence with quencher

Ksv ! Stern-Volmer coefficient

[Q] ! concentration of quencher

EXPERIMENTAL:

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 time-

based scan with the scan length of 30 seconds and data collection at 1 point/second.

! &!

DATA and RESULTS:

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

" 0.05 mol/L H2SO4 (blank) absorption measurements:

blank no. avg. absorption

2 -0.00002519

3 0.0004038

4 -0.000813

5 -0.0004194

6 -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)

210 -0.02449 -0.036575 -0.013137 -0.021957 0.0482330 0.6388854

250 0.011718 0.1137237 1.16567993 0.5001068 0.7757568 0.6425018

316 -0.00025 -0.001266 0.00856018 0.002136 0.0160217 0.0953063

Wave

len

gth

s (n

m)

346 -0.00064 -0.001983 0.00140380 -0.000854 0.0142517 0.1182861

! '!

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

cm-1

.

!


cm-1

.

! (!


cm-1

.


cm-1

.

! )!

" MS 1 Q5:

210 nm: " = 0.2664 ! limit of detection = 3" = 0.7992




{calculations based on data in Table 1.}

! *+!

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

" MS 2 Q1:

concentration (ppm) concentration (mol/L) intensity (counts/s)

0.0001 1.2773 ! 10-10

7869

0.001 1.2773 ! 10-09

9251

0.01 1.2773 ! 10-08

25835

0.1 1.2773 ! 10-07

198065

1 1.2773 ! 10-06

1.79E+06

10 1.2773 ! 10-05

3.77E+06

! **!

" MS 2 Q5: [quinine sulphate] in tonic water:

intensity of fluorescence of tonic water: 347,845

!

347,845 =1"1012 x +10,709

x =quinine sulphate

in tonic water

#

$ %

&

' ( =347845 )10709

1"1012

x =quinine sulphate

in tonic water

#

$ %

&

' ( = 3.371"10

)7molL

)1

! *"!

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

" MS 3 Q1:

! *#!

" MS 3 Q4:

!

Ii

I f=1+ Ksv Q[ ]

Y = c + m( )X

where : m = Ksv; X = Q[ ]

! *$!

" MS 3 Q5:

!

If Q[ ] = 0

then

Y =190.2 0( ) + 0.952

Y =Ii

I f=191.5

I f =Ii

191.5=1.785 "10

6

191.5= 9321counts /s

! *%!

DISCUSSION:

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.

! *)!

CONCLUSION:

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).

ms 1 - 3 report

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