1

FLUORESCENCE SPECTROSCOPY

1 ABSTRACT

This experiment uses the fluorescence capability of Rhodamine B and 8-

hydroxyquinoline both for its qualitative and quantitative analysis. Rhodamine B is
widely used in chemistry and biology due to its high fluorescence quantum yield. The
experimentally obtained fluorescence spectrum of Rhodamine B revealed that the
compound excites at around 450 – 452 nm (λ ex) and emits at 575 nm (λ em). Divalent
mercury ion (Hg2+) evidently marked its quenching capacity in this experiment to quench
the fluorescence of Rhodamine B as indicated in the subsequent decrease in fluorescence
intensity after the addition of known concentrations of Hg2+. This experiment also able to
quantitatively analysed the fluorescence of 8-hydroxyquinoline aside from Rhodamine B.
A non-fluorescent divalent magnesium metal ion (Mg2+) showed potential to induced
chelation-assisted fluorescence with 8-hydroxyquinoline as shown in the increase of
fluorescence intensity upon addition of Mg2+ to the system and forming a more stable,
fluorescent Mg2+-8-hydroxyquinoline complex.

2 INTRODUCTION

Fluorescence occurs when a molecule absorbs light photons from the UV -

Visible light spectrum, known as excitation, and then rapidly emits light photons as it
returns to its ground state. (Matthews, 1979) Fluorescence spectroscopy characterizes the
relationship between absorbed and emitted photons at specified wavelengths. It is a
precise quantitative analytical technique that is inexpensive and easily mastered. All
chemical compounds absorb energy which causes excitation of electrons bound in the
molecule such as increased vibrational energy or, under appropriate conditions,
transitions between discrete electronic energy states (Lingemann, 1985). For a transition
to occur, the absorbed energy must be equivalent to the difference between the initial
electronic state and a high-energy state. This value is constant and characteristic of the
molecular structure. This is termed the excitation wavelength. If conditions permit, an
excited molecule will return to ground state by emission of energy through heat and/or
emission of energy quanta such as photons. (Alarie, 1993) The emission energy or
wavelengths of these quanta are also equivalent to the difference between two discrete
 2

energy states and are characteristic of the molecular structure. Fluorescence occurs when
a molecule absorbs photons from the UV - Visible light spectrum (200-900 nm), causing
transition to a high-energy electronic state and then emits photons as it returns to its
initial state, in less than 10-9 sec. (Fink, 1982)

The emission spectrum provides information for both qualitative and quantitative
analysis. As shown in Figure 1, when light of an appropriate wavelength is absorbed by a
molecule (i.e., excitation), the electronic state of the molecule changes from the ground
state to one of many vibrational levels in one of the excited electronic states. The excited
electronic state is usually the first excited singlet state, S1 (Figure 1). Once the molecule
is in excited state, relaxation can occur via several processes. Fluorescence is one of these
processes and results in the emission of light. (Skoog, 2004)

Figure 1. Electronic transition energy level diagram. (Skoog, 2004)

Fluorescence corresponds to the relaxation of the molecule from the singlet

excited state to the singlet ground state with emission of light. Fluorescence has short
lifetime (~10-8 sec) so that in many molecules it can compete favourably with collisional
 3

deactivation, intersystem crossing and phosphorescence. (Krahn, 1993) The wavelength

(and thus the energy) of the light emitted is dependent on the energy gap between the
ground state and the singlet excited state. An overall energy balance for the fluorescence
process could be written as:

Efluor = Eabs – Evib - Esolv.relax (Eq. 1)

where Efluor is the energy of the emitted light, E abs is the energy of the light absorbed by
the molecule during excitation, and E vib is the energy lost by the molecule from
vibrational relaxation. The Esolv.relax term arises from the need for the solvent cage of the
molecule to reorient itself in the excited state and then again when the molecule relaxes
to the ground state. As can be seen from Equation (1), fluorescence energy is always less
than the absorption energy for a given molecule. Thus the emitted light is observed at
longer wavelengths than the excitation (Lakowicz, 1983).

Fluorescence quantitative analysis can be made for both excitation and emission
spectrum. In this experiment, fluorescence quantitative analysis was performed using
Rhodamine B. Rhodamine B belongs to a family of fluorone dyes that all share the same
basic fluorone skeleton (Figure 2) that is commonly used as an active medium in tunable
lasers due to its high fluorescence quantum yield. It also finds a wide range of
applications in materials science, chemistry and biology as a sensitizer in solar cells, as a
molecular probe, as an electrochemical luminescence sensitizer, as a water-tracing agent,
as a biological stain, etc. (Setiawan, 2010)

Figure 2. The chemical structure of Rhodamine B.

(Source: Fikry, M., Omar, M. M. & Lotfi, Z. I., (2009). Effect of host medium on the fluorescence
emission intensity of Rhodamine B in liquid and solid phase. J. Fluores., 19, 741–746Mahasin F. Al-
Kadhem, Journal of Physical Science, Vol. 22(2), 77–86, 2011)
 4

One important phenomenon which can simultaneously cause additive and

multiplicative interference during fluorescence analysis is called Quenching. The
fluorescence signal generated by an analyte may be altered - perhaps even totally
suppressed - by other sample constituents (Hofstraat, et.al, 1993). Quenching is any
process in which a sample constituent decreases the fluorescence quantum yield for the
analyte. In this experiment, fluorometric method will be employed to examine the
capability of Hg2+ as a quencher to the fluorescence of Rhodamine B.

Aside from quenching, another process called Chelation affects fluorescence

analysis. Chelators are compounds that bind to metal ions forming a complex and some
of them are also fluorescent. If the complex has fluorescence properties different from
those of the free chelator, it can be used as an ion indicator. In this experiment, chelation-
induced fluorescence will be studied. Magnesium ion is not fluorescent. However,
alcohol solution of Mg2+ - 8-hydroxyquinoline exhibits a fluorescence at λ ex at 420 nm
and λem 530 nm. Thus, Mg2+ will be analysed through fluorescence technique after
chelating Mg2+ with 8-hydroxyquinoline.

3 MATERIAL AND METHODS

3.1 Quantitative Fluorescence Analysis

3.1.1 Laboratory Apparatus and Instrument

 Pipet, 25 mL and 10 mL
 Beaker, 250 mL
 Volumetric flasks, 50 mL and 100 mL
 Stirring rod
 Wash Bottle

All sample absorbance were analysed using JASCO FD-777 Spectrofluorometer.

The system was designed to hold one (1) sample vessel. All fluorescence intensities were
taken at wavelength of 452 nm and 575 nm for excitation and emission spectra
respectively.
 5

3.1.2 Reagents and Solutions

All chemicals used were of analytical-reagent grade or the highest purity

available. Distilled water was used throughout this experiment. Glass vessels were
cleaned by soaking in a luke-warm detergent solution, followed by washing and rinsing
several times with distilled water. Stock solutions were kept in a clean 250mL amber
bottle. In addition, volumetric flasks and pipettes were used throughout the preparations
of standard solutions.

3.1.2.1 Primary Stock Solution, 100 ppm

A 100-mL solution containing 100 ppm Rhodamine B was prepared by dissolving

10.0 milligrams (0.0100 grams) of solid Rhodamine B in distilled water in a 100 mL
volumetric flask and diluting to the mark. The resulting solution was shaken and stored
into a properly labelled, clean amber bottle.

3.1.2.2 Secondary Stock Solution, 10 ppm

From the above primary stock solution, a 50-mL of 10 ppm Rhodamine B

solution (secondary stock) was prepared by diluting to the mark 5 mL of primary stock
solution in a 50 mL volumetric flask. This was then diluted to mark with distilled water.
The resulting solution was shaken and stored into a properly labelled, clean amber bottle.

3.1.2.3 Standard Solutions (0.2, 0.4, 0.6, 0.8 ppm)

From the above secondary stock solution, a 100 mL of 0.8 ppm Rhodamine B
solution was prepared by appropriate dilution in a 100 mL volumetric flask. Then from
the 0.8 ppm, the remaining standards (0.6, 0.4, 0.2 ppm) were prepared by serial
dilutions, all in 100 mL volumetric flasks. Each of these standard solutions was carefully
transferred into a properly labelled, clean amber bottle.

3.1.3 Experiment Procedure

All fluorescence intensity readings were determined using the instrument JASCO
FD-777 Spectrofluorometer. Initially, the instrument was pre-heat for about fifteen (15)
minutes prior to use. A peak search on Rhodamine B was performed using 0.6 ppm
 6

standard Rhodamine B solution. The excitation spectrum of the 0.6 ppm standard
Rhodamine B solution was taken. From the excitation spectrum, the λ ex was determined.
The emission spectrum was also taken and λem was determined from the obtained
emission spectrum. Then, the fluorescence intensity of each of the standard solutions of
Rhodamine B (0.2, 0.4, 0.6, 0.8 ppm) was measured. Three cycles for each trial was
performed and three trials were done for intensity measurements of each standard
solution. The fluorescence of the blank (distilled water) was also determined. The
obtained blank’s intensity corrected the measured intensity of fluorescence of the
standard solutions. A calibration curve (concentration vs. fluorescence intensity) was then
constructed. The intensity of the unknown Rhodamine B solution was also taken and the
concentration of the unknown was calculated based on the constructed calibration curve.

3.2 Quenching Method of Analysis

3.2.1 Laboratory Apparatus and Instrument

 Pipet, 25 mL and 10 mL
 Buret, 25 mL
 Beaker, 250 mL
 Volumetric flasks, 100 mL
 Test tubes, 10 mL
 Stirring rod
 Vortex
 Wash Bottle

All sample absorbance were analysed using JASCO FD-777 Spectrofluorometer.

The system was designed to hold one (1) sample vessel. All fluorescence intensities were
taken at wavelength of 450 nm and 575 nm for excitation and emission spectra
respectively.

3.2.2 Reagents and Solutions

All chemicals used were of analytical-reagent grade or the highest purity

available. Distilled water was used throughout this experiment. Glass vessels were
cleaned by soaking in a luke-warm detergent solution, followed by washing and rinsing
 7

several times with distilled water. Stock solutions were kept in a clean 250mL amber
bottle.

3.2.2.1 Rhodamine B Solution, 0.8 ppm

From previously prepared secondary stock solution (section 3.1.2.2), the 0.8 ppm
Rhodamine B solution was prepared by quantitatively transferring 8 mL of secondary
stock (10 ppm) into 100-mL volumetric flask and diluting it to mark with distilled water.
The resulting solution was shaken and stored into a properly labelled, clean amber bottle.

3.2.2.2 Hg(II) Solution, 5 ppm

Initially, a stock solution of Hg2+ having a concentration of 100 ppm was

prepared. This was done by accurately weighing 13.53 milligrams of solid HgCl 2 and
quantitatively transferred into 100-mL volumetric flask. Dilution was done with distilled
water up to 100-mL mark. The resulting solution was shaken and stored into a properly
labelled, clean amber bottle.

A volume of 5 mL of 100 ppm Hg2+ stock solution was quantitatively transferred

through a pipette into a 100-mL volumetric flask. This was then diluted with distilled
water up to 100-mL mark. The resulting solution was shaken and stored into a properly
labelled, clean amber bottle.

3.2.2.3 Unknown Solution Containing Hg(II)

The unknown solution containing unknown concentration of Hg 2+ was freshly

prepared by laboratory technician. This was stored in a plastic canister.

3.2.3 Experiment Procedure

Initially, all reagents as stated above were prepared. A set of 16 solutions (Table
1) with different ratios of Rhodamine B and Hg(II) was then prepared.
 8

Table 1. Reagents’ Volume for the Calibration Curve Preparation

Solution Rhodamine B solution, mL Hg(II) solution, mL mL of H2O

1 1.0 0.0 4.0
2 1.0 0.0 4.0
3 1.0 0.0 4.0
4 1.0 0.5 3.5
5 1.0 0.5 3.5
6 1.0 0.5 3.5
7 1.0 1.0 3.0
8 1.0 1.0 3.0
9 1.0 1.0 3.0
10 1.0 2.0 2.0
11 1.0 2.0 2.0
12 1.0 2.0 2.0
13 1.0 3.0 1.0
14 1.0 3.0 1.0
15 1.0 3.0 1.0
16 1.0 4.0 0.0

The above mentioned 16 solutions were prepared through a burette and were
contained in labelled test tubes. Fluorescence intensity was determined for each solution.
A calibration curve of ppm Hg2+ (x-axis) against fluorescence intensity (y-axis). The
fluorescence intensity of the unknown solution containing Hg2+ was also determined. The
unknown solution was treated as solution no.16 where its composition ratio is 1:4
(volume of Rhodamine B:volume of unknown solution). From the plot, the
concentration of the unknown Hg2+ solution was determined.

3.3 Fluorescence Analysis by Chelation

3.3.1 Laboratory Apparatus and Instrument

 Micropipet, 1000 µL
 Pipet, 25 mL and 10 mL
 Beaker, 250 mL
 Buret, 25 mL
 9

 Volumetric flasks, 250 mL and 100 mL

 Test tubes, 10 mL
 Vortex
 Stirring rod
 Wash Bottle

All sample absorbance were analysed using JASCO FD-777 Spectrofluorometer.

The system was designed to hold one (1) sample vessel. All fluorescence intensities were
taken at wavelength of 420 nm and 530 nm for excitation and emission spectra
respectively.

3.3.2 Reagents and Solutions

All chemicals used were of analytical-reagent grade or the highest purity

available. Distilled water was used throughout this experiment. Glass vessels were
cleaned by soaking in a luke-warm detergent solution, followed by washing and rinsing
several times with distilled water. Stock solutions were kept in a clean 250mL amber
bottle.

3.3.2.1 Alcohol solution of 8-hydroxyquinoline

Alcohol solution of 8-hydroxyquinoline having a concentration of 100 ppm was

prepared. This was done by accurately weighing 25.0 milligrams of solid 8-
hydroxyquinoline and quantitatively transferred into 250-mL volumetric flask. Dilution
was done with absolute alcohol up to 250-mL mark. The resulting solution was shaken
and stored into a properly labelled, clean amber bottle.

3.3.2.2 MgCl2•6H2O Solution

A 100-mL MgCl2•6H2O solution which the concentration is 210 ppm was

prepared by weighing 21 milligrams of solid MgCl 2•6H2O and transferring it into 100 mL
volumetric flask. Dilution was done with distilled water up to 100-mL mark. Such
solution was prepared instead of 200 ppm (as directed on the lab manual) since the exact
solid MgCl2•6H2O weighed was 21 milligrams instead of the calculated 20 milligrams.
Necessary adjustments for the volume needed for the preparation of the standard solution
were also done.
 10

3.3.3 Experiment Procedure

A 6.0-mL portion of the 8-hydroxyquinoline solution was transferred into 3 sets

of three test tubes. The transfer was made through a burette. Using a micropipette, an
amount of the MgCl2 was measured and added by increments to each set of test tubes
such that the Mg2+ concentrations were 0.5, 1.0, 1.5, 2.0, 3.0 ppm. The fluorescence
intensities of the previously prepared solutions, as well as the unknown Mg 2+ solution,
were measured at λex 420nm and results were tabulated. A calibration curve of
fluorescence intensity vs. concentration of Mg2+ was also prepared. From the calibration
curve, the concentration of the unknown Mg2+ solution was determined.

4 DATA AND CALCULATIONS

4.1 Data

4.1.1 Quantitative Fluorescence Analysis

Initially, two basic types of spectra are produced by a fluorescence spectrometer

namely excitation and emission spectrum. In an excitation spectrum, the fluorescence
signal was measured as the wavelength of the exciting radiation is varied. This excitation
spectrum identified the wavelengths of light that the analyte is able to absorb because an
analyte fluoresced only after it has absorbed radiation. In an emission spectrum, on the
other hand, the wavelength of the exciting radiation is held constant (λ ex) and the spectral
distribution of the emitted radiation were measured. Peak search results were tabulated as
shown in the table below.

Table 2. Obtained Wavelength for the Peak Search on Excitation and Emission
Spectrum of Rhodamine B.
Spectrum Wavelength with Maximum Peak Height, nm
Excitation 452
Emission 575
 11

Using the wavelengths obtained during peak search, the fluorescence intensities
were determined for the working calibration curve as well as for the blank and unknown
solution and were shown in the Table 3.

Table 3. Fluorescence Intensity Obtained for the Working Calibration Curve

Solution, Blank and Unknown Solution.
Fluorescence Intensity
Solutions
Cycle 1 Cycle 2 Cycle 3 Average
Blank 0.031 0.094 0.062 0.062
0.2 ppm Rhodamine B 11.2 11.35 11.48 11.34
0.4 ppm Rhodamine B 23.12 23.18 23.12 23.14
0.6 ppm Rhodamine B 36.94 36.15 36.19 36.43
0.8 ppm Rhodamine B 46.96 46.42 46.39 46.59
Unknown 55.32 53.47 53.28 54.02

4.1.2 Quenching Method of Analysis

Again, emission and excitation spectra were initially scanned. Peak search results
were tabulated in Table 4.

Table 4. Obtained Wavelength for the Peak Search on Excitation and Emission
Spectrum of Rhodamine B.
Spectrum Wavelength with Maximum Peak Height, nm
Excitation 450
Emission 575

Using the obtained wavelengths, the fluorescence intensities were measureed for
the working calibration curve as well as for the blank and unknown solution and were
indicated in the Table 5.
 12

Table 5. Fluorescence Intensity Obtained for the Working Calibration Curve

Solution, Blank and Unknown Solution
Fluorescence Intensity Averaged
Solution Fluorescence
Cycle 1 Cycle 2 Cycle 3
Intensity
Blank Solution -0.094 -0.312 -0.157 -0.188
1 10.51 10.51 10.73 10.58
2 10.92 11.04 11.26 11.07
3 11.04 11.13 11.10 11.09
4 10.26 10.57 10.32 10.38
5 9.858 9.888 9.918 9.888
6 10.73 10.76 10.91 10.80
7 10.54 10.79 10.69 10.67
8 9.672 9.827 9.547 9.682
9 9.827 9.890 9.827 9.848
10 10.67 10.85 10.88 10.80
11 9.577 9.545 9.670 9.597
12 9.921 9.890 10.35 10.05
13 9.263 9.295 9.357 9.305
14 9.390 9.640 9.640 9.557
15 9.641 9.700 9.670 9.670
16 9.045 8.952 8.890 8.962
Unknown Solution 10.39 10.51 10.60 10.50

4.1.3 Fluorescence Analysis by Chelation

At λex and λem indicated in the procedure which is 420 nm and 530 nm
respectively, fluorescence intensities were obtained and indicated in Table 6.
 13

Table 6. Measured fluorescence intensity at λex, 420 nm and λem, 530 nm.

Fluorescence Intensity
Solutions Trial Average
cycle 1 cycle 2 cycle 3
Blank 1 0.592 0.592 0.467 0.550
1 6.802 6.770 6.739 6.770
0.5 ppm Mg2+ 2 5.365 5.303 5.21 5.293
3 4.336 4.274 4.275 4.295
1 5.460 5.397 5.367 5.408
1.0 ppm Mg2+ 2 5.241 4.774 4.742 4.919
3 5.147 4.742 4.772 4.887
1 5.147 5.147 5.147 5.147
1.5 ppm Mg2+ 2 4.866 4.866 4.928 4.887
3 4.772 4.773 4.679 4.741
1 5.022 5.085 5.147 5.085
2.0 ppm Mg2+ 2 4.866 5.179 5.116 5.054
3 4.835 4.928 4.836 4.866
1 4.992 5.024 4.992 5.003
3.0 ppm Mg2+ 2 4.835 4.929 4.897 4.887
3 4.991 4.898 4.929 4.939
Unknown 1 4.024 4.055 4.117 4.065
2 4.151 4.306 4.181 4.213
3 3.900 3.774 3.868 3.847

4.2 Calculation

4.2.1 Quantitative Fluorescence Analysis

4.2.1.1 Standard Solutions Preparation

For the preparation of standard solutions, serial dilution was used and to
determine the exact volume needed simple dilution formula was employed:

M1V1 = M2V2

where M1 = concentration of secondary stock solution which is 10 ppm, V 1 = volume

needed, V2 = final volume which is 100 mL and M 2 = final concentration of Rhodamine
 14

B in the solution. As such to prepare 0.8 ppm Rhodamine B solution from secondary
stock (10 ppm), the calculation of needed volume was shown below:

M1V1 = M2V2

(10 ppm) (V1) = (0.8 ppm) (100 mL)

V1 = 8 mL

Same calculation has been done for the rest of the standard solutions. The calculated
volumes required for the preparation of the standard solutions were indicated in Table 7.

4.2.1.2 Working Calibration Curve and Determination of Unknown’s Concentration

All fluorescence intensity observed was at first corrected by the intensity of blank
solution.

Corrected Fluorescence Intensity = Measured Intensity – Blank’s Intensity (Eq.2)

For 0.2 ppm Rhodamine B, its corrected fluorescence intensity is

Corrected Fluorescence Intensity = 11.34 - 0.062 = 11.28

The same calculation has been made for the rest of the standard solutions and corrected
fluorescence intensities were tabulated below (Table 8). Then, a calibration curve (Figure
4) was constructed by plotting the concentration of Rhodamine B against the corrected
fluorescence intensity.

From the equation of the working calibration curve, the concentration of unknown
Rhodamine B solution was determined. The obtained fluorescence intensity for the
unknown Rhodamine B solution was 53.96. Substituting this value (y-variable) to the
equation will finally yield the concentration (x-variable) of the unknown as shown below:

y = 59.513x - 0.444

53.96 = 59.513x – 0.444

x = 0.9142

Thus, unknown Rhodamine B solution has a concentration of 0.9142 ppm.

 15

4.2.2 Quenching Method of Analysis

4.2.2.1 Preparation Hg2+ Solutions

At first, a primary stock solution of Hg2+ which concentration is 100 ppm. The
calculation below showed the exact amount of HgCl 2 quantitatively weighed for the
preparation of primary stock solution.

Hg 2+¿ 1
weight of HgCl2=100 mg x Total volume of solution x ¿
mL 2+¿ 1 mmol HgCl 2
MW , Hg x ¿
1 mmol Hg2+¿ x MW , HgCl 2 ¿
2+¿
Hg mmol
weight of HgCl2=100 mg x 0.100 L x ¿
L 2+ ¿ 1mmol HgCl2
200. 59 mg Hg x ¿
2+¿ 271.496 mg
1 mmol Hg x ¿
mmol HgCl 2

weight of HgCl2=13.53 mg

Then, the calculation below illustrated how 5 ppm of Hg 2+ was prepared from 100 ppm
primary stock solution via simple dilution method:

M1V1 = M2V2

where M1 = concentration of primary stock which is 100 ppm, V 1 = volume needed, V2 =

final volume which is 100 mL and M2 = sought concentration which is 5 ppm.

M1V1 = M2V2

(100 ppm) (V1) = (5 ppm) (100 mL)

V1 = 5 mL

4.2.2.2 Working Calibration Curve and Determination of Unknown’s Concentration

Initially, the concentration of Hg2+ on each test tube was determined using the
simple dilution formula:

M1V1 = M2V2
 16

where M1 = Initial Hg2+ concentration which is 5 ppm, V 1 = volume of transferred Hg2+

into the solution, V2 = final volume which is 5 mL and M 2 = final concentration of Hg2+
in the solution. For instance, solution no. 4 has a concentration of:

M1V1 = M2V2

(5 ppm) (0.5 mL) = M2 (5 mL)

M2 = 0.5 ppm

Same calculation has been done for the rest of the solutions and calculated Hg 2+
concentrations were tabulated below. Next, all fluorescence intensity were corrected by
the intensity of the blank solution which is distilled water. All corrected intensity was
tabulated below (Table 10).

Then, a calibration curve was constructed by plotting concentration of Hg 2+ (x-

axis) against the fluorescence intensity (y-axis). From the equation of the working
calibration curve, the concentration of unknown Hg 2+ solution was determined. The
obtained fluorescence intensity for the unknown Hg2+ solution was 10.50. Substituting
this value (y-variable) to the equation will finally yield the concentration (x-variable) of
the unknown as shown below:

y = -0.4217x + 10.732

10.50 = -0.4217x + 10.732

x = 0.5502 ppm

This obtained value will be further corrected by the dilution factor in such a way that the
final concentration of Hg2+ in the unknown solution will be:

¿¿

¿¿

Thus, unknown Hg2+ solution has a concentration of 0.6877 ppm.

4.2.3 Fluorescence Analysis by Chelation

4.2.3.1 Preparation of Stock Solutions

 17

Two reagents were prepared namely alcohol solution of 8-hydroxyquinoline and

MgCl2•6H2O. The necessary calculations for the preparations of the said solutions were
shown below.

mg 8−hydroxyquinoline
weight of 8−hydroxyquinoline=100 x Total volume of solution
L

mg 8−hydroxyquinoline
weight of 8−hydroxyquinoline=100 x 0.250 L
L

weight of 8−hydroxyquinoline=25.0 mg8−hydroxyquinoline

mg MgCl 2 • 6 H 2 O
weight of MgCl 2 •6 H 2 O=210 x Total volume of solution
L

mg MgCl 2 • 6 H 2 O
weight of MgCl 2 •6 H 2 O=210 x 0.100 L=21.0 g MgCl 2 •6 H 2 O
L

4.2.3.2 Working Calibration Curve and Determination of Unknown’s Concentration

First, the concentration of Mg2+ was calculated in the stock solution of

MgCl2•6H2O.

¿¿¿

¿¿¿

¿¿¿

Next, the volume of MgCl2•6H2O needed for the preparations of the solutions with
known concentrations of Mg2+ was then calculated using simple dilution formula as
shown below:

M1V1 = M2V2

where M1 = initial Mg2+ concentration which is 25.10 ppm, V 1 = needed volume, M2 =

sought concentration, V2 = final volume. As such for the 0.5 ppm Mg2+ solution, the
required volume is:

M1V1 = M2V2

(25.10 ppm) (V1) = (0.5 ppm) (6 mL + V1)

(25.10 – 0.5) (V1) = (0.5) (6)

 18

V1 = 122 µL

Thus, 122 µL of 210 ppm MgCl2•6H2O was pipetted into 6 mL of 8-hydroxyquinoline to

prepare a solution of 0.5 ppm Mg2+. Same calculation has been done for the rest of the
solutions each containing 1.0, 1.5, 2.0 and 3.0 ppm of Mg 2+. All calculated volume was
indicated in Table 13.

Next, all measured fluorescence intensities were corrected by the intensity of the blank
which is the distilled water. That is for the solution containing 0.5 ppm of Mg 2+ (trial 1),
its corrected fluorescence intensity is

Corrected Fluorescence Intensity = Measured Intensity – Blank’s Intensity (Eq.2)

Corrected Fluorescence Intensity = 6.770 - 0.5503 = 6.220

The same calculation has been made for the rest of the calibration solutions and for the
unknown solution as well. All corrected fluorescence intensities were tabulated below
(Table 13). Then, a calibration curve was constructed by plotting concentration of Mg2+
(x-axis) against the fluorescence intensity (y-axis) (Figure 6)

From the equation of the working calibration curve, the concentration of unknown
Mg2+ solution was determined. The obtained fluorescence intensity for the unknown Hg2+
solution was 3.491. Substituting this value (y-variable) to the equation will finally yield
the concentration (x-variable) of the unknown as shown below:

y = -0.3477x + 5.2508

3.491= -0.3477x + 5.2508

x = 5.060 ppm

This obtained value will be further corrected by the dilution factor in such a way that the
final concentration of Mg2+ in the unknown solution will be:

¿¿

¿¿

Thus, concentration of Mg2+ in the unknown solution is 80.96 ppm.

5 RESULTS: FIGURES, TABLES

 19

5.1 Quantitative Fluorescence Analysis

The figure below revealed the excitation and emission spectra of Rhodamine B.

Figure 3. Excitation and Emission Spectra of Rhodamine B.

Then, calculated volumes for the preparation of standard solutions via serial
dilutions were listed in the table below.

Table 7. Calculated volumes for the preparation of standard solutions via serial dilutions.
Standard Solutions, ppm Calculated Volume
0.8 8 mL of secondary stock solution (10 ppm)
0.6 75 mL of 0.8 ppm solution
0.4 66.7 mL of 0.6 ppm solution
0.2 50 mL of 0.4 ppm solution
 20

In addition, the observed and corrected fluorescence intensities of standard

solutions were tabulated in the following tables as shown below.

Table 8. Observed and Corrected Fluorescence Intensity of Standard Solutions.

Observed Fluorescence Corrected Fluorescence
Solutions
Intensity Intensity
0.2 ppm Rhodamine B 11.34 11.28
0.4 ppm Rhodamine B 23.14 23.08
0.6 ppm Rhodamine B 36.423 36.36
0.8 ppm Rhodamine B 46.59 46.53
Unknown 54.02 53.96

Plotting the concentration of Rhodamine B against corrected intensity reading

yields plots as shown below.

50
45 f(x) = 59.5133333333334 x − 0.444000000000003
40 R² = 0.997562187816178

35
30
Corrected Flu- 25
orescence In-
tensity 20
15
10
5
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Concentration of Rhodamine B, ppm

Figure 4. Calibration Curve for the Quantitative Fluorescence Analysis of

Rhodamine B.

Employing the above working calibration curve finally determined the

concentration of Rhodamine B in the unknown solution. Results were tabulated in the
table below.

Table 9. Calculated concentration of Rhodamine B + in the unknown solution.

Solution/Sample Concentration, ppm
 21

Rhodamine B in the Unknown Solutiom 0.9142 ± 0.0277

5.2 Quenching Method of Analysis

Based on the calculations above, the corrected fluorescence intensities as well as

the calculated Hg2+ concentration on each of the solutions needed for the construction of
working calibration curve were indicated in the table below.

Table 10. Calculated Hg2+ Concentration and its Corrected Fluorescence Intensity

Corrected Averaged
Concentration
Solution Fluorescence Fluorescence
of Hg2+, ppm
Intensity Intensity
1 10.58
2 0.0 11.07 10.92
3 11.09
4 10.38
10.36
5 0.5 9.888
6 10.80
7 10.67
10.07
8 1.0 9.682
9 9.848
11 9.557 9.826
2.0
12 10.05
13 9.305
14 3.0 9.557 9.511
15 9.670
16 4.0 8.962 8.962

Plotting the concentration of Hg2+ against corrected fluorescence intensity yields

plots as shown below.
 22

12

10 f(x) = − 0.421749707602347 x + 10.7320249512671

R² = 0.91480735370792

Fluorescence 6
Intensity
4

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Hg2+ concentration, ppm

Figure 5. Calibration Curve for the Fluorescence Analysis of Rhodamine B as

Quenched by Hg2+.

Using the above working calibration curve finally determined the concentration of
Hg2+ in the unknown solution. Result was tabulated in the table below.

Table 11. Calculated concentration of Hg2+ in the unknown solution.

Solution/Sample Concentration, ppm
Hg in the unknown solution
2+
0.6877

5.3 Fluorescence Analysis by Chelation

The calculated weight of 8-hydroxyquinoline and MgCl2•6H2O required to

prepare the stock reagents were tabulated below:

Table 12. Calculated Weight for the Prepartion of 8-hydroxyquinoline and MgCl 2•6H2O
Solutions.
Reagent Calculated weight required to prepare the solution, grams
8-hydroxyquinoline 25.0 x 10-3
MgCl2•6H2O 21.0
 23

Table 13. Calculated Volume of MgCl2•6H2O for Preparation of Calibration Curve

Solutions and its Corrected Fluorescence Intensity.
Calculated
Volume of 8- Corrected
Volume of Averaged
Solutions Trial hydroxuquinoline Fluorescence
MgCl2•6H2O, Fluorescence
solution, mL Intensity
µL Intensity
0.5 ppm 1 6.220
6.0 122.0 5.481
Mg2+ 2 4.742
1.0 ppm 1 4.858 4.613
6.0 249.0
Mg 2+
2 4.369
1.5 ppm 1 4.597
6.0 381.3 4.466
Mg2+ 2 4.336
2.0 ppm 1 4.534
6.0 519.5 4.519
Mg 2+
2 4.503
1 4.452
3.0 ppm
2 6.0 814.5 4.337 4.393
Mg2+
3 4.389
1 3.515
Unknow
2 N/A N/A 3.662 3.491
n
3 3.297

Plotting the concentration of Mg2+ against corrected fluorescence intensity

yields plots as shown below.

5
f(x) = − 0.347720720720722 x + 5.25081981981982
R² = 0.559623098517589
4

Fluorescence 3
intensity
2

0
0 0.5 1 1.5 2 2.5 3 3.5
concentration of Mg2+, ppm

Figure 6. Calibration Curve for the Mg2+ Chelation-Induced Fluorescence

Analysis of 8-hydroxyquinoline.
6 DISCUSSIONS
 24

6.1 Quantitative Fluorescence Analysis

As shown above in Figure 3, two spectra were obtained namely Excitation and
Emission Spectra. These spectra made the fluorescent compounds or fluorophors such as
Rhodamine B to be identified and quantified on the basis of their excitation and emission
properties. It is very prominent in Figure 3 that Rhodamine B has detectable emission
intensity for a broad excitation range (440- 460 nm). Maximum emission occurs at a
unique excitation wavelength of 452 nm. Emitted light is detected for a broad
wavelength range (500–600 nm), however, when excited at 452 nm, maximum emission
occurs at 550 nm. These obtained excitation and emission properties of Rhodamine B are
fixed and can be used for identification and quantification. It is also necessary to conduct
peak search for both spectra since at the absorption maximum the maximum number of
light quanta is absorbed and therefore the maximum number of molecules is excited per
unit of time. It must be noted that the number of molecules fluorescing is proportional to
the number of excited molecules, maximumfluorescence intensity is achieved when the
exciting wavelength is at the absorption maximum.

6.1.1 Statistical Method Analysis

To validate the effectiveness of the prepared calibration, obtained data were

subjected to linear least square test. Statistical analysis was done through Windows Excel
2010. The table below showed the results of linear least square test.

Table 14. Linear least square test’s results.

Regression Equation
slope -0.42175
10.73202
intercept
5
0.550148
concentration x
5
Error Analysis
0.221757
standard error in y
3
N 6
Sxx 11.875
y bar 9.993963
Multiplicity, M 1
standard deviation in -0.596711
 25

concentration
6.2 Quenching Method of Analysis

When a molecule arrives at the lowest vibrational level of the excited electronic
state, the possibility is so high that the energy quantum for the return to the ground state
will be accepted by a molecule present in the solution. In this case, some of the energy
quantum for the return to the ground state was transferred to Hg 2+ as manifested by the
decreasing emission intensity after subsequent addition of Hg2+ into the system. The
divalent only confirmed its good capability as a quencher of fluorescence of Rhodamine
B. There are three major mechanisms for bimolecular quenching being collisional
deactivation, energy transfer and electron transfer.

In addition, quenching occurs via internal conversion and intersystem crossing in

the excited states of aggregates (e.g., dimers) of Rhodamine B (RhB) in solution. Indeed,
xanthene dyes can form aggregates in polar solution due to the strong electrostatic and
dispersion interactions between the dye molecules. The fluorescence quantum yield of
aqueous RhB can be strongly affected by dimerization, because dimers of RhB in water
can only make a weak contribution to fluorescence, whereas they are capable of strong
optical absorption. It was suggested that the intersystem crossing between the singlet and
the triplet excited states can take place in RhB dimeric species thus leading to a
radiationless decay channel that cannot be realised in the monomeric species.

On the other hand, all emission intensity is taken at wavelengths of maximum

peak for both excitation and emission spectra. This can be again rationally explained that
number of molecules fluorescing is proportional to the number of excited molecules,
maximumfluorescence intensity is achieved when the exciting wavelength is at the
absorption maximum. It is also quite noted that the blank solution incurred negative
emission intensity. There is no meaning to negative fluorescence since there is no such
thing. However, what have been displayed on the graphs is no longer fluorescence, it is a
corrected measurement derived from fluorescence. The corrected measurement has been
manipulated by both the instrument firmware, and possibly software (in the case of
compensation), before it is displayed. Thus, emission intensity of blank solution was
treated as zero intensity reading and no correction has been made for the fluorescence
intensity of working calibration solutions and for the unknown solution as well.
 26

6.3 Fluorescence Analysis by Chelation

As indicated in the Table 13, only two out of three trials obtained in emission
intensity were used for the construction of calibration curve. The instrument has been
consistently provided an outlier of the three trials conducted. Sudden drift of emission
intensity (usually in the third trial) has been attributed to the fluctuation current sources.
Thus, spectrofluorometer provided a variation of emission intensity.

6.4 Sources of Error

The biggest source of error in the measurement is probably read out of the
fluorescence spectrometer since the errors in weighting a sample of a substance are
usually quite small and since there is completely confidence those, who prepared the
standard solutions the errors coming from these solutions probably can be ignored. The
micropipettes used to conduct the experiment are so precise, that there as well the errors
not coming from human failure can be ignored. So it is virtually impossible to read the
values any exacter than 1 % intensity. The instrument Spectrofluorometer also initially
provides erratic reading which might be attributed to fluctuation electric current sources.

In addition, the reference cuvette should used to minimise the effect of an

instrumental drift of the fluorescence spectrometer during the measurement. In this
experiment, the reference was measured between every two measurements and the
intensity was readjusted to 100 % so there shouldn't be any error due to instrumental
drift. (Harris, 1982) However there can be seen a certain tendency to lower fluorescence
values in the values of the three measurements that were conducted. Mostly the first
measurement corresponds to the biggest intensity and the last measurement to the
smallest. So obviously there still was some drift. Moreover, the Rhodamine B and 8-
hydroxyquinoline get destroyed by UV-radiation, so the reference was probably not
constant throughout the whole experiment.

Fluorescence is a very sensitive technique. However, it is extremely susceptible to

interference by contamination of trace levels of organic chemicals. Potential sources of
contamination are ubiquitous since any aromatic organic compound can be a possible
source of fluorescence signal (Parker, 1983). For instance, the researcher is a possible
source of this type of contamination since oils secreted by the skin are fluorescent. Good
 27

laboratory procedure is essential in preventing solvents and chemicals from becoming

contaminated with high background fluorescence that could hinder low-level
measurements. Furthermore, care must be taken to eliminate all forms of solid
interference (suspended particulates such as dust and fibers). These will float in and out
of the sampling area of the cuvette via convection currents, and cause false signals due to
light scattering while they remain in the instrument's beam.

7 CONCLUSION

Fluorescence spectroscopy can yield low detection limits, high sensitivity and
high specificity. The high specificity is largely due to the fact that fluorophores exhibit
specific excitation (absorption) and emission (fluorescence) wavelengths. These
wavelengths can be determined via the collection of two spectra, an excitation spectrum
and an emission spectrum. In this experiment, the excitation and emission spectra for the
Rhodamine B and 8-hydroxyquinoline was measured. Fluorescence capability of
Rhodamine B was determined using a standard calibration curve. Quantitative
fluorescence analysis revealed that unknown solution contains 0.9142 ± 0.0277 ppm of
Rhodamine B. Linear least square and other statistical method confirmed that standard
addition method was repeatable and fulfilled precision criteria as indicated in a very low
standard deviation in concentration which corresponds to 0.0277 ppm. In addition, the
effect of divalent mercury ion (Hg2+) to quench the fluorescence of Rhodamine B was
used determine the concentration of Hg2+ in an unknown solution. The quenching
capability of Hg2+ was very evident as the emission intensity decreases after subsequent
addition of Hg2+. Calibration curve from the plot of concentration of Hg 2+ against
emission intensity revealed that the Hg2+ concentration in the unknown solution is 0.6877
ppm. Aside from Rhodamine B, fluorescence activity of 8-hydroxyquinoline was studied.
Magnesium metal, which is non-fluorescent, able to induce a fluorescent by forming
chelate with 8-hydroxyquinoline. Thus, the concentration of Mg2+ in an unknown
solutionwas determined employing the fluorescent complex of Mg2+- 8-
hydroxyquinoline. Calibration curve from the plot of concentration of Mg2+ against
emission intensity revealed that the Mg2+ concentration in the unknown solution is 80.96
ppm.
 28

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