Fluorescence Lifetime Analysis of Anthraquinone Derivatives

1.23

Abdul Zamani, Marco Allard

Department of Chemistry and Biochemistry, La Sierra University, Riverside, CA 92505

Fluorescence lifetime (Tau) can be determined

through an exponential decay function. This
decay curve is represented by the Arrhenius
equation. Fluorescence is one of many paths
for a molecule to reach the ground state after
being electronically excited. Lifetime equates to
the reciprocal sum of all the decay pathway
rate constants. Vibrational relaxation and
intersystem crossing are factor into their rate
constants. This study focuses on the
determination of the rate constant of
fluorescence (kf)

Objective
Assess Anthraquinones viability as a fluorescent dye.
Characterize and compare various AQ derivatives
with respect to excitation lifetimes.
Predict its functionality and stability in a cellular
environment

1
=

Theory

The Franck-Condon principle states that

photon absorption is an instantaneous
process that allows no time for electronic
rearrangement. As it returns to the
lowest energy state that closely
resembles that of the excited state,
energy is lost by means of vibrational
relaxation or another intermolecular
interactions.
Because
of
this
phenomenon, the absorption energy is
always higher than the emission energy.
This energy difference is related to a shift
in emission photon wavelength

Fluorescein Fluorescence Decay @278K

8

A fluorescent compound, such

as fluorescein (FlCn), is often
used as a standard for
comparison due to its long
lifetime.

7
Instrument Response (V)

*
Ph.D.

6
5
4
3

= 0

2
1

0
39

49

59

69

79

89
Time (ns)

Anthraquinone

99

109

119

129

139

Results

Introduction

1-ClAQ: Fluorescence Lifetime v. Temperature

1-ClAQ: Rate Constant v. Temperature

Table 1: 1-ClAQ Lifetime Analysis

2.5
0.9
0.8

0.6
Lifetime (ns)

Rate Constant

Temp (K)

Tau (ns)

kf

kf Error

276

2.31

0.44

23%

286

20.4

0.51

22%

296

1.60.2

0.61

13%

306

2.20.3

0.46

13%

316

1.10.1

0.94

6%

0.7

0.5
0.4

1.5

0.3
0.2

0.5

0.1
0

0
270

275

280

285

290

295
300
Temperature (K)

305

310

315

270

320

275

280

285

290

295
300
Temperature (K)

305

310

315

320

Table 2: 1-AmAQ Lifetime Analysis

1-AmAQ: Rate Constant v. Temperature

1-AmAQ: Fluorescence Lifetime v. Temperature

0.8

2.5

Temp (K)

Tau (ns)

kf

kf Error

2.0

273

2.00.6

0.51

33%

1.5

277

1.50.1

0.68

5%

287

1.90.1

0.52

6%

297

1.30.1

0.75

8%

307

1.70.1

0.59

8%

0.7

0.6

Lifetime (ns)

0.5
Rate Constant

Anthraquinones may be used in biological imaging

applications as fluorescent indicator dyes. These
inexpensive dyes are considered to be fluorophores
organic molecules or compounds that have the ability
to emit light after being promoted to an electronic
excited
state
through
external
radiation.
Anthraquinones make for good fluorophores due to
their highly conjugated pi systems and resonance
stability. This allows for the molecule to undergo
vibrational relaxation in addition to fluorescencethe
emission of photons. Through exponential decay curve
analysis, the fluorescence lifetimes can be calculated.
These lifetimes will provide insight into the duration
that the electrons spend in the excited state. The data
gathered from this research study will also aid in
determining the feasibility of the anthraquinone dyes
and allow for a better understanding of their
limitations and chemical properties.

0.4

0.3

The fluorescence lifetimes of the following

anthraquinone compounds will be investigated:

1.0

0.2
0.5
0.1

0.0
270

275

280

285

290
Temperature (K)

295

300

305

310

270

275

280

285

290
Temperature (K)

295

300

305

310

Table 3: 2-AmAQ Lifetime Analysis

2-AmAQ: Rate Constant v. Temperature

2-AmAQ: Fluorescence Lifetime v. Temperature

0.9

3.5

Temp (K)

Tau (ns)

kf

kf Error

275

3.20.6

0.31

19%

285

1.60.2

0.64

14%

0.8
3.0

1-Chloroanthraquinone 2-Aminoanthraquinone

0.7
2.5

Lifetime (ns)

Rate Constant

0.6
0.5
0.4

1.5

1.0
0.2

295

1.40.2

0.73

14%

305

1.50.2

0.66

10%

315

1.40.2

0.72

16%

kf

kf Error

0.5
0.1
0

0.0
270

275

280

285

290

295
300
Temperature (K)

305

310

315

The results indicate a trend between temperature

and fluorescence lifetime for each of the
antraquinone compounds. As temperature increased
in increments of 10 Kelvin, lifetime () decreased.
The drop in lifetime was significant with a 10 K
increase from the initial near-freezing temperature.
Subsequently, the lifetime reduction was more subtle
as temperature was raised. Since lifetime is inversely
proportional to the fluorescence rate constant (kf),
its value increased with a rise in temperature. For
comparison, Fluorescein was evaluated and
produced a significantly broader lifetime-- as
expected from its characteristics as standard
fluorophore. The 1-ClAQ and 1-AmAQ fits retained
substantial error. It is suspected that the compounds
that were dissolved in acetonitrile had been
quenched by oxygen. Quenching reduces the
fluorescence intensity by directing electronic
relaxation towards non-radiative means. It is unlikely
that self-absorption caused significant error in the
data output, since the AQ solutions were not
concentrated. However, temperature is known to
quench fluorophores by increasing the number of
collisions of the analyte with the solvent molecules.
If the experimentation was performed under
intervals of subfreezing temperatures (etc. 77K) it is
quite possible that the error would be reduced and
the lifetimes would be broadened. To conclude, the
fluorescence lifetime analysis of anthraquinone
derivatives characterized the effect of temperature
on the lifetime and further demonstrated the
relationship between fluorescence rate constant and
lifetime.

Future Work
It is known that anthraquinones absorb wavelengths
in the UV-Vis region and fluoresce in the Near
Infrared spectrum. These chemical and physical
properties demonstrate the potential favorability of
anthraquinone as a choice dye in Near-IR
spectroscopy. This is advantageous due to the fact
that human tissue is transparent in the Near-IR
spectral region . This can allow for in vivo imaging of
cells and tissues as opposed to still images produced
by MRI scans.

2.0

0.3

320

270

FlCn: Rate Constant v. Temperature

275

280

285

290

295
300
Temperature (K)

305

310

315

320

FlCn: Fluorescence Lifetime v. Temperature

0.0295
38.5

Table 4: FlCn Lifetime Analysis

To simulate dye function in a biological cell, large

organic substituents (like a membrane protein)
would be attached to the chlorinated or aminated
anthraquinone compounds. They can
also be
somewhat miscible in water with an addition of a
sulfate group, simulating intracellular solubility. It is
proposed that larger substituents will reduce lifetime
of the dye. Future testing will involve the application
of the dyes to various cell cultures.

0.029
38
0.0285
37.5

Temp (K)

0.028

0.0275

0.027

36.5

36

0.0265

35.5

0.026

35

0.0255

278

383

0.026

9%

288

34.88

0.029

21%

References
[1] Inoue, H.; Hida, M.; Nakashima, N.; Yoshihara, K.
Picosecond Fluorescence Lifetimes of Anthraquinone
Derivatives. Radiationless Deactivation via Intra- and
Intermolecular Hydrogen Bonds. The Journal of
Physical Chemistry J. Phys. Chem. 1982, 86, 3184
3188.

34.5
276

Figure 1: Jablonski Diagram

Tau (ns)

37
Lifetime (ns)

Rate Constant

1-Aminoanthraquinone

Conclusions

278

280

282
284
Temperature (K)

286

288

290

276

278

280

282
284
Temperature (K)

286

288

290

[2] Federici, J.; Helman, W.; Hug, G.; Kane, C.;

Patterson, L. A Work Station for Laboratory Data
Acquisition: Flourescence Lifetime Apparatus.
Computers & Chemistry. 1985, 9, 171177.