ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY

SCHOOL OF APPLIED NATURAL SCIENCE

DEPARTMENT OF APPLIED MATHEMATICS

FLUORESCENCE SPECTROSCOPY (Phys8522)

ASSIGNMENT ONE

By: Alemayehu Getahun

ID No.: PGR/21749/13

Submitted to:
Abebe Belay (Asso. prof.)

Adama, Ethiopia

30th March 2021

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1. Estimation of Fluorescence and Phosphorescence Quantum Yields:

The quantum yield for fluorescence is determined by the radiative and
non-radiative decay rates. The non-radiative rates are typically similar for
fluorescence and phosphorescence states, but the emissive rates (Γ) vary
greatly. Emission spectra, lifetimes (τ ) and quantum yields (Q) for eosin
and erythrosin B (ErB) are shown in Figure 1.13.
A. Calculate the natural lifetime(τn ) and the radiative and non-radiative
decay rates of eosin and ErB. What rate accounts for the lower quantum
yield of ErB?
B. Phosphorescence lifetimes are typically near 1–10 ms. Assume that the
natural lifetime for phosphorescence emission of these compounds is 10 ms,
and that the non-radiative decay rates of the two compounds are the same
for the triplet state as for the singlet state. Estimate the phosphorescence
quantum yields of eosin and ErB at room temperature.

ANSWER # 1.A
From figure 1.3 we have that τ = 3.1,Q = 0.65 for Eosin and τ = 0.61, Q = 0.12 for Er B using
this we can calculate τn for both Eosin and Er B as flows.
For Eosin
τ
τn = . (1)
Q
3.1
= . (2)
0.65
After simplification we have that

τn = 4.77ns. (3)

For Er B
τ
τn = , (4)
Q
0.61
= , (5)
0.12
After simplification we have that
τn = 5.08ns. (6)

The radiative and nonradiative can be calculated using the life time and quantum yield formulas
of
1
τ= (7)
Γ + knr
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Where Γ is for radiative and knr is for nonradiative.

And The quantum yield is
Γ
Q= . (8)
Γ + knr
We know that
τ
τn = . (9)
Q
Substituting Eqs.(7)and (8) into Eq.(9), we have

1
τn = . (10)
Γ

From Eq.(10), we can determine the radiative decay of Eosin and as

1
Γ= . (11)
τn

Using Eqs.(11) and (3), we can calculate the radiative decay for Eosin as

Γ = 0.21ns. (12)

Similarly for Er B we have

Γ = 0.196ns. (13)

The nonradiative decay can be calculated using

1
τ = , (14)
Γ + knr
1
Γ + knr = (15)
τ

From this we can easily simplify to find the nonradiative decay as

1
knr = − Γ. (16)
τ

With help of Eqs.(3) and (12) into Eq.(16) we can calculate the nonradiative decay of Eosin as,

1
knr = − 0.21, (17)
3.1
= 0.322 − 0.21 (18)

Finally we have
knr = 0.11ns. (19)

Similarly for Er B
knr = 1.44ns (20)
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ANSWER # 1.B
The quantum yield can be denoted by
Γ
Q= . (21)
Γ + knr
Using Eqs.(13) and (20), into Eq.(21), we find
0.196
Q = , (22)
0.196 + 1.44
0.196
= (23)
1.636
After more simplification we have that

Q = 0.12. (24)

2. Estimation of Emission from the S2 State: When excited to the second

singlet state (S2 ) fluorophores typically relax to the first singlet state within
1013 s. Using the radiative decay rate calculated for eosin (problem 1.1),
estimate the quantum yield of the S2 state.

ANSWER # 2
Similar to question# 1B we can calculate the quantum yield using radiative decay as
0.21ns
Q = , (25)
0.21ns + 0.11ns
0.21
= . (26)
0.32
After more simplification, the quantum yield of radiative decay can be put as

QEosin = 0.65. (27)

From Eqs.(24) and (27) one can explains why it is difficult to observe phosphorescence at room
temperature: most of the molecules that undergo intersystem crossing return to the ground
state by non-radiative paths prior to emission.

3. Thermal Population of Vibrational Levels: The emission spectrum of

perylene (Figure 1.3) shows equally spaced peaks that are due to various
vibrational states, as illustrated. Use the Boltzmann distribution to estimate
the fraction of the ground-state molecules that are in the first vibrationally
excited state at room temperature.
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ANSWER # 3.
The energy spacing between the various vibrational energy levels is revealed by the emission
spectrum of perylene (Figure 1.3). The individual emission maxima (and hence vibrational
energy levels) are about 1500 cm−1 apart. The Boltzmann distribution describes the relative
number of perylene molecules in the 0 and 1 vibrational states. The ratio (R) of molecules in
each state is given by
R = e∆E/kT (28)

where ∆E is the energy difference, k is the Boltzmann constant, and T is the temperature
in degrees kelvin (K). Assuming a room temperature of 300 K, this ratio is about 0.01. Hence
most molecules will be present in the lowest vibrational state, and light absorption results mainly
from molecules in this energy level. Because of the larger energy difference between S0 and S1 ,
essentially no fluorophores can populate S1 as a result of thermal energy.

4. Anisotropy of a Labeled Protein: Naphthylamine sulfonic acids are widely

used as extrinsic labels of proteins. A number of derivatives are available.
One little known but particularly useful derivative is 2-diethylamino-5-
naphthalenesulfonic acid (DENS), which displays a lifetime near 30 ns,
longer than that of most similar molecules. Absorption and emission spectra
of DENS are shown in Figure 1.36.
A. Suppose the fundamental anisotropy of DENS is 0.30 and that DENS is
bound to a protein with a rotational correlation time of 30 ns. What is the
anisotropy?
B. Assume now that the protein is bound to an antibody with a molecular
weight of 160,000 and a rotational correlation time of 100 ns. What is the
anisotropy of the DENS-labeled protein?

ANSWER # 4
Anisotropy is given by
r0
r= . (29)
1 + ( τθ )
From the given we have that τ = 30ns and r0 = 0.3, for steady state (τ = θ) then we have

0.3
r = , (30)
1+1
0.3
= , (31)
2
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After more simplification we have

r = 0.15. (32)

Similarly For # 4B, we find

r = 0.23. (33)

Comparing Eqs.(32) and (33) we can state the increases in anisotropy upon antigen–antibody
binding are the basis of the fluorescence polarization immunoassays, which are used to detect
drugs, peptides, and small proteins in clinical samples.

5. Effective Distance on the Efficiency of FRET: Assume the presence of

a single donor and acceptor and that the distance between them (r) can be
varied.
A. Plot the dependence of the energy transfer efficiency on the distance
between the donor and the acceptor.
B. What is the transfer efficiency when the donor and the acceptor are
separated by 0.5R0 , R0 , and 2R0 ?

ANSWER # 5
The dependence of transfer efficiency on distance (r) between a donor and acceptor can be
denoted by
R06
E= . (34)
R06 + r6
From the given we have that 0.5R0 , R0 and 2R0 . In line with this taking r = R0 we can calculate
the transfer efficiency as

R06
E = ,
R06 + R06
= 0.5. (35)

The efficiency is 50 %. For r = 0.5R0

R06
E = ,
R06 + r6
R06
= 6
R0 + (0.5R0 )6
R06
= 6 (36)
R06 + (R640 )

After simplification we have that

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E = 0.98. (37)

The efficiency is 98 %.
For r = 2R0
R06
E = ,
R06 + r6
R06
= 6
R0 + (2R0 )6
R06
= 6 . (38)
R0 + (64R0 )6
After simplification we have that

E = 0.015. (39)

The efficiency is 1.5 %.

With help of Eq.34-38 we can plot the graph which shows dependence of energy transfer efficiency
on distance between donor and acceptor.

Even without Approximating value of r, R0 , we can plot and investigate the relation between
transfer efficiency and distance. The efficiency can be rewritten in more compact form of
1
E= . (40)
1 + ( Rr0 )6
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Using above relation we can graph tranisfer efficiency versus r/R0 graph as shown bellow.

Fig.(2) plot of Tranisfer efficiency versus r/R0 .

From Fig.(2), one can state that transfer efficiency is inverse proportion to the distance.

6. Calculation of a Distance from FRET Data: The protein human serum

albumin (HSA) has a single tryptophan residue at position 214. HSA was
labeled with an anthraniloyl group placed covalently on cysteine-34. Emission
spectra of the labeled and unlabeled HSA are shown in Figure 1.37. The
Förster distance for Trp to /anthraniloyl transfer is 30.3 Å. Use the emission
spectra in Figure 1.37 to calculate the Trp to anthraniloyl distance.

ANSWER # 6
To calculate the distance of trp to anthraniloylfirst we have to apply the Beer-Lambert Law.
Which is given by
P
A = −log10 ( ). (41)
P0
Where P is the maximum pick and P0 is the minimum pick. From Figure 1.37, we have that
p0 = 0.26 and P = 0.7, then we have that
 
0.26
A = log10 (42)
0.7
= −0.430. (43)

We know that the maximum pick(P = I) and the minimum pick(P0 = I0 ). Applying laws of
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logarithmic function we have that

I
= 10−A , (44)
I0
= 0.37. (45)

We know that the transfer efficiency and Intensity are releted by

I
E = 1− , (46)
I0
= 0.63. (47)

Finally using transfer efficiency one can calculate the distance of Trp to anthraniloyl as
r
6 1 − E
r = R0 . (48)
E
From the given we have that R0 = 30.3Å, and using transfer efficiency we have that
r
0 6 1 − 0.63
r = 30.3A , (49)
0.63
= 30.3(0.587)0.166 , (50)

After simplification we have that

r = 27.7Å. (51)

7. Interpretation of Tryptophan Fluorescence from a Peptide: Figure 1.38

shows a summary of spectral data for a peptide from myosin light-chain ki-
nase (MLCK). This peptide contained a single tryptophan residue, which was
placed at positions 1 through 16 in the peptide. This peptide binds to the
hydrophobic patch of calmodulin. Explain the changes in emission maxima,
Stern-Volmer quenching constant for acrylamide (K), and anisotropy (r)2

ANSWER # 7
The changes in λmax , K, and r shown in Figure 1.38 are the result of the tryptophan residue
being exposed to or shielded from the water. Increases in λmax and K indicate increased exposure
to water, and decreases in λmax and K indicate decreases in exposure to water. Increases and
decreases in r indicate a less mobile and more mobile tryptophan residue, respectively. The three
parameter values show a cyclical behavior with a period of about 3.5 amino acid residues per
cycle. This suggests that the MLCK peptide is in an α-helical state when bound to calmodulin
(Figure 1.42). The spectral changes seen in Figure 1.38 are the result of the tryptophan residue
being alternately exposed to water or shielded between the MLCK peptide and calmodulin as
its position is shifted along the peptide chain.