Fundamentals of Analytical Chemistry: 8th ed.
Chapter 27
Chapter 27
27-1
(a) Resonance fluorescence is observed when excited atoms emit radiation of the same
wavelength as that used to excite them.
(b) Vibrational relaxation occurs when excited species collide with molecules, such as
the solvent, and in doing so lose energy without emission of electromagnetic radiation.
The energy of the excited species is decreased by an amount equal to the quantity of
vibrational energy transferred.
(c) Internal conversion is the nonradiative relaxation of a molecule from a low energy
vibrational level of an excited electronic state to a high energy vibrational level of a
lower electronic state.
(d) Fluorescence is a photoluminescence process in which atoms or molecules are
excited by absorption of electromagnetic radiation and then relax to the ground state,
giving up their excess energy as photons.
(e) The Stokes shift is the difference in wavelength between the radiation used to excite
fluorescence and the wavelength of the emitted radiation.
(f) The quantum yield of fluorescence is the ratio of the number of fluorescing molecules
to the total number of excited molecules.
(g) Self-quenching occurs when the fluorescent radiation from an excited analyte
molecule is absorbed by an unexcited analyte molecule. This process results in a
decrease in fluorescence intensity.
27-2
For spectrofluorometry, the analytical signal, F, is given by F = 2.3K’εbcP0. The
magnitude of F, and thus sensitivity, can be enhanced by increasing the source intensity,
P0, or the transducer sensitivity. For spectrophotometry, the analytical A is given by A =
Fundamentals of Analytical Chemistry: 8th ed.
Chapter 27
P / P0. Increasing P0 or the detector’s response to P0 is accompanied by a corresponding
increase in P. Thus, he ratio does not change nor does the analytical signal.
Consequently, no improvement in sensitivity accompanies such changes.
27-3
(a) Fluorescein because of its greater structural rigidity due to the bridging –O– groups.
(b) o,o’-Dihdroxyazobenzene because the –N=N– group provides rigidity that is absent in
the –NH–NH– group.
27-4
Compounds that fluoresce have structures that slow the rate of nonradiative relaxation to
the point where there is time for fluorescence to occur. Compounds that do not fluoresce
have structures that permit rapid relaxation by nonradiative processes.
27-5
Organic compounds containing aromatic rings often exhibit fluorescence. Rigid
molecules or multiple ring systems tend to have large quantum yields of fluorescence
while flexible molecules generally have lower quantum yields.
27-6
Excitation of fluorescence usually involves transfer of an electron to a high vibrational
state of an upper electronic state. Relaxation to a lower vibrational state of this electronic
state goes on much more rapidly than fluorescence relaxation. Fluorescence almost
always occurs from the lowest vibrational level of the excited electronic state to various
vibrational levels of the ground electronic state. Such transitions involve less energy than
the excitation energy. Therefore, the emitted radiation is longer in wavelength than the
excitation wavelength.
27-7
See Figure 27-8. A filter fluorometer usually consists of a light source, a filter for
selecting the excitation wavelength, a sample container, an emission filter and a detector.
Fundamentals of Analytical Chemistry: 8th ed.
Chapter 27
A spectrofluorometer uses monochromators instead of filters for excitation and emission.
There are also hybrid instruments that use an excitation filter and an emission
monochromator. In corrected spectrofluorometers, there is also a reference detector for
monitoring and correcting for fluctuations in the light sources intensity. Emission is
usually detected at right angles to the incident radiation to maximize the fluorescence
signal.
27-8
Most fluorescence instruments are double beam to compensate for fluctuations in the
analytical signal due to variations in source intensity.
27-9
Because fluorometers don’t disperse the emitted radiation the instrument can detect the
entire spectrum of fluorescence emission (as long as a suitable filter is used to remove
scattered light from the excitation source). Thus, a fluorometer can provide lower limits
of detection than a spectrofluorometer. In addition, fluorometers are substantially less
expensive and more rugged than spectrofluorometer, making them particularly well
suited for routine quantitation and remote analysis applications.
Fundamentals of Analytical Chemistry: 8th ed.
1
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19
A
27-10 Determination of NADH
Part (a)
Concentration in M
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
unknown
Part (b)
Regression equation
Slope
Intercept
Concentration of unknown
Parts (c), (d), (e), and (f)
Error Analysis
Chapter 27
B
C
D
E
F
I
22.346
4.E-04
0.54
Spreadsheet Documentation
B15=SLOPE(B4:B11,A4:A11)
0.175
8
B16=INTERCEPT(B4:B11,A4:A11)
B17=(B12-B16)/B15
22 Sxx
0.42
B20=STEYX(B4:B11,A4:A11)
sm
y bar (average fluoresence)
M for part (e)
M for part (f)
Standard deviation in c for part (e)
RSD in c for part (e)
Standard deviation in c for part (f)
RSD in c for part (f)
H
Fluorescence
2.24
4.52
6.63
9.01
10.94
13.71
15.49
17.91
12.16
20 sr (standard error in y)
21 N
23
24
25
26
27
28
29
30
31
32
G
0.27
10.056
1
3
0.008
0.015
0.005
0.010
B21=COUNT(B4:B11)
B22=B21*VARP(A4:A11)
B23=SQRT(B20^2/B22)
B24 =AVERAGE(B4:B11)
B25= Replicates part (e) (entry)
B26=Replicates part (f)
B27 =B20/B15*SQRT(1/B25+1/B21+((B12-B24)^2)/((B15^2)*B22))
B28=B27/B17
B29=B20/B15*SQRT(1/B26+1/B21+((B12-B24)^2)/((B15^2)*B22))
B30=B29/B17
Fundamentals of Analytical Chemistry: 8th ed.
Chapter 27
27-11 In the first printing of the text, volumes of the standard solution were presented in both
the text and the table. Using only the data in the table, we find …
A
B
2+
C
1 V (Zn )
5.00
2 cS, ppm
3
4 Part (a)
1.10E+00
5
6
7
8
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13
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15
16
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19
E
F
G
Volume Standard Fluorometer
Solution, mL
Reading
0.00
6.12
4.00
11.16
8.00
15.68
12.00
20.64
Part (b)
Slope
Intercept
Part (c)
1.202000
6.1880
sr
SD m
SD b
Part (d)
2+
c (Zn ), ppm
0.154402
0.017263
0.129182
20 Part (e)
2+
21 SD c (Zn ), ppm
22
23
24
25
26
27
28
29
30
31
32
D
1.13258
LINEST Values
m
1.20200
6.1880
SD m
0.017263 0.129182
0.02870
R
F
2
SSregr
0.999588 0.154402
4848.336
2
115.5843
0.04768
b
SD b
sr
DOF
SSresid
Spreadsheet Documentation
B12 = SLOPE(B6:B9,A6:A9)
B13 = INTERCEPT(B6:B9,A6:A9)
E18:F22 = LINEST(B6:B9,A6:A9,TRUE,TRUE)
B15 = F20
B16 = E19
B17 = F19
B19 = B13*B2/(B12*B1)
B21 = B19*SQRT((B16/B12)^2+(B17/B13)^2)
27-12 cQ = 100 ppm  288 / 180 = 160 ppm
160 ppm 
100 mL
1 mg quinine
1 g solution


 500 mL = 533 mg quinine
3
15 mL 1  10 g solution
1 mL
H
Fundamentals of Analytical Chemistry: 8th ed.
27-13 cQ =
A1c sVs
(540)(50 ppm)(10.0 mL)

= 225 ppm
 A2  A1 VQ 600  540(20.0 mL)
225 ppm 
1 mg quinine
1 g solution

 1000 mL = 225 mg quinine
3
1 mL
1  10 g solution
0.225 g Q
 10 6 = 1.02105 ppm
2.196 g Tablet
Chapter 27