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Lab 9 & 10
UV-Vis spectroscopy
Introduction
Ultraviolet-visible light spectroscopy is an analytical technique used to find both the
electronic transitions of different species but also the concentration of a species with a known
extinction coefficient. The instrument works by sending light through a wavelength selector (in
this case a prism), through the sample, and to a detector. The output of a uv-vis spectrometer is
either in percent transmittance or absorbance. Percent transmittance is the ratio of what light
makes it through a sample compared to the light that enters it multiplied by 100 as in figure 1.
Absorbance is just the negative logarithm of the transmittance, and absorbance tends to be used
because it is directly proportional to the concentration of a sample using beers law. Beers law
includes an extinction coefficient and the path length which for this sample cell is 1 cm. The
extinction coefficient has the units of cm-1M-1 which gives absorbance no units (also known as
absorbance units). See figures 2 and 3 for the relationship between absorbance along with
absorbance and concentration. This experiment used both a spectronic-20 (spec-20) and the
agilent UV-Vis to identify the max of different colors along with different metal complexes. The
spec-20 uses a monochromator while the UV-Vis uses a polychromator which allows for it to
record the entire spectrum at once.
P
%T = 100
Figure 1: % transmittance.
P0
A=log ( T )
Figure 2: Absorbance and transmittance
A= bc
Figure 3: Absorbance and concentration.
Procedure
Two food coloring solutions were made by placing two drops of green into 50 mL
of water and two drops of blue into a separate 50 mL solution. Then a small volume of
each solution was placed in a 100 mm cuvet (round) along with a mixture that was
Prater
formed by both the colors in approximately a 1:1 ratio. These three solutions were used to
find the %Tmin from 390 nm to 750 nm in increments of 10 nm (390 nm was the lowest
limit of the instrument). The data from this is illustrated in figures 4, 5, and 6. The
Agilent UV-Vis was also used on these three solutions with a two-fold dilution. The other
solutions (metal complex solutions) were also analyzed by the UV-Vis to find the max for
absorbance. The data for each of these spectra appears in the results section.
Results
It was found that the %Tmin which correlates to the max for absorbance was 630
nm for the green food coloring, and 630 nm for blue food coloring, thus the mixture also
had a max of 630 nm. Figures 4, 5, and 6 represent the data from the spec-20 graphically.
Green (spec-20)
150
100
% Transmitance
50
0
395 445 495 545 595 645 695 745
Wavelength (nm)
Blue (spec-20)
150
100
% Transmittance
50
0
395 445 495 545 595 645 695 745
Wavelength (nm)
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Mixed-Blue/green (spec-20)
200
% Transmittance
100
0
395 445 495 545 595 645 695 745
Wavelength (nm)
The absorbance data that was collected with the Agilent UV-Vis for the three food color
solutions is below, shown as absorbance data and then overlapped with the transmittance
to see the correlation in the data.
Green (UV-Vis)
Absorbance
355 405 455 505 555 605 655
Wavelength (nm)
Green T
Green Abs
0
375
475
575
675
Wavelength (nm)
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Blue (UV-Vis)
3
2
Absorbance 1
0
345 395 445 495 545 595 645 695
Wavelength (nm)
Blue T
Blue Abs
0
375
475
575
675
775
Wavelength (nm)
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Mix T
Mix Abs
0
375425475525575625675725775
Wavelength (nm)
Ni(NO_3)_2
1
Absorbance
0.5
0
345 395 445 495 545 595 645 695
Wavelength
KMnO4
Absorbance
350
400
450
500
550
Wavelength
600
650
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K_2CrO_4
4
Absorbance
2
0
250
300
350
400
450
500
Wavelength
Cu(NO_3)_2
Absorbance
550 600 650 700 750 800 850 900
Wavelength (nm)
Cr(NO_3)_3
0.75
0.5
Absorbance 0.25
0
265 315 365 415 465 515 565 615 665
Wavelength (nm)
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CoCl_2
Absorbance
400
450
500
550
600
Wavelength (nm)
Fluorescein
1
Absorbance
0.5
0
400
-0.5
450
500
550
600
Wavelength (nm)
max (nm)
484
511
252
806
371
526
384
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Wavel
ength
(nm)
400
410
420
430
440
450
460
470
480
490
500
510
520
530
540
550
560
570
580
590
600
610
620
630
640
650
660
670
680
Gr
ee
n
%T
4
3
3
3
4
5
7
15
29
51
70
76
73
66
53
45
30
19
12
8
4
3
2
2
3
8
22
46
71
Blu
e
%T
27
29
41
54
62
60
54
45
38
33
29
27
25
23
21
19
14
9
6
4
2
2
2
2
3
5
15
36
64
Mi
xt
ur
e
%T
9
8
10
12
13
16
19
24
32
38
42
42
41
37
33
29
21
14
8
5
3
2
2
2
3
6
18
41
68
Prater
690
84
80
700
92
89
710
96
95
720
97
97
730
98
97
740
99
99
750
99
99
Table 3: Absorbance of each food color.
Wavel
ength
(nm)
Gr
ee
n
Ab
s
1.3
97
94
00
1
1.5
22
87
87
5
1.5
22
87
87
5
1.5
22
87
87
5
1.3
97
94
00
1
1.3
01
03
400
410
420
430
440
450
Blu
e
Ab
s
0.5
68
63
62
4
0.5
37
60
2
0.3
87
21
61
4
0.2
67
60
62
4
0.2
07
60
83
1
0.2
21
84
87
82
91
96
97
98
99
99
Mi
xtu
re
Ab
s
1.0
45
75
74
9
1.0
96
91
00
1
1
0.9
20
81
87
5
0.8
86
05
66
5
0.7
95
88
00
Prater
460
470
480
490
500
510
520
530
540
1.1
54
90
19
6
0.8
23
90
87
4
0.5
37
60
2
0.2
92
42
98
2
0.1
54
90
19
6
0.1
19
18
64
1
0.1
36
67
71
4
0.1
80
45
60
6
0.2
75
72
5
0.2
67
60
62
4
0.3
46
78
74
9
0.4
20
21
64
0.4
81
48
60
6
0.5
37
60
2
0.5
68
63
62
4
0.6
02
05
99
9
0.6
38
27
21
6
0.6
77
78
0.7
21
24
64
0.6
19
78
87
6
0.4
94
85
00
2
0.4
20
21
64
0.3
76
75
07
1
0.3
76
75
07
1
0.3
87
21
61
4
0.4
31
79
82
8
0.4
81
48
10
Prater
550
560
570
580
590
600
610
620
630
640
41
3
0.3
46
78
74
9
0.5
22
87
87
5
0.7
21
24
64
0.9
20
81
87
5
1.0
96
91
00
1
1.3
97
94
00
1
1.5
22
87
87
5
1.6
98
97
1.6
98
97
1.5
07
1
0.7
21
24
64
0.8
53
87
19
6
1.0
45
75
74
9
1.2
21
84
87
5
1.3
97
94
00
1
60
6
1.6
98
97
1.6
98
97
1.6
98
97
1.6
98
97
1.5
0.5
37
60
2
0.6
77
78
07
1
0.8
53
87
19
6
1.0
96
91
00
1
1.3
01
03
1.5
22
87
87
5
1.6
98
97
1.6
98
97
1.6
98
97
1.5
11
Prater
650
660
670
680
690
700
710
720
22
87
87
5
1.0
96
91
00
1
0.6
57
57
73
2
0.3
37
24
21
7
0.1
48
74
16
5
0.0
75
72
07
1
0.0
36
21
21
7
0.0
17
72
87
7
0.0
13
22
82
7
22
87
87
5
1.3
01
03
0.8
23
90
87
4
0.4
43
69
75
0.1
93
82
00
3
0.0
96
91
00
1
0.0
50
60
99
9
0.0
22
27
63
9
0.0
13
22
82
7
22
87
87
5
1.2
21
84
87
5
0.7
44
72
74
9
0.3
87
21
61
4
0.1
67
49
10
9
0.0
86
18
61
5
0.0
40
95
86
1
0.0
17
72
87
7
0.0
13
22
82
7
12
Prater
730
740
750
0.0
08
77
39
2
0.0
04
36
48
1
0.0
04
36
48
1
0.0
13
22
82
7
0.0
04
36
48
1
0.0
04
36
48
1
13
0.0
08
77
39
2
0.0
04
36
48
1
0.0
04
36
48
1
Figure 24
Figure 25
=26 400 c m M 1
1
Discussion
The above data clearly illustrates that the agilent UV-vis is a much better
instrument for acquiring spectra than the spec-20 because of the efficiency that occurs
and the number of data points that can be observed. As shown in figures 8, 10, and 12, the
qualitative relationship between absorbance and transmittance is inversely proportional
(ignoring the logarithm). As absorbance increases, the transmittance decreases. This is
because absorbance is the amount of light that the analytes absorb while transmittance
represents the amount of light that makes it through the sample cell and reaches the
detector. These are fundamentally opposite qualitatively but are inverse lograthmically
proportional mathematically.
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The exact spectra of the different metal complexes is due to a few specific things:
the oxidation state of the metal ion along with how many electrons are in the d orbitals,
the coordination number, and the strength of the ligand that is bound to the metal. Each of
these affect the o or t depending on the molecular geometry. The larger the value, the
higher the energy will be of a transition (smaller wavelength), whilst the opposite is also
true.
The molar absorptivities of both the copper (II) nitrate solution and the
fluorescein solution were found and for fluorescein, it was quite low, which could be due
to the instrument being off by about 10 nm because the literature states the value to be
494 nm instead of 484 nm.1 The molar absorptivity for fluorescein is quite low compared
to the literature values of more than 50,000.2 The molar absorptivity of a copper (II)
solution is about 20,000 which is very different than the value discovered in this
experiment.3
Due to the detection limits of the spec-20, the max for the absorbance was found
to be over a broad range of up to 20 nm. If the detector was more sensitive, this would
have been distinguished.
The specific colors of the metal solutions relates to the max via the color wheel
such that the complimentary color is absorbed. For example, if the solution appears blue,
it will absorb in the red region of the spectrum. The color for each solution is determined
by the energy of the absorbance and the energy of the emission.
Conclusion
UV-Vis absorption spectra are very useful for a number of fields in chemistry in order to
find the energy of the electronic transitions present and to find the concentration of a
given analyte if the molar absorptivity is known. The values obtained for the molar
absorptivities, however, were very different than the literature values. Therefore, the
instrument needs to be checked to verify that it is calibrated to ensure proper data
analysis.
References
(1)
Fluorescein- Learn Chemistry http://www.rsc.org/learnchemistry/resource/rws00015968/fluorescein (accessed Feb 11, 2015).
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(2)
Spectrophotometric analysis of sodium fluorescein aqueous solutions.
Determination of molar absorption coefficient.pdf.
(3)
SciFinder - Synthesis and c...
https://scifinder.cas.org/scifinder/view/scifinder/scifinderExplore.jsf (accessed Feb 11,
2015).
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