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Matt Prater

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

Materials and reagents:


Spectronic-20
Agilent UV-Vis
Green Food coloring
Blue food coloring
Distilled water
Cuvets for both instruments
Copper nitrate solution

Cobalt (II) nitrate solution


Potassium chromate solution
Cobalt (II) chloride solution
Nickel nitrate solution
Chromium (III) solution
Potassium permanganate solution
Fluorescein solution

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

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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)

Figure 4: % transmittance vs. wavelength for green.

Blue (spec-20)
150
100
% Transmittance

50
0
395 445 495 545 595 645 695 745
Wavelength (nm)

Figure 5: % transmittance vs. wavelength for blue.

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Mixed-Blue/green (spec-20)
200
% Transmittance

100
0
395 445 495 545 595 645 695 745
Wavelength (nm)

Figure 6: % transmittance vs. wavelength for the mixed color.

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)

Figure 7: Absorbance vs. wavelength for green with uv-vis.

Abs and T for Green


2
Abs and T

Green T

Green Abs

0
375

475

575

675

Wavelength (nm)

Figure 8: Absorbance and Transmittance (not %) compared.

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Blue (UV-Vis)
3
2
Absorbance 1
0
345 395 445 495 545 595 645 695
Wavelength (nm)

Figure 9: Absorbance of blue food coloring.

Abs and T for Blue


4
Abs and T

Blue T

Blue Abs

0
375

475

575

675

775

Wavelength (nm)

Figure 10: Absorbance and Transmittance (not %) compared.

Mix (Blue/Green) by Uv-Vis


1.5
1
Absorbance 0.5
0
345 395 445 495 545 595 645 695
Wavelength (nm)

Figure 11: Absorbance spectrum of the mixture.

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Abs and T for Mix


1.5
1

Mix T

Abs and T 0.5

Mix Abs

0
375425475525575625675725775
Wavelength (nm)

Figure 12: Absorbance and Transmittance (not %) compared)


As it is apparent in the figures above, the correlation between absorbance and
transmittance are opposite of each other. This relationship will be discussed in more
detail below. The following figures show the UV-Vis absorbance data for the different
metal ions and fluorescein.

Ni(NO_3)_2
1
Absorbance

0.5
0
345 395 445 495 545 595 645 695
Wavelength

Figure 13: The absorbance spectrum of nickel (II) nitrate.

KMnO4
Absorbance
350

400

450

500

550

Wavelength

600

650

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Figure 14: The absorbance spectrum of potassium permanganate.

K_2CrO_4
4
Absorbance

2
0
250

300

350

400

450

500

Wavelength

Figure 15: The absorbance spectrum of potassium chromate.

Cu(NO_3)_2
Absorbance
550 600 650 700 750 800 850 900
Wavelength (nm)

Figure 16: The absorbance spectrum of copper (II) nitrate.

Cr(NO_3)_3
0.75
0.5
Absorbance 0.25
0
265 315 365 415 465 515 565 615 665
Wavelength (nm)

Figure 17: The absorbance spectrum of chromium (III) nitrate.

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CoCl_2
Absorbance
400

450

500

550

600

Wavelength (nm)

Figure 18: The absorbance spectrum of cobalt (II) chloride.

Fluorescein
1
Absorbance

0.5
0
400
-0.5

450

500

550

600

Wavelength (nm)

Figure 19: The absorbance spectrum of fluorescein.


From the above figures, the max of fluorescein was 484 nm, that of cobalt (II) chloride
was 511 nm; that of chromium (III) nitrate was 252 nm; that of copper (II) nitrate was
806 nm; that of potassium chromate was 371 nm; that of potassium permanganate was
526 nm with some ligand to metal charge transfer appearing; that of nickel (II) nitrate
was 384 nm; that of the green food coloring was 630 nm along with blue and the mixture.
Table 1: Summary of absorbance spectra.
Species
Fluorescein
Cobalt (II) chloride
Chromium (III) nitrate
Copper (II) nitrate
Potassium chromate
Potassium
permanganate
Nickel (II) nitrate

max (nm)
484
511
252
806
371
526

384

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Green food coloring


620-630
Blue food coloring
610-630
Mixed food coloring
610-630
Table 2: spec-20 data for food coloring.

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

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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

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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

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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

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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

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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

Calculation of copper (II) nitrate extinction coefficient


A= bc
Figure 20
1.2069=10.1
Figure 21
1.2069
=
Figure 22
0.1
Figure 23
=12 c m1 M 1
Calculation of Fluorescein extinction coefficient
0.8755= bc

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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