Transcript Spectrophotometry Lab: Determination of the absorption

Spectrophotometry Lab:
Determination of the absorption
spectra of colored dyes.
Steve Harris
Instructor
SPASH – Biotechnical Engineering
Violet
Indigo
Blue
Cyan
Green
Yellow
Orange
Red
Relationship between %
transmittance and Absorbance
%T
A = - log
100
Purpose:

To understand the principles of
spectrophotometry.


Absorbance, Transmittance & Wavelength.
To understand the variety of uses for
spectrophotometry.

Qualitative vs. quantitative
To generate an absorbance and
transmittance spectrum graph using excel.
 To determine the maximum absorbance of
the four test dyes.

Procedure – Part I
Construct an excel data table to record your data.
Using the Spec 20 and the slanted chalk determine
the wavelengths of visible light.
1.
2.
Data Table 1: Transmittance of Visible Light in nanometers
Wavelength Range
Visible Light
Start
End
Experimenta
l Average
Experimental
Expected
Wavelength Range
Start
End
R
Red
625
740
O
Orange
590
625
Y
Yellow
565
590
G
Green
520
565
C
Cyan
500
520
B
Blue
440
500
I
Indigo
420
440
V
Violet
380
420
Expected
Average
% Error
Data Table 1: Transmittance of Visible Light in nanometers
Visible Light
R
Red
O
Orange
Y
Yellow
G
Green
C
Cyan
B
Blue
I
Indigo
V
Violet
Start
End
Expected
Wavelength
Range
Start
End
Expected
Average
Wavelength
Range
Experimental
Average
Experimental
% Error
380-420 nm — wavelength of violet light
 420-440 nm — wavelength of indigo light
 440-500 nm — wavelength of blue light
 500-520 nm — wavelength of cyan light
 520-565 nm — wavelength of green light
 565-590 nm — wavelength of yellow light
 590-625 nm — wavelength of orange light
 625-740 nm — wavelength of red light

Procedure – Part II





Obtain a sample of Red, Blue, Green & Yellow
dye.
Using Excel prepare a data table.
Set 0% T to 0.0%
Set 100%T/0A to 100%
Check Absorbance and Transmittance from
380nm – 680nm in 20nm increments.



At each new wavelength 100%T/0A needs to be reset
to 100%
Negative absorbance's are recoded as 0.0.
Transmittances above 100% are recorded as 100%.
Following the rules of graphing and using Excel,
graph the absorbance result of your experiment.
Data Table 2: Absorbance and Transmitance of Colored dyes.
Dye Color
Wavelength
in
nm
Red
Absorbance
380
400
420
440
460
480
500
520
540
560
580
600
620
640
660
680
FLIP
Transmittance
Blue
Absorbance
Transmittance
Green
Absorbance
Transmittance
Yellow
Absorbance
Transmittance
N.D.H
Graph 1: Light Absorbance Vs. Light Wavelength of Colored Dyes
1.6
1.5
1.4
1.3
1.2
1.1
Absorbance (A)
1
Key
0.9
Red Dye
0.8
Blue Dye
0.7
Green Dye
0.6
Yellow Dye
0.5
0.4
0.3
0.2
0.1
0
-0.1
-0.2
360
380
400
420
440
460
480
500
520
540
560
Wavelength (nm)
580
600
620
640
660
680
700
N.D.H
Graph 2: Light Transmittance Vs. Light Wavelength of Colored Dyes
120
110
100
90
Transmittance
80
Key
70
Red
Blue Dye
60
Green Dye
Yellow Dye
50
40
30
20
10
0
340
360
380
400
420
440
460
480
500
520
540
Wavelength (nm)
560
580
600
620
640
660
680
700
Questions



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
What is the max absorbance values and
corresponding wavelengths of each colored
dye?
What is the max transmittance values and
corresponding wavelengths of each colored
dye?
Does each absorbance spectrum make sense
for what is expected? Why or why not?
What is the practical value of knowing the max
and min for each solution?
What is the relationship between transmittance
and absorbance? (look up on-line if you missed it last week
when we started the lab)