1. Steve Harris Instructor SPASH – Biotechnical Engineering
Spectrophotometry Lab: Determination of the absorption spectra of colored dyes.
Steve Harris
Instructor
SPASH – Biotechnical Engineering

5. Red
Orange
Yellow
Green
Cyan
Blue
Violet
Indigo

8. Relationship between % transmittance and Absorbance
100
A = - log

9. 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.

10. 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.
Data Table 1: Transmittance of Visible Light in nanometers
Visible Light
Experimental
Experimental Average
Expected
Expected Average
% Error
Wavelength Range
Start
End R
Red
625
740 O
Orange
590 Y
Yellow
565 G
Green
520 C
Cyan
500 B
Blue
440 I
Indigo
420 V
Violet
380

11. Data Table 1: Transmittance of Visible Light in nanometers
Experimental
Experimental Average
Expected
Expected Average
% Error
Wavelength Range
Start
End R
Red O
Orange Y
Yellow G
Green C
Cyan B
Blue I
Indigo V
Violet

12. 380-420 nm — wavelength of violet light
nm — wavelength of indigo light
nm — wavelength of blue light
nm — wavelength of cyan light
nm — wavelength of green light
nm — wavelength of yellow light
nm — wavelength of orange light
nm — wavelength of red light

14. 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.

15. Data Table 2: Absorbance and Transmitance of Colored dyes.
Wavelength in nm
Dye Color
Red
Blue
Green
Yellow
Absorbance
Transmittance
380
400
420
440
460
480
500
520
540
560
580
600
620
640
660
680
FLIP

16. N.D.H
Key

17. N.D.H
Key

18. Questions
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)