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

Red Orange Yellow Green Cyan Blue Violet Indigo

Relationship between % transmittance and Absorbance 100 A = - log

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. 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

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

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. 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

N.D.H Key

N.D.H Key

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)