Spectrophotometry Lab:Spectrophotometry Lab: Determination of the absorption Determination of the absorption

spectra of colored dyes.spectra of colored dyes.

Steve HarrisSteve Harris

InstructorInstructorSPASH – Biotechnical EngineeringSPASH – Biotechnical Engineering

Red

Ora

nge

Yel

low

Gre

en

Cya

n

Blu

e

Vio

let

Indi

go

Relationship between % Relationship between % transmittance and Absorbancetransmittance and Absorbance

A = - log A = - log %T%T100100

Purpose:Purpose:To understand the principles of To understand the principles of

spectrophotometry.spectrophotometry.Absorbance, Transmittance & Wavelength.Absorbance, Transmittance & Wavelength.

To understand the variety of uses for To understand the variety of uses for spectrophotometry.spectrophotometry.Qualitative vs. quantitativeQualitative vs. quantitative

To generate an absorbance and To generate an absorbance and transmittance spectrum graph using excel.transmittance spectrum graph using excel.

To determine the maximum absorbance of To determine the maximum absorbance of the four test dyes. the four test dyes.

Procedure – Part IProcedure – Part I1.1. Construct an excel data table to record your data.Construct an excel data table to record your data.

2.2. Using the Spec 20 and the slanted chalk determine Using the Spec 20 and the slanted chalk determine the wavelengths of visible light.the wavelengths of visible light.

Data Table 1: Transmittance of Visible Light in nanometers

Data Table 1: Transmittance of Visible Light in nanometers

380-420 nm — wavelength of 380-420 nm — wavelength of violetviolet light light 420-440 nm — wavelength of 420-440 nm — wavelength of indigoindigo light light 440-500 nm — wavelength of 440-500 nm — wavelength of blueblue light light 500-520 nm — wavelength of 500-520 nm — wavelength of cyancyan light light 520-565 nm — wavelength of 520-565 nm — wavelength of greengreen light light 565-590 nm — wavelength of 565-590 nm — wavelength of yellowyellow light light 590-625 nm — wavelength of 590-625 nm — wavelength of orangeorange light light 625-740 nm — wavelength of 625-740 nm — wavelength of redred light light

Procedure – Part IIProcedure – Part II Obtain a sample of Obtain a sample of RedRed, , BlueBlue, , GreenGreen & & YellowYellow

dye.dye. Using Excel prepare a data table. Using Excel prepare a data table. Set 0% T to 0.0%Set 0% T to 0.0% Set 100%T/0A to 100%Set 100%T/0A to 100% Check Absorbance and Transmittance from Check Absorbance and Transmittance from

380nm – 680nm in 20nm increments.380nm – 680nm in 20nm increments. At each new wavelength 100%T/0A needs to be reset At each new wavelength 100%T/0A needs to be reset

to 100%to 100% Negative absorbance's are recoded as 0.0. Negative absorbance's are recoded as 0.0.

Transmittances above 100% are recorded as 100%.Transmittances above 100% are recorded as 100%. Following the rules of graphing and using Excel, Following the rules of graphing and using Excel,

graph the graph the absorbanceabsorbance result of your experiment. result of your experiment.

Data Table 2: Absorbance and Transmitance of Colored dyes.

Wavelength in nm

DDyyee CCoolloorr

RedRed BlueBlue GreenGreen YellowYellow

AbsorbanceAbsorbance TransmittanceTransmittance AbsorbanceAbsorbance TransmittanceTransmittance AbsorbanceAbsorbance TransmittanceTransmittance AbsorbanceAbsorbance TransmittanceTransmittance

380                

400                

420                

440                

460                

480                

500                

520                

540                

560                

580                

600                

620                

640                

660                

680                

FLIP

Graph 1: Light Absorbance Vs. Light Wavelength of Colored Dyes

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

Wavelength (nm)

Ab

so

rba

nc

e (

A)

Red Dye

Blue Dye

Green Dye

Yellow Dye

N.D.H

Key

Graph 2: Light Transmittance Vs. Light Wavelength of Colored Dyes

0

10

20

30

40

50

60

70

80

90

100

110

120

340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

Wavelength (nm)

Tra

ns

mit

tan

ce

Red

Blue Dye

Green Dye

Yellow Dye

N.D.H

Key

QuestionsQuestions What is the What is the maxmax absorbance values and absorbance values and

corresponding wavelengths of each colored corresponding wavelengths of each colored dye?dye?

What is the What is the maxmax transmittance values and transmittance values and corresponding wavelengths of each colored corresponding wavelengths of each colored dye?dye?

Does each absorbance spectrum make sense Does each absorbance spectrum make sense for what is expected? Why or why not?for what is expected? Why or why not?

What is the practical value of knowing the What is the practical value of knowing the maxmax and and minmin for each solution? for each solution?

What is the relationship between transmittance What is the relationship between transmittance and absorbance? and absorbance? (look up on-line if you missed it last week (look up on-line if you missed it last week when we started the lab)when we started the lab)