BMI_Lab 4 Photometry

BMI_Lab 4 Photometry - Biomed I, Lab 4 Photometry and...

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Biomed I, Lab 4 Photometry and Writing Lab Reports
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Objectives To demonstrate the principles of spectrophotometry To determine The molar extinction coefficient of a weak acid, para-nitrophenol The pKa for the dissociation of p-nitrophenol To learn how to write a GOOD lab report
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Introduction As light passes through a substance, one or more of the following phenomena can occur: reflection, refraction, absorption , interference, diffraction, fluorescence and ionization Spectrophotometric methods are based on absorption. Absorbance is the log 10 of % total light (100%) / % of light transmitted. Absorbance (A) = 2 - log %T If 10% of violet light is transmitted than 90% violet light is absorbed. A = Log 100/10 = 2-1 = 1 All atoms and molecules are capable of absorbing various types of energy. The amount of energy absorbed depends on the structure of the molecules and the number of molecules present.
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Visible Light Today’s lab will focus on the visible spectrum of light. Visible light can be broken down into the colors we observe with the human eye. A compound will absorb light at one wavelength, but transmit light at another wavelength. The color of a compound that we see is due to the transmitting wavelengths of the compound. Type Wavelength (λ) absorbed Far UV 100-200nm UV 200-400nm Visible 400-750nm Near Infrared 750-2000nm Infrared 2000-25,000nm
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Visible Light The molecules that make up chlorophyll absorb red light, but transmit green light. That is why we observe most vegetation to be green in color. Wavelength Color absorbed Apparent (transmitted) color 400-450nm Violet Yellow ** 450-480 Blue Orange 480-490 Green-blue Red-orange 490-500 Blue-green Red 560-575 Yellow-green Violet 575-590 Yellow Blue* 590-625 Orange-red Green-blue** 625-750 Red Green Jones and Atkins, Chemistry: Molecules, Matter and Change , 2008. 947
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Spectrophotometer Data values recorded for analysis Sample Light source 100% % Transmitted % Absorbed = 100 -%Transmitted Absorbance is different from % Absorbed !
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Beer-Lambert Law A=εcl A=absorbance ε= absorption coefficient (when c is M) c= concentration l= length of light path A=abc A=absorbance a=absorption coefficient (when c is g/L) b= length of light path c=concentration Beer-Lambert law states that the absorption of light energy at any wavelength is dependent on the concentration of the absorbing compound (molar or gms/L) and the path length of light ( 1 cm) through the absorbing compound.
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BMI_Lab 4 Photometry - Biomed I, Lab 4 Photometry and...

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