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CHMB16H3 (16)
Chapter 17

CHMB16 Chapter 17

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Kagan Kerman

Chapter 17: Fundamentals of Spectrophotometry Properties of Light • Spectrophotometry: any technique that uses light to measure chemical concentrations. • Colorimetry: a procedure based on absorption of visible light. • Wavelength (λ): the crest-to-crest distance between waves. • Frequency (v): the number of complete oscillations that the wave makes each second. • Relation between frequency and wavelength: λv = c, where c is the speed of light at 2.998 x 10 m/s. o In a medium other than a vacuum, the speed of light is c/n, where n is the refractive index. o For visible wavelengths in most substances, n > 1, so visible light travels more slowly through matter than through vacuum. • Photons: particles of light with regard to energy. • Relation between energy and frequency: E = hv, where h is Planck’s constant of 6.626 x 10 -34Js. o E = hc/v = hcṽ, where ṽ is 1/ λ or the wavenumber. o Energy is inversely proportional to wavelength and directly proportional to wavenumber. o Electromagnetic Spectrum: the visible spectrum spans the wavelength range of 380-780 nm. Absorption of Light • Excited State: when a molecule absorbs a photon, the energy of the molecule increases and the molecule is promoted to an excited state. • Ground State: if a molecule emits a photon, the energy of the molecule is lowered and this is the lowest energy state of a molecule. • Irradiance/Intensity/Radiant (P): the energy per unit time per unit area in the light 2 beam (W/m ). • Monochromator: a wavelength selector in which light passes through and it selects one wavelength. • Transmittance (T): the fraction of original light that passes through the sample (T = P/Po) and has a range of 0 to 1. • Percent Transmittance: 100T and ranges between 0 to 100%. • Absorbance: directly proportional to the concentration of the light-absorbing species in the sample. o A = log(P oP) = -logT -1 - o Beer’s Law: A = Ԑbc, where Ԑ is the molar absorptivity and has units of M cm . o The greater the molar absorptivity, the greater the absorbance. o Absorption Spectrum: a graph showing how A or Ԑ varies with wavelength. o Chromophore: the part of a molecule responsible for light absorption. • Any substance that absorbs visible light appears colored when white light is transmitted through it or reflected from it; the substance absorbs certain wavelengths of the white light and our eyes detect the wavelengths that are not absorbed. • When Beer’s Law Fails o At very high concentration, the solute becomes the solvent so the properties of a molecule are not exactly the same in different solvents. o Nonabsorbing solutes in a solution can also interact with the absorbing species and alter the absorptivity. o Beer’s Law works for monochromatic radiation passing through a dilute solution in which the absorbing species is not participating in a concentration- dependent equilibrium. Measuring Absorbance • Cuvet: where liquid samples are usually contained and has flat, fused-silica faces. • Do not touch the clear faces of a cuvet because fingerprints scatter and absorb light. • Gases are more dilute than liquids and require cells with longer pathlengths (10cm to many meters). • For spectrophotometric analysis, we choose the wavelength of maximum absorbance because: o The sensitivity of the analysis is greatest at maximum absorbance; we get the maximum response for a given concentration of analyte. o The curve is relatively flat at the maximum, so there is little variation in absorbance if the monochromator drifts a little or if the width of the transmitted band changes slightly. • Modern spectrophotometers are most precise at intermediate levels of absorbance, around 0.3 to 2. Beer’s Law in Chemical Analysis • Proteins are normally assayed in the ultraviolet region at 280 nm because aromatic groups present in virtually every protein have an absorbance maximum at 280 nm. • Reagent Blank: contains all reagents but with analyte replaced by distilled water. • Subtract the blank absorbance from the absorbance of samples and standards before doing any calculations. • Supernate: the liquid layer above the solid that collects at the bottom of a tube during centrifugation. Spectrophotometric Titrations • Spectrophotometric Titration: we monitor changes in absorbance during a titration to tell when the equivalence point has
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