UV-VIS Molecular Spectroscopy Chapter 13 From 190 to 800 nm!Reflection and Scattering LossesThe solution ….Beer-Lambert Law ionconcentr atpat hl engthtyabsorptiviloglog00=====⎟⎟⎠⎞⎜⎜⎝⎛−=−===cbakcabcAPPTAPPPPTsolv entsoluti onPower of radiation after passing through the sample solution Power of radiation before passing through the sample solutionAbsorption VariablesBeer’s law and mixtures The total absorbance of a solution at a given wavelength is equal to the sum of the absorbances of the individual components present. • Each analyte present in the solution absorbs light! • The species do not interact. • The magnitude of the absorption depends on its ε• A total = A1+A2+…+An • A total = ε1bc1+ε2bc2+…+εnbcn • If ε1 = ε2 = εn then simultaneous determination is impossibleAssumptions of the absorption law • The incident beam is monochromatic. • The absorbers absorb independently of each other. • Incident radiation consists of parallel rays perpendicular to the surface of the absorbing medium. • Path length traversed is uniform over the cross section of the beam. • Absorbing medium is homogenous and does not scatter the radiation. • The incident beam is not large enough to cause saturation effects.Deviations from Beer’s Law • Real%limita)ons%• Chemical%factors%• Instrumental%factors%Deviations from Beer’s Law • Real%limita)ons%%%%% 1. Deviations in absorptivity coefficients at high concentrations (> 0.01 M) due to electrostatic interactions between molecules in close proximity. 2. Low absorber concentration but high electrolyte ion conc. 3. Change in refractive index of the medium when concentration changes. 4. Scattering of light due to particulates in the sample. 5. Fluorescence or phosphorescence of the sample. Successful at low analyte concentrations (0.01M)!Deviations from Beer’s Law • Chemical%factors%%%%% 1. Shifts in the position of a chemical or physical equilibrium involving the absorbing species. 2. Deviations can only be observed when concentrations are changed. 3. A common example of this behavior is found with acid/base indicators.Chemical Equilibria Consider the equilibrium: A + C AC If ε is different for A and AC then the absorbance depends on the equilibrium. [A] and [AC] depend on [A]total. ∴ A plot of absorbance vs. [A]total will not be linear.Chemical deviation of acid and base forms of phenolDeviations from Beer’s Law • Instrumental%factors%–%nega)ve%absorbance%errors%%Unsatisfactory performance of an instrument may be caused by fluctuations in the power-supply voltage, an unstable light source, or a non-linear response of the detector-amplifier system.• Polychroma)c%radia)on%• Stray%radia)on%Instrumental deviation with polychromatic radiation Ideal: measurements with monochromatic source radiation In practice, polychromatic sources + a grating or with a filter A nearly symmetric band of wavelengths surrounding the wavelength to be employed.Radiation of dichromatic beamDeviation of polychromatic radiation B%kcabcAPPPPATAPPPPTPSSSSS==⎟⎟⎠⎞⎜⎜⎝⎛++−=−=++==00logloglightstray Instrumental deviation with stray radiationDeviations from Beer’s Law • Mismatched%cells%%Path length and optical characteristics of cells are not equal.solutions…• Using carefully matched cells • Using a linear regression procedure • Using single-beam instrumentsInstrument Noise • Uncertainties • Noise associated with the instrument: - instrumental noise - Slit width - Scattered radiationConcentration uncertainty and transmittance The%uncertainty%in%concentra)on%as%a%func)on%of%the%uncertainty%in%transmi?ance%can%be%sta)s)cally%represented%as:%scc=0.434sTT logTAbsolute%standard%devia)on%of%transmi?ance%measurement.%Rela)ve%standard%devia)on%of%concentra)on%measurement.%Best%precision:%absorbance%value%%in%the%range%from%0.290.7%Instrument Noise Johnson/Thermal%noise%Shot%noise%Flicker%noise%Case I: thermal detector – dark current and amplifier noise (Johnson/thermal nose) Case II: photo-type detectors such as photomultiplier tube (shot noise) Case III: source – the slow drift in the radiant output of the source (source flicker noise)Instrument Noise Johnson/Thermal%noise%Shot%noise% Flicker%noise%RelaBve%concentraBon%uncertainBes%arising%from%various%categories%of%instrumental%noise.%• Slit%width%Instrument NoiseInstrument Noise • Sca?ered%radia)on%Useful%range%from%340%I780%nm,%be%careful%with%absorbance%below%380%nm.%Components of instrumentation: • Sources • Wavelength selector • Sample Containers • Radiation transducers • Signal processors and readout devicesComponents of instrumentation: • Sources: Agron, Xenon, Deuteriun, or Tungsten lamps • Monochromators: Quarts prisms and all gratings • Sample Containers: Quartz, Borosilicate, Plastic • Transducers: PhotomultipliersDeuterium (D2) Lamps 29%Discharge of excited deuterium gas. Continuum of UV light (190 – 400 nm). Ultraviolet source for UV-Vis and HPLC.Deuterium and hydrogen lamps (a continuum spectrum in the ultraviolet region) D2 + Ee → D2* → D’ + D’’ + hν Excited deuterium molecule with fixed quantized energy Dissociated into two deuterium atoms with different kinetic energies Ee = ED2* = ED’ + ED’’ + hv Ee is the electrical energy absorbed by the molecule. ED2* is the fixed quantized energy of D2*, ED’ and ED’’ are kinetic energy of the two deuterium atoms.31%Tungsten Filament Heated to 2870 K. Useful Range: 350 – 2500nmTungsten-halogen lamps (350-2500 nm) Blackbody type, temperature dependent Why add I2 in the lamps? W + I2 → WI2 • Weak intensity in UV range • Good intensity in visible range • Very low noise • Low drift • Glass envelop • Low limit – 350 nm.33%Source: Tungsten / Halogen….why? Iodine added. Reacts with gaseous W near the quartz wall to form WI2. W is redeposited on the filament. Smooth spectral curve, stable output and little UV radiation Gives longer lifetimes Allows higher temperatures (~3500 K).Intensity Spectrum of the Xenon Arc Lamp 34%• High intensity, smooth continuum from UV-Vis range • Small radiating arc