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31 Cards in this Set
- Front
- Back
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Absorption Experiments
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• An incident beam impinges upon a sample and the light passes through it is monitored for its decrease in power due to absorption by the sample.
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Measurement of Transmittance and
Absorbance |
• The absorption process is measured in one of two ways:
Transmittance: T = P/P0 Absorbance: A = -log10 T = log10 (P0/P) • In order to account for the numerous losses other than absorption → measure P0 with a cell containing everything (solvent) except the analyte of interest. Thus: Transmittance: T=Psample/Psolvent Absorbance: A=-log10 T=log10(Psolvent/Psample) |
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Beer´s Law
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Radiation of initial radiant power P0 is attenuated to transmitted power P by a solution containing c moles per liter of absorbing solution with a path length of b centimeters.
For application see lecture! • Assumes: - monochromatic radiation - system not saturated in light - absorbers (=analytes) behave independently and are distributed homogenously |
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Limitations to Beer´s Law
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Is the absorbance really linear with respect to the
variables? - Path length (b): Essentially this is always found to be linear - Concentration (c): Nonlinearity can arise from: > intermolecular interactions > shifting chemical equilibria - Molar absorptivity (E thingy): Non-linearity can arise from: the solution´s index of refraction. • The instrument itself can skew the behaviour away from linearity in a number of ways. |
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Intermolecular Interactions
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• Beer´s Law is strictly a limiting law for dilute solutions
• At high concentrations (> 0.01 M) the average distance between analyte molecules is small enough that the charge distributions around one affects that around another • Some organic molecules show deviations even at 10-6 M concentrations • Need to be aware of concentration linearity |
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Chemical Equilibrium
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• When a substance is involved in a chemical reaction, the extent of that reaction is concentration dependent
• If the alternate form of the molecule has a different absorption spectrum, there will be non-linear distortion away from Beer´s Law |
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Index of Refraction
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• The molar absorptivity depends upon the index of
refraction of the solution, n. • In some cases, the index of refraction can change with concentration. • When a concentration change causes a significant change in n, then this can cause a deviation in Beer´s Law away from linearity. • In practice, this correction is never very large and is rarely significant at concentrations below 0.01 M |
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Instrument (due to polychromatic
radiation) |
• Beer´s Law is strictly applicable only for monochromatic radiation
• Analyte will have a different absorptivity at each wavelength. If variation is large, then the non-linearity can be observed Remedy: - Choose a spectral range where the absorptivity changes slowly with wavelength - Select an excitation radiation bandwidth that is <0.1 of the analyte´s spectral FWHM |
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Instrument (due to stray radiation)
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• Stray radiation (stray light) radiation from the instrument that is outside the wavelength band chosen for determination
• Stray radiation is the result of scattering and reflection of instrument´s components • Stray radiation´s wavelength differs greatly from that of the principal radiation and it may not have passed the sample |
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Instrument (in the presence of stray
radiation) |
• Increased light reaching detector
• Contributes most when P << P0 • Causes negative deviation at high concentration (High Abs.) • Decreasing bandpass lowers stray light and increases linearity |
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Effect of Instrumental Noise on Spectrophotometric Analyses
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• Experimental noise leads to an uncertainty in determining absorbance. Partial differentiation leads to a relationship between error in T and error in A.
• This error in absorbance is connected to an error in concentration |
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Effect of Instrumental Noise on Spectrophotometric Analyses
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• Different noise sources can contribute differently to T error
• Three general cases have been identified: - T error is constant - T error varies as (T2 + T)1/2 - T error varies as T |
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T error is constant
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• Arises in inexpensive spectrometers which suffer from limited readout resolution
• Experiments where source intensity is low or detector sensitivity is low will be limited by dark current and amplifier noise |
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T error varies as (T2 + T)1/2
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• High quality UV/Vis spectrometers are susceptible to this case
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T error varies as T
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• High quality UV/Vis and IR spectrometers will be subject to cell positioning errors
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Choose Absorbance Range Carefully
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• The take-home message is that just because a machine gives you a number, doesn´t mean you have to believe it
• When making spectrometric measurements, you need to adjust the concentration of the sample so that the absorbance range covered falls in the region which will minimize the instrumental error • Absorbance range between A = 0.1 and A = 1 should give reliable results with almost all instruments |
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Slit Width Affects Absorbance Measurements
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• If a significant variation in absorptivity occurs over the spectral bandwidth admitted by the slot, a non-linear variation (non-Beer´s Law) with concentration will be observed
• This arises because the spectrometer measures the average transmissivity over the spectral bandwidth, but transmissivity and concentration are not linearly related |
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Slit Width Affects Absorbance Measurements
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• Keep slit width large to increase S/N ratio but must keep it small enough to maintain a linear relationship with concentration changes
• This effect is minimized if the absorptivity changes slowly with wavelength • Select a wavelength near a peak maximum. Use a slit width to provide a bandwidth that is about 1/10 of the spectral feature´s width |
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Other Problems
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• Stray Light: it is a problem when working at the limits of a spectrometer's range
• Cells and Solvents: everything besides the analyte should be as transparent as possible • Sample Preparation: if two samples are prepared so that one carries along a greater concentration of insoluble particulates, then additional scattering will lead to an apparent greater absorption |
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Instrument Components (Typical UV-VIS)
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Source -> Wavelength Disperser -> Sample (Blank) -> Detector -> Readout
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UV-Vis Sources
• Typically continuum sources: UV Range |
UV Range: Hydrogen and Deuterium arc lamps
– Electrical excitation at low pressure, low voltage – Forms molecular excited state that undergoes dissociation and photoemission |
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UV-Vis Sources
• Typically continuum sources: Visible Range |
Visible Range: Tungsten Filament Lamps
– Resistively heated wire – Emits from ~350-3000 nm – ~15% of radiation falls in the visible @3000K |
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UV-Vis Sources
• Typically continuum sources: Emmison Spanning UV-VIS (Xe arc lamps) |
Emmison Spanning UV-VIS: Xe arc lamps
– High pressure Xenon gas – Emit from ~200-1000 nm – Generate significant heat, need external cooling |
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Line Sources in the UV and Vis
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• Hollow Cathode Lamp
– Cathode is coated with atom of interest – Tube is filled with Ar or Ne – High voltage ionizes gas, charged ions are accelerated toward electrodes - Produces sputtering of atoms (ground and excited) - Excited atoms emit light at atomic lines |
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Wavelength Dispersion and Selection
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• Most instruments use a monochromator to separate light form the source into discrete wavelength segments
• Components: – Entrance slit – Collimating/focusing device - mirror or lens, nonideal – Dispersing device -filter, grating or prism – Collimating/focusing device - mirror or lens – Exit slit |
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Optical Elements and Wavelength
Dispersion: Optical components are not ideal! - Lenses and Mirrors |
Lenses: Chromatic aberration because refractive index changes with wavelength
- focal length changes with wavelength Mirrors: Reflective losses. Lenses and inefficiencies in mirrors contribute to ~4% loss per element. |
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Optical Elements and Wavelength
Dispersion: Optical components are not ideal! - Dispersive Elements: Filters |
Construction determines what fixed range of wavelength will be allowed to pass.
Interference Filters: - "sandwich" containing reflective material and dielectric layer - only wavelength that result in in-phase reflections: depends on thickness and dielectric Absorption Filters: - "colored" plates - light that is not absorbed by the filters is transmitted - often used in combination |
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Wavelength Dispersion: Gratings
Wavelength Dispersion: Prisms |
see lecture
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Sample Considerations
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• Several possible fates for photon
– Reflection – Scattering – Absorption • Choose cell and sample composition carefully • “Match” |
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Detectors for UV-Vis
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• Photon Transducers: Convert photon energy to electrical signal (current, voltage, etc.)
• Detectors based on photoelectric effect: Phototubes, Photomultiplier tubes • Phototube: – Incident photon causes release of an electron – Photocurrent proportional to Plight |
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A phototube
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The photocurrent induced by the radiation causes a voltage drop across R, which appears as frequencyo at the output of the current-to-voltage converter. This voltage may be displayed on a meter or acquired by data-acquisition system
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