Chapter 13 An Introduction to Ultraviolet- Visible Molecular Absorption Spectrometry

input power P o b concentration C (Molarity) pathlength (cm) output power P

P o = power of incident radiation P = power of output radiation b = sample thickness n = number of absorbing particles a = “capture cross-section” (cm 2 /molecule) photon of light (h ) h probability of absorption = probability of absorption also =

Sometimes absorbance A is plotted on the y-axis, or sometimes  is plotted instead (  = A/bc). The molar absorptivity (  ) is wavelength dependent

1. Concentration Effect In the derivation of Beer's Law, dilute solutions were used so there would be no interactions between particles. At higher concentrations - especially if charged electrolytes are present - there are electrostatic interactions that distort the electron cloud in a molecule and therefore its absorbance. The result is a negative deviation from Beer's Law. Beer's Law holds for C < 0.01 M photon of light (h ) h h → A → → K+K+ K+K+

2. Chemical Deviations Commonly occur when the analyte dissociates. For example, the acid- base indicator HIn - HIn = H + + In - color 1 color 2 A =  HIn bC HIn +  In- bC In-

3. Deviations Due to Polychromatic Radiation Consider light composed of wavelengths 1 and 2 passing through a sample. Using Beer's Law - Rearranging and solving for P( 1 ) and P( 2 ) The power entering the solution will be P o ( 1 ) + P o ( 2 ), and the power exiting will be P( 1 ) + P( 2 ). So the total measured absorbance will be -

Case I:  1   2 (Band A) The bandpass of the spectrometer is passing light where the absorbance spectrum is flat. Case II:  1   2 (Band B) The bandpass is set in a region of rapidly changing absorbance - negative deviations increase as the difference between the two molar absorptivities (see figure at left).

4. Deviations Due to Stray Light Usually there is a small amount of stray light exiting the spectrometer due to higher diffraction orders, reflections, etc. The measured absorbance is then given by - Appreciable amounts of stray light can cause negative deviations as well - Absorbance appears to be lower because the extra stray light makes it seem like there’s more transmittance through the sample.

Light Sources - need a continuous, stable source of radiation. UV - D 2 or H 2 LampsVisible - Tungsten Filament Lamps nm arc discharge in a few Torr of gas quartz D 2 slightly brighter than H nm blackbody radiation K long lifetime

increasing cost & quality single-beam uses a filter instead of a monochromator, or a manual scan monochromator photodiode detector large D -1 and low resolution double-beam motorized scanning monochromator PMT detector small D -1 and high resolution

PoPo P A = -logT → 1.00 solvent band spectrum obtained by reference cuvette = solvent and other background components spectrum obtained by sample cuvette with an absorbance feature Transmittance - ratios out features common to the sample and reference (T = 1.00) Absorbance Spectrum PoPo PPoPo PPoPo PPoPo PPoPo PPoPo P Detector Signal time (scan)  P PoPo

Double-Dispersing Instruments These instruments pass the source light through two monochromators in a row, eliminating much of the stray light in the system. Consequently detection limits are lower with these instruments.

Multichannel Instruments (Diode-Array Spectrophotometers) Resolution: no need to scan – entire spectrum obtained at once