Beers Law log P o /P = ebc = A The derivation of this law assumes a) That the incident radiation is monochromatic. b) The absorption occurs in a volume of uniform cross-section. c) The absorbing substances behave independently of each other in the absorbing process.
Beers Law Limitations to Beers Law Real Limitations to Beers Law Apparent Chemical Deviations Apparent Instrumental Deviations with Polychromatic Radiation Instrumental Deviations in the Presence of Stray Radiation
The Effects of Instrumental Noise on Spectrophotometric Analyses Types of Noise Shot noise – This noise is generated by current flowing across a P-N junction and is a function of the bias current and the electron charge. The impulse of charge q depicted as a single shot event in the time domain can be Fourier transformed into the frequency domain as a wideband noise. Thermal noise – In any object with electrical resistance the thermal fluctuations of the electrons in the object will generate noise. White noise- The spectral density of thermal noise is flat with frequency. Burst noise – Occurs in semiconductor devices, especially monolithic amplifiers and manifests as a noise crackle.
The Effects of Instrumental Noise on Spectrophotometric Analyses Types of Noise Avalanche noise – Occurs in Zener diodes are reversed biased P-N junctions at breakdown. This noise is considerably larger than shot noise, so if zeners have to be used as part of a bias circuit then they need to be RF decoupled. Flicker noise – This noise occurs in almost all electronic devices at low frequencies. Flicker noise is usually defined by the corner frequency FL. Sources of Noise Case I: s T = k 1 Case II: s T = k 2 (T 2 + T) ½ Case III: s T = k 3 T
The Effects of Instrumental Noise on Spectrophotometric Analyses Effect of Slit Width on Absorbance Measurements
Instrumentation Instrument Components Sources Wavelengths selectors Sample containers Radiation detectors Signal processors and readout devices
Instrumentation Most common spectrophotometer: Spectronic 20. 1.On/Off switch and zero transmission adjustment knob 2.Wavelength selector/Readout 3.Sample chamber 4.Blank adjustment knob 5.Absorbance/Transmittanc e scale