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Food Analysis
Lecture 7 (2/12/2013)
Basic Principles of Spectroscopy
Qingrong Huang
Department of Food Science
http://foodsci.rutgers.edu/huang/Food_Analysis/FA2013.html
Spectroscopy-Basic Concepts
Spectroscopy: deals with the production, measurement, and interpretation
of spectra arising from interaction of electromagnetic radiation with matter.
Properties of Light (1)
Dual Nature: Particulate and Wavelike
Wave properties: wave’s frequency, wavelength, and amplitude
Frequency = speed c/wavelength
The amplitude of the wave (A) represents the magnitude of the electric
vector at the wave maxima.
The radiant power/intensity: are proportional to A2
Properties of Light (2)
Energy of Light
What are the energies of photons of different wavelengths,
blue light (480 nm) and near infrared (800 nm)? Which one
has a higher energy?
Interference
• Principle of superposition:
-Maximum constructive
interference
-Maximum destructive
interference
Energy States
• Quantum nature of matter
• Electronic, vibrational, and rotational
energy levels
Eatom=Eelectronic
Emolecule=Eelectronic+Evibrational+Erotational
Energy Level Transitions
• Absorption of radiation
- Ground state Excited state
e.g. UV, Vis, IR
• Emission of radiation
- Excited stateground state
e.g. fluorescence or
phosphorescence
Typical Absorption Spectrum
UV/Vis Spectroscopy
T = P / P0
Where T: Transmittance
P: Radiant power of beam exiting
the cell
P0: Radiant power of beam
incident on absorption cell
Beer’s Law
Absorbance (A) = log(P0/P)=-log T (1)
Beer’s Law:
A=abc (2)
A: absorbance
a: absorptivity
b: pathlength through solution (cm)
c: concentration of absorbing species
If M is used in concentration, the a is called molar absorptivity ()
A= bc
Deviation from Beer’s Law
• Beer’s law is only valid for low concentration, up to 10 mM;
• The intermolecular distances in a given solution will decrease,
eventually reach a point at which neighboring molecules
mutually affect the charge distribution of the other affect
• Chemical processes such as the reversible association-dissociation
of analyte molecules, or the ionization of a weak acid in an
unbuffered solvent.
• Instrumentation limitation-incident beam may be polychromatic .
Background Correction
-Processes other than analyte absorption
result in significant decrease in the power
of the incident beam;
- Reference cell is used to correct
these processes;
- Reference cell is often prepared by
adding distilled water to an absorption
cell;
- The reference cell is then placed in the
path of the light beam, and the power
of the radiation exiting the reference cell
is measured and taken as P0 for the sample cell.
UV Experiment
• Sample-holder: Quartz
• Choose appropriate wavelength
Calibration Curves
• Linear calibration curve;
• Nonlinear calibration