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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