Spectrophotometry Chapter 17, Harris. Spectrophotometry is the use of the measurement of the interaction of Electromagnetic radiation (EMR) with matter to quantize the concentration of an analyte. There are many different types of
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Spectrophotometry is the use of the measurement of the interaction of Electromagnetic radiation (EMR) with matter to quantize the concentration of an analyte. There are many different types of
spectrophotometers, based on the wavelength region of the EMR they measure. Examples are uv-vis, IR, microwave, x-ray, etc.
Electromagnetic Radiation travels at the speed of light (c), 2.997 x 108 m/s
Monochromatic light has a very small wavelength spread or narrow bandwidth; one
Polychromatic light has several wavelengths or in its beam.
Frequency (, Greek nu): Number of peaks that pass a given point per unit time.
Wavelength (, Greek lambda): Distance from one wave peak to the next.
= 1/, cm-1
Amplitude: Height measured from the center of the wave. The square of the amplitude gives intensity.
Plane polarized EMR consists of the sinusoidal electric field vectors in one plane with magnetic field vectors orthogonal to the electric field vectors. The above wave is traveling in the x direction in the above diagram.
Frequency and wavelength are related by
c = = 2.997 X 108m/s
Energy and frequency are related by the expression
E = h
where h (Planck’s constant = 6.626 X 10-34 J s)
Depending on the specific region of the EMR, various units are used to express the
symbolRegion of EMR
meter m radio
millimeter mm microwave
micrometer m infrared
nanometer nm visible/ultraviolet
Angstrom (10-10 m) Å X-ray
picometer pm -ray
Different regions of the EM spectrum produce different types of transitions in molecules. Note the inverse relationship between wavelength and energy, i.e., the shorter the the greater the Energy.
The absorption of EM radiation increases the energy of the molecule in one of the ways described on the proceeding slide. Emission results when the molecule loses energy.
The visible spectrum is a very narrow region of the EMR spectrum. Note that the shortest is at the violet end of the visible spectrum. The ultraviolet region is to its left. The longest at the red end of the visible spectrum, and the infrared region is to its right.
Fundamental to all of the spectroscopic methods is the quantization of energy.
Now, for a molecule,
Energy TOTAL =Eelectronic + Evibrational + Erotational
Where Eelectronic occursin the UV-Vis
Evibrational in the IR, and
Erotational in the microwave region of the EMR
Electronic transitions are accompanied by fine structure, i.e., vibrational and rotational transitions.
The fine structure of vibrational transitions (v0 – v4 ) on the electronic E 0 – E1. Not shown would be the fine structure of rotational transitions within each vibrational level.
There are two quantities that relate the change in
the intensity of EM before and after interaction
A = log10 (P0 / P ) = - log10 (P/ P0 )
Simple diagram of a single-beam spectrophotometer
Beer’s Law (or the Beer-Lambert Law) expresses the relationship between absorbance (A), the length of cell(b), and the concentration of the absorbing species (c). The proportionality constant is and is known the molar absorptivity. This relationship is given by the equation
A = b c
Plot of Transmittance vs interval number
Plot of –log T or Absorbance vs interval number
The absorption spectrum of a sunscreen lotion showing A as a function of of the EM radiation. This spectrum is in the ultraviolet (UV) region. UV-B is the shorter or higher energy and thus more harmful to skin.
Various typical cells (cuvets) used in spectrophotometry. The faces (part in the light path) may be silica, quartz, glass, or plastic, but must be transparent to the radiation of interest.
Obtaining minimum error in spectrophotometric measurements.
Analysis of analytes by spectrophotometry involves converting the analyte to a light-absorbing species. Pictured here is the chemistry for the analysis of the nitrite ion, NO2-.
A portion of the visible spectrum of the colored complex in the determination of nitrite.
Calibration curve for the analysis of nitrite. The of 543 nm and b (the pathlength of the cuvet) are kept constant