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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 chapter 17 harris

SpectrophotometryChapter 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

spectrophotometers, based on the wavelength region of the EMR they measure. Examples are uv-vis, IR, microwave, x-ray, etc.


Electromagnetic radiation

Electromagnetic Radiation

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.

Electromagnetic radiation1

Electromagnetic Radiation

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.

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

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)

Spectrophotometry chapter 17 harris

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

Electromagnetic radiation2

Electromagnetic Radiation

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

  • Fundamental to all of the spectroscopic methods is the quantization of energy.

  • Let’s consider an atom first.

  • Atoms exist in discrete (or quantized) potential energy (PE)

  • levels.

  • The PE depends on the electronic configuration of the atom

  • Transitions of outer shell electrons between definite levels

  • occur at definite “sized” energy according to E = h 

    • if E absorbed, the electron is excited to a higher level.

    • if E emitted, the electron falls to a lower level.

  • Thus, each transition occurs with a specific energy, or since

  • E = h = hc/, each transition gives rise to a specific spectral

  • line, either absorption or emission depending on the process.

Stopped monday jan 19 2004

Stopped, Monday, Jan 19, 2004

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

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.

Transmittance and absorbance

Transmittance and Absorbance

There are two quantities that relate the change in

the intensity of EM before and after interaction

with matter.

  • Transmittance = P / P0, and

  • Absorbance, defined as

    A = log10 (P0 / P ) = - log10 (P/ P0 )

Spectrophotometry chapter 17 harris

Simple diagram of a single-beam spectrophotometer

Beer s law

Beer’s Law

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

Spectrophotometry chapter 17 harris

Plot of Transmittance vs interval number

Spectrophotometry chapter 17 harris

Plot of –log T or Absorbance vs interval number

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

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.

Spectrophotometry chapter 17 harris

Obtaining minimum error in spectrophotometric measurements.

Spectrophotometry chapter 17 harris

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-.

Spectrophotometry chapter 17 harris

A portion of the visible spectrum of the colored complex in the determination of nitrite.

Spectrophotometry chapter 17 harris

Calibration curve for the analysis of nitrite. The  of 543 nm and b (the pathlength of the cuvet) are kept constant

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