FUNDAMENTALS OF MODERN UV-VISIBLE SPECTROSCOPY

 Principles and applications of UV-Visible Spectroscopy
Basic Principles
  1. The electromagnetic spectrum
  2. Wavelength and frequency
  3. Origin of UV-visible spectra
  4. Transmittance and absorbance
  5. Derivative spectra
  • Obtaining derivative spectra
  • Applications
  • Signal-to-noise
  • Instrumental considerations 
Qualitative Analysis
  1. Identification-spectra and structure
  2. Confirmation of identity
  3. Color
  4. Other qualitative information
  • Protein and nucleic acid melting temperature 
  • Enzyme activity
  • Instrumental considerations
Quantitative Analysis
  1. Beer's law
  • Sample requirements
  • Multicomponent analysis
  • Principle of additivity
  • Simple simultaneous equations method
  • Least squares method
  • Other method
  • Sample requirements
  • Instrumental requirements
Indirect quantification
  • Chemical derivatization
  • Spectrophotometric titrations
  • Enzyme kinetic assays

This chapter outlines the basic theories and principles of UV-Visible spectroscopy. These provide valuable insight into the uses and limitations of this technique for chemical analysis. The primary applications of UV-visible spectroscopy are also briefly reviewed.

Basic Principles

The electromagnetic   Ultraviolet (UV) and visible radiation comprise only a small part of the 
                  spectrum    electromagnetic spectrum, which includes such other forms of radiation as                                             radio, infrared (IR), cosmic, and X rays.
Figure 1
The electromagnetic spectrum

The energy associated with electromagnetic radiation is defined by the following equation:

                                                                    E  =  hv

where E is energy (in joules), h is Planck's constant (6.62 x 10-34 Js), and v is frequency (in seconds).

Wavelength and  Electromagnetic radiation can be considered a combination of alternating electric 
          frequency  and magnetic field that travel through space with a wave motion. Because                                              radiation acts as a wave, it can be classified in term of either wavelength or                                          frequency, which are related by the following equation:

                                                                   V   =   C / Î»
                                    Where V  is frequency (in seconds), c is the speed of light (3 x 10-8 ms-1), and Î»                                    is wavelength (in meters). In UV-visible spectroscopy, wavelength usually is                                        expressed in nanometers (1 nm  =  10-9 m).

                              It follows from the above equations that radiations with shorter wavelength has                                    higher energy. In UV-visible spectroscopy, the low-wavelength UV light has the                                    highest energy. In some cases, this energy is sufficient to cause unwanted                                             photochemical reactions when measuring sample spectra (remember, it is the UV                                 component of light that causes sunburn).

Origin of             When radiation interacts with matter, a number of processes can occur, including     
UV-visible           reflection, scattering absorbance, fluorescence/phosphorescence (absorption and 
spectra                reemission), and photochemical reaction (absorbance and bond breaking). In                                        general, when measuring UV-visible spectra, we want only absorbance to occur.

                            Because light is a form of energy, absorption of light by matter causes the energy                                  content of the molecules (or atoms) to increase. The total potential energy of a                                      molecule generally is represented as the sum of its electronic,
                            
Etotal  =  Eelectronic + Evibrational + Erotational

                           The amount of energy a molecule possesses in each form is not a continuum but a                                 series of discrete levels or states. The differences in energy among the different                                     states are in the order:
                                                        Eelectronic + Evibrational + Erotational

                           In some molecules and atoms, photons of UV and visible light have enough energy                             to cause transitions between the different electronic energy levels. The wavelength                             of light absorbed is that having the energy required to move an electronic form a                                 lower energy level to a higher energy level. Figure 2 shown an example of                                           electronic transition in formaldehyde and the wavelengths of light that cause them.

Figure 2

Electronic transitions in formaldehyde

                             These transitions should result in very narrow absorbance bands at wavelengths                                   highly characteristic of the difference in energy levels of the absorbing species.                                     This is the true line for atoms, as depicted in Figure 3.

Figure 3
Electronic transitions and spectra of atoms

                         However, for molecules, vibrational and rotational energy levels are superimposed                               on the electronic energy levels. Because many transitions with different energies can                           occur, the bands are broadened (See figure 4). The broadening is even greater in                                   solutions owing to solvent-solute interactions.  
Figure 4
Electronic transitions and UV-visible spectra in molecules 

Transmittance and When light passes through or is reflected from a sample, the amount of light 
             absorbance absorbed is the difference between the incident radiation (Io) and the transmitted
                                          radiation (I). The incident radiation (Io) and the transmitted radiation (I). The                                                    amount of light absorbed is expressed as either transmittance or absorbance.                                                      Transmittance usually is given in terms of a fraction of 1 or as a percentage and is                                              defined as follows:
                                                                       T  = I / Io   or % T  =(I/Io)  X  100
                                              Absorbance is defined as follows: 
                                                                               A  = -log T
                                  
                                              For most applications, absorbance values are used since the relationship between                                             absorbance and both concentration and path length normally is linear.