You've got a new job as a scientist in a lab, and now you learn the latest secrets of spectroscopy. This article will give you the secrets to spectroscopy

                                 SPECTROSCOPY

    The branch of science concerned with the investigation & measurement of spectra produced when matter interact with or emits electromagnetic radiation.

PRINCIPLE:

      All spectroscopic techniques is to shine a beam of electromagnetic radiation onto a sample & observe how it respond to such a stimulus. The response is usually recorded as a function of radiation wavelength.

TYPES OF SPECTROSCOPY 

    1.Fluorescence spectroscopy

    2.X - ray spectroscopy

    3.Flame spectroscopy 

•  Atomic spectroscopy 

• Atomic absorption spectroscopy

•  Atomic fluorescence spectroscopy

   4.Plasma  emission spectroscopy 

   5. Spark or arc spectroscopy 

   6.UV/ VIS spectroscopy 

   7.IR spectroscopy 

   8.Raman spectroscopy 

   9.NMR spectroscopy

  10.Photo thermal spectroscopy 

  11.Thermal infra red spectroscopy 

  12.Mass spectroscopy

     UV absorption spectroscopy deals with the measurement of energy absorbed when electron are promoted to higher energy levels.

   The UV spectrum is simply a plot of wavelength of light absorbed versus the absorption intensity ( absorbance or transmittance ) & is conveniently recorded by plotting molar absorptivity( ϵ ) against wavelength (λ).

    UV spectroscopy requires "electromagnetic radiation of higher energy"

      Basic principle of spectroscopy  is Beer's Lambert law.

Beer's law :

     Beer law states "absorbance" is proportional to the concentration of material sample.

lambert law 

Lambert law states that 'absorbance' of a material is directly proportional to its thickness ( path length ).

Beer's Lambert law :

A - absorbance

 - molar absorptivity 

b - length of light path

C - concentration 

INSTRUMENT ASSOCIATED WITH UV VISIBLE SPECTROSCOPY

• Source of light 
• Monochromator
• Sample solution in cuvette
• Photo detector 
• Readout device

SOURCE OF LIGHT 

•  UV & visible radiation source is tungsten lamp
•  UV radiation source is deuterium or hydrogen lamp
•  Range of wavelength 200 - 400nm 

MONOCHROMATOR 

      It is device that breaks the polychromatic radiation into component wavelength .

 Monochromator unit consist of 

1. Entrance slit - Narrow beam of radiation from source 
2. Collimating mirror - ( polished surface ) collimates the light
3. Diffraction grating or prism - Disperses the light into specific wavelength .
4. Foassing mirror - Capture the dispersed light & sharpens the same to the sample via exist slit
5. Exist slit - Allows the corrected wavelength of light to the sample.

SAMPLE SOLUTION IN CUVETTE 

• Liquid sample is usually contained in a cell called cuvette
• Fingerprints & droplets of water disrupt light rays during measurement 
• Cuvette from Quartz can be used in UV as well as in visible spectroscopy 
• Cuvette from Glass is suitable for visible but not for UV radiation.       

PHOTO DETECTOR 

         A photo detector is semiconductor device which converts light energy to electrical energy .

       It consist of a simple P-N junction diode & is designed to work in reverse biased condition.

     The photons approaching the diode are absorbed by the photodiode & current is generated.

READOUT DEVICE 

    Digital screen to record an UV spectroscopy with absorbance against the wavelength.

CHOICE OF SOLVENTS & SOLVENT EFFECT 

        

Effect of solvent:

     A solvent is a liquid that dissolves another solid,liquid or gaseous solute, resulting in a solution at specified temperature 

   Solvent can classified into 2 categories:

• Polar 
• Non polar                      

      These appeared changes in the spectrum are exclusively due to various characteristic feature namely

1. Nature of solvent
2. Nature of absorption bond 
3. Nature of solute          

EFFECT OF SOLVENT 

       The solvent exerts a profound influence on the "quality" &  shape of spectrum.

    A drug may absorb a maximum radiation energy at particular wavelength in one solvent but shall absorb partially at the same wavelength in another solvent.

  eg : Acetone in n-hexane λ_{max  at 279nm}

         _{Acetone in water} λ_{max  at 264.5nm}

_{NATURE OF SOLVENT} 

    _{Most commonly used solvent is 95% ethanol.}

  _{It is best solvent as} 

• _{It is cheap} 
• _{Has good dissolving power}
• _{Doesn't absorbs radiation above 210nm.}

  In choosing a solvent, consideration must be given not only to its transparency, but also to its possible effect on absorbing system 

   There are other solvent which are transparency, but also to its possible effect on absorbing system.

   There are other solvent which are transparent above 210nm.

Benzene ,chloroform can't be used because they absorb in the range of 240-280nm.

EX :Common solvent used in recording UV-spectra.

            Solvent                                                 wavelength(nm)

1. water                                                            205
2. Methanol                                                     210
3. ethanol                                                         210
4. ether                                                             210
5. cyclohexane                                                 210
6. dichloroethane                                             220

CHOICE OF SOLVENT:

        A suitable solvent for UV spectroscopy should meet the following requirement:

      It shouldn't itself absorb itself absorb radiation in the region under investigation

      It should be less polar so that it has minimum interaction with the solute molecule.

POLARITY:

      It play an important role in the position & intensity of absorption maximum of a particular chromophore.

1. In case of non-polar solvent eg :Iodine solution (purple color) the absorption maxima occur at almost the same wavelength as in iodine vapor(5180A)
2. Incase of polar ,a brownish color is obtained instead of purple color is obtained instead of purple ,because the absorption occurs at shorter wavelength .
3. color change polarization of I2 by the electric field of solvent dipoles.     

 PURITY OF SOLVENT:

            Purified & certified solvent for spectroscopy should be used as we are looking for the 'smooth' absorbance curve of solvent.

RESOLUTION & INTERPRETATION OF SPECTRUM PROBLEM :

          These are resulted when solvent is used for measurement of near/below its UV cutoff that is the approximate wavelength below which they can't be used because of absorption.

DIPOLE MOMENT:

       Absorption band of many substance are relatively sharpes & may also exhibit fine structure when measured in solvent of low dipole moment.

EFFECT OF CHROMOPHORE ON UV-VISIBLE SPECTROSCOPY:

       The term chromophore was previously used to denote a functional group,the presence of which gives color to the compound.

       Chromophore is defined as any group which exhibits absorption of electromagnetic radiation in the visible or UV region.

      EX: ethylene, carbonyl acids ,ester, nitrogen

     

Chromophore in which the group is having π electron undergo π→π*

transition.  

     Ex: ethylene ,acetylenes

    Chromophore in which the group both π electron & n electron undergoes 2 types of transitions

       π→π* & n→π* 

ex: carbonyl ,nitryl

TYPES OF CHROMOPHORE

• Conjugated  π bond system

• Metal complexes.