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Shahin*,Vemavarapu Satish kumar1
*Shadan Women’s College of Pharmacy. khairtabad, Hyderabad. A.P
1IPQC team member at GRANULES INDIA LIMITED, M.Pharmacy (pharmaceutics) Deevena College of Pharmacy.

A drug may be defined as a substance meant for diagnosis, cure, mitigation, prevention or treatment of diseases in human beings or animals or for alternating any structure or function of the body of human being or animals. Pharmaceutical chemistry is a science that makes use of general laws of chemistry to study drugs i.e. their preparation, chemical nature, composition, structure, influence on an organism and studies the physical and chemical properties of drugs, the methods of quality control and the conditions of their storage etc. The family of drugs may be broadly classified as.
1. Pharmacodynamic agents.
2. Chemotherapeutic agents.

It is necessary to find the content of each drug either in pure or single, combined dosage forms for purity testing. It is also essential to know the concentration of the drug and it’s metabolites in biological fluids after taking the dosage form for treatment.

The scope of developing and validating analytical methods is to ensure a suitable method for a particular analyte more specific, accurate and precise. The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation.

A survey of literature reveals that even though good analytical methods have been available for the drugs like Metronidazole, as a part of B.Pharm project we have selected the same drug for which method development and validation has been carried out and the results obtained were all within the limits. The method which we followed has good accuracy, sensitivity and it was best suited for the drug i.e.; Metronidazole.

For the method development and validation of Metronidazole we have selected UV-Visible double beam spectrophotometer and the best suited solvent for the determination of Metronidazole was found to be 0.1 N HCl.


1.2 Introduction to Ultraviolet (UV) Spectroscopy
Ultraviolet spectroscopy is one of several forms of spectroscopy that we will study this semester. Accordingly, it is important that you understand the capabilities and limitations of each of these forms of spectroscopy. The word spectroscopy implies that we will use the electromagnetic spectrum to gain information about organic molecules. The modifier ultraviolet means that the information will come from a specific region of the electromagnetic spectrum called the ultraviolet region.  The electromagnetic spectrum includes all radiation that travels at the speed of light c (3 x 1010 cm/sec). The electromagnetic spectrum includes radio waves, which have long wavelengths, x-rays, which have short wavelengths, and visible light, which has wavelengths between those of radio waves and x-rays.  All of these waves travel at the speed of light.  We normally describe these waves in terms of their energy.  Of the three kinds mentioned, x-rays are most energetic, visible light next, and radio waves least energetic.  Thus, the shorter the wavelength, the greater the energy of an electromagnetic wave.

Electromagnetic radiation (EMR) has a dual nature; it has the characteristics of both waves and particles. These particles are so tiny that they are indistinguishable from a wave. Both forms of EMR are important.  From the wave nature of the waves we get the wavelength or distance between two crests. The wavelength is related to the frequency, how many wavelengths pass a given point in a given time, by the velocity of the wave c.  From the particulate nature of EMR, we get the energy E of a given wave, which is proportional to its frequency. Plank’s constant h turns the proportionality into an equation. The mathematical relationships among these variables are shown below.

Visible light includes the rainbow colors red, orange, yellow, green, blue, indigo, and violet. A handy acronym for these colors is ROY G BIV, said like the name of a person called Roy. Note red is at the low-energy end of the visible spectrum and violet is at the high-energy end. These facts allow us to quickly understand the terms infrared and ultraviolet.  The prefix infra means below, and the prefix ultra means above. Thus, infrared radiation is outside the visible range and lies just below red on the energy scale. That is, infrared radiation is less energetic than visible light.  Ultraviolet radiation is outside the visible range and is just above violet on the energy scale. Thus, infrared literally means “below” red (in terms of energy), and ultraviolet means “above” violet (in terms of energy).  We know UV is more energetic than visible light or IR because UV light gives us sunburns.

We learned in general chemistry that visible yellow light is observed when sodium ions are heated in a Bunsen burner. The heat excites some ground-state electrons to higher energy levels, then when the electrons “fall” back to the ground state, they “emit” energy that corresponds to the energy difference between the energy states (orbitals) where the electrons are found. When this energy difference falls within the energy range of visible light, we can see it as a color.  In the case of sodium, we see yellow light. Note that it takes the same amount of energy to make the electrons jump from the lower to higher states as the amount of energy the electrons emit when they fall from higher to lower states. We generally add more energy than is absolutely necessary for the transition to ensure that the transition occurs. When we add energy to a system, we give it a positive sign (endoenergetic). When a system gives off energy, we give it a negative sign (exoenergetic).

Just as heat causes some of sodium’s electrons to move to higher energy states, ultraviolet radiation causes electrons in certain organic compounds to move from their ground state locations to orbitals of higher energy. The energy of the ultraviolet light acts just like the energy of the heat. In this case, the molecules are said to “absorb” ultraviolet radiation. A measurement of this phenomenon is called an absorption spectrum as opposed to an emission spectrum. When electrons move from lower to higher energy levels, we call the movement an electronic transition. Thus, the basic interaction between UV light and organic compounds is that UV light causes electronic transitions in certain organic structures.

The organic compound is dissolved in a solvent that does not absorb UV light.  Such a solvent is said to be transparent to UV light. The sample (compound in its solvent) is placed in a cuvette. A cuvette is a sample holder that has very precise dimensions. The cuvette is placed in an ultraviolet spectrophotometer. The instrument produces ultraviolet light over a range of wavelengths between 200 and 400 nanometers (nm), and the UV light is directed through the solution of the organic compound.

We will then make use of certain relationships that govern how much UV light can be absorbed by a sample. Namely, that the amount of light absorbed (A) is proportional to how many molecules or the concentration (c) of molecules that are absorbing light, and how far the UV light must pass through this concentration or the path length l.

A= εcl

This last proportionality says that the absorbance A is directly proportional to the concentration of the sample and to the path length (width of the cuvette). This is why the dimensions of the cuvette must be precise. The proportionality is useful for one given concentration or sample. A far more useful form of the relationships above is the Beer-Lambert equation, which makes the proportionality into an equation by addition of the proportionality constant.


Spectrophotometry is generally preferred especially by small-scale industries as the cost of the equipment is less and the maintenance problems are minimal. The method of analysis is based on measuring the absorption of a monochromatic light by colorless compounds in the near ultraviolet path of spectrum (200-380nm). The photometric methods of analysis are based on the Bouger-Lambert-Beer’s law, which establishes the absorbance of a solution is directly proportional to the concentration of the analyte. The fundamental principle of operation of spectrophotometer covering UV region consists in that light of definite interval of wavelength passes through a cell with solvent and falls on to the photoelectric cell that transforms the radiant energy into electrical energy measured by a galvanometer.



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