By Sunil Bhardwaj

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Many compounds absorb ultraviolet or visible light and undergo electronic transition from low electronic energy levels to high electronic, energy levels. The instant re-emission of the absorbed energy is called Fluorescence. That instrument with the help of which we can measure the florescence phenomenon is known as Fluorimeter.

In this case beam of light travels in a single optical path is most commonly used light from mercury vapor lamp is allowed to pass through primary filter. It allows only UV light to pass through it and absorb the visible light then UV light is incident on the sample holder which is also known as Cuvette. Here the UV light is absorbed by the sample molecules. The light which is coming out from sample holder contains fluorescent light and may contain original UV light. The emergent light from sample holder is then analyzed at the 90o from incident in order to avoid in mixing of reflected or transmitted light with the emergent light. This emergent (fluorescent) light is then allowed to pass through secondary filter. This filter will absorb any of UV light. Now the emergent light is only fluorescent light and is then incident at photocathode Digital display which gives us the intensity of fluorescent light.

First we have to standardize (Calibrate) the instrument by taking blank (solvent) in the cuvette. With the blank, we adjust the dial reading to zero with the help of calibration knob. Now the instrument is standardized and then we perform the experiment with the sample solution. For sample solution it we known dials readings then we can have quantitative analysis by plotting a graph of F (intensity of fluoresce) Vs C.

Here the UV light is absorbed by the sample molecules. The light which is coming out from sample holder contains fluorescent light and may contain original UV light. The emergent light from sample holder is then analyzed at the 90o from incident in order to avoid in mixing of reflected or transmitted light with the emergent light. This emergent (fluorescent) light is then allowed to pass through secondary filter. This filter will absorb any of UV light. Now the emergent light is only fluorescent light and is then incident at photocathode Digital display which gives us the intensity of fluorescent light.

First we have to standardize (Calibrate) the instrument by taking blank (solvent) in the cuvette. With the blank, we adjust the dial reading to zero with the help of calibration knob. Now the instrument is standardized and then we perform the experiment with the sample solution. For sample solution it we known dials readings then we can have quantitative analysis by plotting a graph of F (intensity of fluoresce) Vs C.

Applications:

1. Quantitative analysis of organic, inorganic and biochemical compounds can be done with this phenomenon.

2. Boron can combine with Benzoin forming fluorescent complex. And then we can find out the amount of boron present.

3. Al, Be, Zn ions combine with 8-hydroxy quinoline forming complexes which are Fluorescent active and can be quantitatively estimated.

4. Uranium present in the sample can be determined by fluorimetry. The uranium sample is fused with sodium Fluoride to convert it into a melt containing uranium and sodium fluoride. This melt solidifies to a glass when cooled. The florescence intensity of this glass is measured in a fluorimeter. The uranium content can be determined to the order of 5 x 10-9 gm in one (1gm) of sample.

5. Vitamin B1 (Thiamine) is non Fluorescent but its oxidation product show intense blue colour fluorescence with the help of it we can estimate the amount of Vitamin B1 in food samples.

6. Many aromatic compounds e.g. steroids and enzymes have been analyzed by using Fluorescence method.

7. There are Fluorescent indicators which are used to detect, correctly the change in colour of coloured solutions in titrimetry. E.g. Eosin shows a green fluorescence in the pH of 3 - 4.

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