Spectrophotometry

single beam spectrometer, double beam spectrometer, beer lambert's law, uv spectrometer, visible spectrometer, ir spectrometer, absorption spectrum, mechanism and diveces

1. SPECTROPHOTOMETRY PRESENTED BY : Sushma P. R 2 nd M. Sc. Biotech Brindavan college PRESENTED TO : Mrs. Divya Dept. of Chemistry Brindavan College

2. INTRODUCTION Compounds absorbs light radiation of a specific wavelength. The light absorbed by the sample is directly proportional to the concentration of sample in the solution. As concentration increases , absorption increases exponentially .

3. SPECTRPHOTOMETRY A method in which the absorption or transmission properties of a material is quantitatively measured as a function of wavelength. The basic principle behind this method is that : “Each compound absorbs or transmits light over a certain range of wavelength”

4. A spectrophotometer is an instrument that measures the amount of photons absorbed by a sample after it is passed through its solution. With the spectrophotometer , the amount of a known chemical substance can be determined by measuring the absorbance.

5. Spectrophotometer can be classified into two different types : SINGLE BEAM SPECTRMETER : To measure the intensity of the incident light the sample must be removed so that the reference can be placed each time. This type of spectrometer is usually less expensive and less complicated.

6. DOUBLE BEAM SPECTOMETER: In this type, before it reaches the sample, the light source is split into two separate beams. From these one passes through the sample and second one is used for reference. This gives an advantage because the reference reading and sample reading can take place at the same time.

7. Based on the wavelength of light used it can be classified into : VISIBLE SPECTROMETER Uses visible range (400 – 700nm) of electromagnetic radiation spectrum. Visible spectrophotometers vary in accuracy. Plastic and glass  cuvettes  can  be used for visible light spectroscopy . UV SPECTOMETER Uses light over the UV range (180 - 400 nm). UV spectroscopy is used for fluids, and even solids. Cuvettes, only made of quartz, are used for placing the samples. IR SPECTROPHOTOMETER Uses light over infra red range (700 -15000) of electromagnetic radiation spectra.

8. DEVICE AND MECHANISM The spectrophotometer, in general, consists of two devices. They are the following : 1. SPECTROMETER : A device that produce, typically disperse and measure the light. 2. PHOTOMETER : Indicates the photoelectric dictator that measures the light.

9. The spectrometer consists of the following parts : 1 . Light source : it produce a desired range of wavelength of light. 2 . Collimator : transmits a straight beam of light. 3. Monochromator : split the light into its component wavelength. 4. Wavelength selector : transmits only the desired wavelength .

10. The photometer detects the light absorbed by the sample as the light from the slit is passed through the solution. And then it sends signal to the galvanometer or digital display.

11. BEER – LAMBERT LAW It states that the absorbance of light by a material in a solution is directly proportional to its concentration in that solution. A = ϵ lc Where, A – absorbance ϵ - molar absorptivity l – length of solution c – concentration

12. STANDARDIZATION GRAPH Standards (solutions of known concentration) of the compound of interest are made, treated, and their absorbances (ABS) and concentration values are used to create a Standardization Graph .

13. Absorption spectra : A spectrum of electromagnetic radiation transmitted through a substance, showing dark lines or bands due to absorption at specific wavelengths.

14. APPLICATIONS Concentration measurement Detection of impurities Chemical kinetics Detection of functional group Molecular weight determination

15. REFERENCES F undamentals of UV –Visible spectrophotometry , Tony Owen, 1996 UNIT : spectrophotometry : Clinical chemistry lab manual www.wikipedia.com www.colorado.edu www.chemwiki.com www.chemguide.co.uk www.slideshare.com

16. THANK YOU