spectrophotometer

Spectrophotometer
Working principle and Application

1. SPECTROPHO T OMET ER M.Sc Zoology (1 st Semester) Roll . No . 2313177450003 Saraswati Vidya Mandir Mahila PG College, Gorakhpur Aastha Singh

2. Index Meaning Spectrophotometer Types of Spectrophotometer Principle of Spectrophotometer Lembert’s Law Beer’s Law Working of Spectrophototmeter Application of Spectrophotometer Reference

3. Meaning of spectrophotometer Colorimetry Spectrum Photometry Spectrophotometer

4. S pe c t r oph o t omet e r Spectrophotometer is an instrument that measures the amount of light that passes through (is transmitted through) a sample. Uses a type of light to detect molecules in a solution . Light is a type of energy , and the energy is reported a s wavelengths in nanometers (nm).

5. Sample Holder D i sp l ay Knobs or buttons used to calibrate the spec to measure the designated molecule W a ve l e n gt h Selection

6. Two different types of s pectrophotometer Ultraviolet (UV ) Spectrophotometers Uses ultraviolet light of wave lengths from 200 nm to 350 nm. Visible ( V L S ) Light Spectrum Spectrophotometers Uses visible light (white light) of wave lengths from 350 nm to 700 nm.

7. The V isible L ight S pectrum R O Y G B I V

8. Working principle of s pectrophotometer When a beam of monochromatic light passed through a solution it may transmitted as a such or some of may be absorbed. Proportional the transmitted light can be represented by intensity of the incident radiation. T=I/Io Absorbance(A) of light through a solution in inversely proportional to log10 of %T.

9. A = Log (1/T) = Log (I/Io) Where I = Intensity of transmitted light Io = Intensity of incident light The quantitative determination of compounds by spectrometeric technique is based on two law. 1. Lamberts Law 2. Beers Law

10. Lambert’s Law Its state that light absorbed by solution is directly proportional to length of the light through the solution. Thus, A = Log (Io/I) = E.C.l Where, A = Absorbance E = Molar absorptivity coefficient l = Path length of the sample C = Concentration of solution

11. Calculating the absorbance of a sample using the equation depends on the absorbance is directly proportional to the path length of the sample.

12. Beer’s Law Its state that amount of light absorbed is directly proportional to concentration of absorbing solute in the solution. Thus, A = Log (Io/I) = E.C.l Where, C = Concentration of solution mole per liter E = Molar absorptivity coefficient l = Path length of the sample

13. Calculating the absorbance of a sample using the equation depends on the absorbance is directly proportional to the concentration of the sample.

14. How a spectrophotometer works

15. How a spectrophotometer works 1. White light hits the prism or grating, it is split into the colors of the rainbow (Visible Spectrum). 2. The wavelength knob rotates the prism/grating, directing different color of light toward the sample. 3. The wavelength of light produced by the tungsten lamp range from about 350 nm (Violet light) to 700 nm (red light).

16. 3. The molecules in the sample either absorb or Transmit the light energy of one wavelength or another . 4. The detector measures the amount of light being transmitted by the sample and reports that value directly (% transmittance) or converts it to the amount of light absorbed in absorbance units (au) using Beers Law.

17. Applications of a spectrophotometer Determines the presence and concentration of samples. Determines the purity of a sample. Look at the change of samples over time. Molecular weight determination of compounds. Respiratory gas analysis in hospitals. The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations.

18. Reference Spectrophotometer- Principle, Instrumentation, Applications January 8, 2022 by S agar Aryal . By Anne Marie Helmenstine , Ph.D. Updated on February 2020. https://www.slideshare.net/ssmvjunwani/spectrophotometer https://byjus.com/chemistry/spectrophotometer-principle S pectrophotometer Dictionary, Thesaurus, Acronyms, Encyclopedia, Wikipedia.

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