Question

Write 3 paragraphs for each of the following

• Define spectrophotometry?
• Why is it important as an analytical chemical method of analysis?
• What are the main components of spectrophotometer?
• List the main types of spectrophotometer and differentiate between them?
• How does matter absorb radiation?
• How does a spectrophotometer measure absorbance?

Answer 1

1. Spectrophotometry is a quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is a quantitative analysis of molecules depending upon how much light absorbed by the colored compounds. It uses photometers which measure a light beam intensity as a function of color or in other words wavelength. It is commonly used for measurement of transmittance or reflectance of solutions. It can also be used to measure the equillibrium constant of a solution.
2. Use of spectrophotometers span various scientific fields such as physics, material science, bio chemistry, molecular engineering etc. It is an efficient and fast quantitative analtical method. Even colorless compounds can be analysed by converting them to colored compound first. its versatility in biochemistry is remarkable. Spectrophotometry is an unavoidable tool regarding RNA, DNA  synthesis, protein isolation, enzyme kinetics etc. As this method only require sample in minute quantities and biochemical samples are usually in micro gram regime, it is very useful. Due to this majority of the sample can be saved for future used and sometimes analyzed samples are reusable.
3. The main components of spectrophotometer is as given
   1) A suitable multispectrum light source
   2) Monochromator - It extracts required wavelegth from the light source. It consist of a narrow slit, a dispersion device and an exit slit
   3) A sample holder in which we place the sample.
   4) An electronic detector.
4. The main types of spectrophotometers are
   1) Atomic absorption spectrometer - It analyzes concentration of elements in a liquid sample based on energy absorbed from certain wavelength of light. it consist of a flame burner to atomize  the sample, and a monochromator and detector.
   2) UV-VIS double beam spectrophotometer - It uses visible light and UV light in the light source. Usual method. It uses a reference beam and a sampling beam.
   3) UV - VIS single beam spectrophotometer - uses uv or visible rediation but only use one beam.
   4) Spectroflurometer - It is used to quantify flurophores. Flurophores absorb visible light and show fluroscence and is detected and analyses.
5. When radiation incident on material atoms or molecules, it excites it from its ground energy state to excited energy state. The energy states can be electronic level, vibrational or roational which are solely depend upon the frequency of the radiation. When excitation happens photons get absorbed and the transmitted light is devoid of this frequency.
6. The detector measures this electronically. Actually it is based on an equation known as absorbance equation.
   A = Log10(I0/I)
   I0 is the initial intensity and I is the transmitted intensity. The intensity can be measured from Lux meter.