What is Spectrophotometer?

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Spectrophotometry is a quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the common term electromagnetic spectroscopy which deals with visible light near ultraviolet and near infra-red. It does not cover the time resolved spectroscopic techniques which means that "anything that allows to measure temporal dynamics and kinetics of photo physical processes". Spectrophotometry involves the use of spectrophotometer.


A spectrophotometer is an instrument which is used to measure the intensity of electromagnetic radiation at different wavelengths. Important features of spectrophotometers are spectral band with and the range of absorption or reflectance measurement. Spectrophotometers are generally used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids such as polished gases or glass. They can also be designed to measure the diffusivity on any of the listed light ranges in electromagnetic radiation spectrum that usually covers around 200 nm-2500 nm using different controls and calibrations.


What are the different types of Spectrophotometers?

There are 2 major classifications of spectrophotometer. They are single beam and double beam.

  • A double beam spectrophotometer compares the light intensity between 2 light paths, one path containing the reference sample and the other the test sample.
  • A single beam spectrophotometer measures the relative light intensity of the beam before and after the test sample is introduced.

Even though, double beam instruments are easier and more stable for comparison measurements, single beam instruments can have a large dynamic range and is also simple to handle and more compact.  


How does a spectrophotometer work? 

Light source, diffraction grating, filter, photo detector, signal processor and display are the various parts of the spectrophotometer. The light source provides all the wavelengths of visible light while also providing wavelengths in ultraviolet and infra red range. The filters and diffraction grating separate the light into its component wavelengths so that very small range of wavelength can be directed through the sample. The sample compartment permits the entry of no stray light while at the same time without blocking any light from the source. The photo detector converts the amount of light which it had received into a current which is then sent to the signal processor which is the soul of the machine. The signal processor converts the simple current it receives into absorbance, transmittance and concentration values which are then sent to the display.


How to use a spectrophotometer?

Spectrophotometer is used to determine absorbency at certain wavelengths of a solution. This can be used to determine the concentration of a solution or to find an unknown substance. 

  • First, clean out the cuvette (a small rectangular transparent tube that holds the solution) in the machine making sure that throughout the experiment, we face it the same way into the machine each time. Any fingerprints or dirt on the cuvette can affect the results of the experiment. So, it is always essential to wear gloves
  • Then, add the solute which would be the blank for the experiment. It is important not to add water to the blank so that it won’t affect the accuracy of the results.
  • As a next step, the spectrophotometer is set to the desired wavelength. Insert the blank cuvette, making sure that the arrow is aligned each time.
  • Then  press the “set zero” to calibrate the spectrophotometer for that wave length.
  • Now, introduce the solution in order to calculate its absorbency. Each time we change the wavelength, it is important to change blank for that wavelength.
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