Theory and Practice of Spectrophotometric Measurements

In this application note, we discuss the theory behind the measurements based on spectrophotometry

 · 2 min read

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UV-visible spectroscopy is the measurement of the absorbance of light at a specific wavelength in a sample. This is used to identify the presence and concentration of molecular entities within the sample. The Beer-Lambert law is used to relate the absorption of light to the properties of the sample through which the light is travelling through. The Beer-Lambert law states that: 

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This law shows that absorbance is linear to concentration but this is only true for low concentrations. For absorbance levels above 3 the concentration starts to move away from the linear relationship. Transmittance is the proportion of the light which passes through the sample:

Spectroscopy Measurement

There are four main components of a spectrophotometer. These are a light source to emit a high and constant amount of energy over the full wavelength range; a method for separating the light into discreet wavelengths; a sample holder and a light detector.  

The light from the pre-focused tungsten halogen (7310) or pre-aligned xenon (7315) lamp is focused onto the grating, with 1200 lines per millimeter, which separates the light into discreet wavelengths. The diffracted spectrum of light then passes through a further slit and lens arrangement before passing through the sample in the sample chamber from left to right. The light which is not absorbed by the sample is transmitted through a collecting lens and onto the signal detector. The photo-diode detector used is mounted directly onto the detector PCB and the output is used to calculate the % transmittance. The result is displayed either as % transmittance or absorbance on the instrument display. 

For more information on our spectrophotometers range, please visit here

In our upcoming blogs, we will discuss the best practice guidelines of properly using your spectrophotometers and the most probable causes of issues of operation.


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