Theory and Practice of Spectrophotometric Measurements
In this application note, we discuss the theory behind the measurements based on spectrophotometry
· 2 min read
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:
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:
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.
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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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