This article summarises UV-visible spectroscopy and the Beer-Lambert equation.
UV-VIS Spectrophotometer Configuration
The diagram below shows a typical UV visible spectrophotometer. This is one of the simplest spectrophotometer designs. Continue.
Standard Spectrophotometer parts
The first component is the source light, which may be a scooter headlight, a Deuterium lamp or a xenon arc lamp.
The monochromator has two slits separated by a prism or diffraction grid—more on slits below.
Next, a beam splitter separates the light beam into two parallel beams. It contains two prisms.
The sample compartment contains reference and sample cuvettes.
Computer-monitored detectors convert photons into electric current.
5 Parts of Spectrophotometer Design
We may now move on to the smaller parts of our spectrophotometer. Let’s try the instrument.
Let’s ignite the lamp, which will produce a spectrum of light. After passing through the first monochromator slit, this light is refracted into a rainbow of colours.
Light goes to the other slit, allowing just one wavelength to pass. To make my monochromator work, I have two slits on it that link to a beam splitter. These two powerful beams will pass through the cuvettes’ sample chambers.
As the beam leaves these cuvettes and strikes the detecting device, which causes an electric current, the reference and sample cuvettes have identical light intensities.
Identical Exit Current for Both Cuvettes
Spectrophotometer data implications
Both detectors record comparable current intensities. When the sample cuvette’s intensity is compared to the reference cuvette’s, we can see that their transmissions are identical. We’ll map 100% transmission at zero concentration.
Specify a small sample to absorb light. This lowers the light intensity of the sample cuvette. So, its detector’s current decreases.
At a specific concentration X, the intensity ratio reduces to 50%.
Intensity decreases more if I add another equal sample to the sample cuvette. Each sample concentration reduces the light intensity passing through the sample cuvette by 50%.
The new current intensities of the sample and reference cuvettes demonstrate a 25% increase in transmittance and concentration.
Re-sample. A sample cuvette lowers transmittance by 50%, to 12.5%. Now, three components are highlighted.
We know enough to recognize anything amazing.
Sample concentration and transmission percent are not linear. It’s exponential.
Researchers and spectroscopists seek to examine linear connections within data sets whenever possible since it simplifies the discourse and simplifies future forecasting.
Here comes Beer-Lambert. You’d believe transmission percent and my attention are exponentially related.
Concentration is an exponent in this equation (conc).
August Beer renamed transmittance absorbance. The logarithm or negative logarithm of transmittance yields the following equation:
The exponential function is logarithmically converted to linear. This makes the numbers much easier to understand. Extrapolating or interpolating data makes estimating absorption simpler.
Absorbance is calculated using UV visible spectroscopy. Here’s how Beer-Lambert is implemented.