Spectroscopy 101

Intro

Molecular spectroscopy is an effective and long-standing method to gain information about matter by exploring how it interacts with light of different wavelengths. The technique relies on the fact that molecules exist in a series of discrete energy states, with specific energy gaps in between. If the energy of an incoming photon matches an allowed transition between an occupied state and an empty one, the molecule can absorb the photon. This increases the energy of the molecule, and it is now refereed to as being “excited”. In the UV-vis range, the transitions are typically electronic transitions, moving one electron from one orbital to the next one.

Different molecules have different energy states, and when the molecule or its environment changes, so do the energy states. Because of this, spectroscopy can reveal a lot about the molecules in a sample. Next we will have a look at the two main modes of spectroscopy.

Absorbance spectroscopy

Absorbance spectroscopy is the art of looking at what is no longer there. The researcher sends broad-spectrum (white) light through the sample and records what comes through on the other side. This data only makes sense if there is something to compare with, so the first step is typically to record a reference spectrum, or blank. Most often, this is done using a cuvette that contains everything except the molecules of interest. The absorbance spectrum is then calculated by taking the logarithm of the ratio between the light transmitted by the reference and the sample.

Absorbance spectroscopy is very useful for identifying different types of molecules, and for measuring the concentration.

Fluorescence spectroscopy

Fluorescence spectroscopy focuses on the photons that are sent out when the molecule relaxes back to the ground state. First the molecule is excited by sending in light of a specific wavelength that will be readily absorbed. The emitted fluorescence light is typically recorded at an angle away from the path of the excitation light.

The fluorescence spectrum of a molecule can change depending on the state of the molecule and its surroundings, making fluorescence spectroscopy a useful tool for following molecular processes in detail. Fluorescence is also a very sensitive technique, allowing researchers to investigate molecules at very low concentrations.

Summary

Different molecular spectroscopy techniques provide windows into the molecular details of a sample. Depending on which technique is used, different details can be revealed, and different types of samples can be investigated. Having access to a several different techniques and combining them can really multiply the level of knowledge a researcher can gain.

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