Lab Procedures, Tips & Safety


What is a spectrophotometer?

A spectrophotometer measures the amount of light that can pass through a sample.  Spectrophotometry is a standard technique in many chemistry labs.  From the data collected, a computer plots an absorbance spectrum for the sample.  Spectrophotometry is the action of using a spectrometer to take a measurement

The basic idea of spectrophotometry is that light passes through a sample and the intensity of the beam is compared before and after the sample. Different samples will absorb light differently and allow different amounts to pass-through of different colors of light.

Spectrophotometer model commonly found in chemistry labs
A typical spectrophotometer instrument seen in a lab (Source: Wikipedia Commons)

This technique works because each molecule absorbs light. Depending on the molecules it will absorb certain colors more than others.

As an example, spectrometry at visible wavelengths is the easiest kind to visualize. A black sample will absorb generally absorbs all colors of visible light. That is the reason it appears black. On the other side, if all wavelengths of visible light pass through the sample then the sample most likely appears white.

We can break down the parts of the word spectrophotometer to help remember what the word means. ‘Spectro’ comes from radiant energy or light energy. The next part, ‘photo’ references light. And finally, ‘meter’ refers to an instrument or measurement tool.

How does a spectrophotometer work?

The simplest spectrometer includes a light source, a sample holder, and a detector.

Simple diagram of a spectrophotometer
Simple diagram of an absorption spectrophotometer

The light source produces the photons that will pass through the sample. The exact type of light source will depend on the wavelength of light needed. Depending on the source, a collimator and prism select the correct wavelength.

The light interacts with the sample next. A quartz cuvette holds the samples. A cuvette is a specialized piece of glassware with a very precise width and material. The material of the sample holder is important. You don’t want to use a sample holder that will also absorb at the wavelength of light you are investigating. This is also why it is best practice to run a blank/background in the instrument with an empty cuvette. The path length through the cuvette is also an important parameter for some calculations. The path length is the amount of sample the light passes through. So a cuvette that is 1 cm wide has a path length of 1 cm.

Cuvettes used in a spectrophotometer
Two different sizes of cuvette used in a spectrophotometer (Source: Wikipedia Commons)

After the light passing through the sample, it travels to the detector. Similar to the light source, the exact detector will depend on the wavelength of light. Some of the most common detectors are photomultiplier tubes and photodiodes. The detector counts the number of photons reaching it. The detector connects to a computer that plots this data. This plot is called an absorption spectrum. It is a plot of light intensity verse the wavelength of light. An example of what an absorption spectrum looks like is seen below.

Absorbance Spectrum of Rhodamine B from a spectrophotometer
Absorbance spectrum of Rhodamine B taken with a spectrophotometer

Most Common Types of Spectrophotometers

Spectrometers are generally classified based on the wavelength of light the source is. There are two main classifications:

UV-Vis Spectrophotometer: The UV-Vis spectrophotometer is commonly referred to as just ‘UV-Vis’. This instrument measures absorbance in the ultraviolet and visible range. Generally, that means 200 nm – 700 nm.

IR Spectrophotometer: The IR spectrophotometer measures samples using infrared light, 700-15000 nm.

Spectrophotometry Calculations

There are a few key calculations for absorption spectroscopy.

The formula for transmission:

Calculating transmission

Here T is transmission, Lt is light intensity after passing through the sample, and L0 is the light intensity before the sample.

The absorbance is related to the transmission by the following formula:

Calculating absorption based on transmission measurements

Applications of Absorption Spectra

Absorption spectra have a lot of different possible uses:

  • Determining an Unknown Concentration: Absorption spectra can be used to determine the concentration of a solution of unknown concentration. Absorption data of a series of solutions of known concentrations creates an absorbance curve. Then, the unknown solution can be compared to this curve to determine the concentration. This is also known as Beer-Lambert Law.
  • Identifying a Material: Each pure material will have a unique absorption spectrum depending on the structure of the molecule. Therefore, an absorption spectrum helps to identify an unknown.
  • Identifying Functional Groups Present: Some functional groups will have distinct spectral signatures. Identifying these in a spectrum can inform if that functional group is present and to test if a reaction was successful.

Common labs with spectrophotometers include chemistry, biochemistry, physics, clinical, and materials labs.

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