Core Concepts
In this article, you will explore the components of UV-Vis spectrophotometry, its discovery, how it works, and some applications.
What is UV-Vis spectrophotometry?
UV-Vis spectrophotometry is an analytical technique that measures the wavelengths of light, from the ultraviolet (UV) range to visible range, that a sample absorbs and emits. You’ll often see UV-Vis spectrophotometry abbreviated as UV-Vis.
“UV-Vis” refers to the range of wavelengths that are involved in this type of spectroscopy: ultraviolet through visible light. Here, let’s distinguish between UV-Vis spectrophotometry and its close cousin, UV-Vis spectroscopy. UV-Vis spectroscopy is the study of how matter interacts and/or emits radiation, specifically radiation within the ultraviolet range to visible light range. Encompassing UV-Vis is the larger field of spectroscopy, which is includes all forms of radiation.
The key distinction here is that UV-Vis spectrophotometry involves quantitatively measuring the radiation, whereas UV-Vis spectroscopy is a more qualitative approach. Valued for its ease of use, UV-Vis spectrophotometry is a highly versatile, highly accurate tool. To better understand UV-Vis, let’s first discuss where the concept originated.
Origin of UV-Vis Spectrophotometry
The first experiment in spectroscopy was done by Isaac Newton, a household name across all of science. Newton was curious about a world of things, but in 1666, he took an interest in light, which laid the groundwork for UV-Vis spectrophotometry. With a prism bought at a market, he attempted to allow only a small sliver of slight peek through from the window into the prism. When the light hit the prism, it was split into a rainbow of color upon the wall!
Newton innocuously reported his findings via a 1672 letter that the Royal Society published, there coining the term spectrum. From then on, a long string of impeccable physicists, chemists, and astronomers from across the world continued Newton’s work, improving and advancing the world of spectroscopy to what we have today! But what exactly does a prism tell us about how light works and why it matters?
The Visible Light Spectrum
To understand UV-Vis, we also need to discuss the visible light spectrum, a rather small portion of the electromagnetic spectrum. As a reminder, the electromagnetic spectrum (EM spectrum) encompasses the wavelengths, frequencies, and energies of various types of electromagnetic radiation. Most of the electromagnetic spectrum is actually invisible to the human eye, with us being able to see only the visible light section.
As we can see in the image above, the visible spectrum is quite small compared to the other regions of the EM spectrum. It overlaps slightly with the UV range to its left and the infrared (IR) range to its right. However, this small section makes up the colors of the entire visible world! Within this range, encompassing wavelengths of 400 nm to 700 nm, are all the colors we can see. The light that produces each color is associated with a certain range of wavelengths, as shown in the image below.
For example, we can estimate that the light producing a lemon’s yellow color has a wavelength between 575 nm and 650 nm. Because the EM spectrum is a spectrum, the distinction between adjacent colors can be ambiguous. Someone could argue that a light with a wavelength of 575 nm appears yellow, or they could argue that it appears green, and it’s hard to determine who’s correct. For this reason, these wavelength ranges are just an estimate; you may see slightly different ranges in other sources.
The fact that every color corresponds to a wavelength forms a massive portion of the UV-Vis spectrophotometer’s functionality, which we will explore momentarily. Even so, the visible light spectrum is only one aspet of the UV-Vis. Let’s discuss how this incredible analysis tool came to be!
UV-Vis Spectrophotometer
Among scientists striving for accuracy in analytical chemistry equipment was Arnold Beckman, a chemist who made the first commercially available UV-Vis spectrophotometer. His goal was improve chemical analyses, which, at that time, were extremely slow and inaccurate. This goal became more crucial due to World War II, when there was an urgent need for accurate analytical chemistry equipment to help measure the amount of vitamins in soldiers’ rations.
So, in 1940, Beckman and his team at the National Technologies Laboratories, with an amplifier, a glass prism, and a vacuum tube photocell, made the first spectrophotometer! Beckman and his team continued to improve this model and eventually produced the Model D (also known as the DU spectrophotometer). Nobel Laureate Bruce Merrifield labeled this innovation, commercially available from 1941 to 1976, as “probably the most important instrument ever developed towards the advancement of bioscience.”
How does the UV-Vis work?
UV-Vis spectrophotometers have come a long way since Newton looked at that secondhand prism all those centuries ago! A standard UV-Vis has four main components: a source, a monochromator, a sample, and a detector.
The Source
In a UV-Vis, the source refers to a continuous light source. Typically, the source is a lamp made out of either deuterium (D or 2H) or tungsten (W). The element chosen for the lamp is based on the wavelength region being measured.
The lamp itself is a gas discharge lamp. In this format, electric discharge is sent through an ionized gas. Then, as the gas molecules are excited, they begin to emit radiation. The deuterium lamp emits within the UV range, from roughly 180 nm to 400 nm. Tungsten lamps, by contrast, emit in a range from approximately 350 nm to 2000 nm. This is a big difference that makes tungsten lamps reliable for assessing samples in the visible and IR ranges. Furthermore, deuterium lamps generally have shorter lifespans, clocking in at approximately 1,000 hours. A tungsten lamp has a longer life span of approximately 3,000 hours.
As we can see, the type of lamp a UV-Vis spectrophotometer uses depends on the instrument’s specific scientific applications. For instance, if a scientist wants to analyze light within the UV range, they would probably choose a deuterium lamp over a tungsten one. If they place more value on the lamp’s long-term use, a tungsten lamp’s extensive life expectancy would be the way to go. Better yet, a UV-Vis spectrophotometer can use both of these lamps simultaneously to gain a stable look at the entire UV to visible light spectrum!
The Monochromator
The monochromator is the device that splits the light into its separate wavelengths. It achieves this basic, yet fascinating, task using a mirror and some form of light separator.
In older UV-Vis models, the light separator was a simple prism, reminiscent of Newton’s original experimental setup. Now, a light separator can be made using either a prism or diffraction gratings. A diffraction grating has a flat surface with many identical, parallel grooves. These grooves are arranged in such a way that they separate light by its wavelength, rather than using optical dispersion, as Newton’s prism did.
The Sample
The sample is held in a cuvette or other small container that will be fitted into the UV-Vis. Often, there is also a second cuvette that has a reference. The purpose of the reference is to calibrate the machine before running the sample. This way, whatever the sample’s readout is, the scientist can compare it to the reference’s readout.
Scientists’ needs determine the material of the cuvette. Each material is best suited for assessing a certain wavelength range, so the cuvette’s material is chosen based on the sample’s hypothesized absorption. Quartz, for example, has a range of 199 nm and above, making it ideal for solutions in the UV range. Glass ranges from 300 nm to 2000 nm, lending a wider applicability. Plastic is common and inexpensive, enabling readings in a range of 350 nm and above.
The Detector
In a UV-Vis, one or more detectors collect the data from the sample. Most commonly, a photomultiplier tube (PMT) is used because of its high sensitivity to light within the UV and visible ranges. A spectrophotometer frequently couples it with an amplifier, which makes the results easer to see on the readout.
Understanding UV-Vis Results
While the inner workings of the UV-Vis are fascinating, it’s also important to know how to understand the results! As an example, the readout from a sample of oenin will look something like this:
Across any readout, some features will remain the same. For instance, the x-axis of the readout will always represent wavelength, while the y-axis shows intensity:
What does not remain the same is the actual curve on the readout. This is unique for each substance! Even though we can’t examine every potential substance as a sample, we can know what we should look for in the readout.
The readout of any given sample may have more than one peak, which is true for oenin. When this is the case, the peak that is most important is the tallest one. We can call the highest point of the tallest peak λmax (lambda, λ, is one way to symbolize wavelength).
Recall that every color of light has a corresponding range of wavelengths. We can use the wavelength at λmax to determine the solution’s color. Once we determine the range that the λmax value falls into, the complementary color is the color of the solution. For this part, we can rely upon the color wheel as a guide. Complementary colors are located directly across from each other on the color wheel.
For example, if the sample is yellow, we can expect to see a peak at a wavelength of about 380 nm to 420 nm. This means that the sample is absorbing in the violet range. It emits the color that is complementary to violet on the color wheel, which is yellow.
Now that we know how to interpret a UV-Vis readout, we can look back at our spectrum of oenin and predict the sample’s color! Oenin’s λmax value is 531 nm, which falls within the range of green color on the visible light spectrum. Thanks to our handy color wheel, we can see that the color complementary to green is red. Therefore, the sample’s color is most likely red!
So far, we’ve highlighted UV-Vis’s amazing utility and colorful scope! However, unfortunately, science is not a perfect subject. All experiments are subject to uncertainty, error, and inaccuracies, and UV-Vis spectrophotometry is no exception. Next, we’ll investigate some of the key disadvantages of UV-Vis and how it makes a difference in our world.
Issues of UV-Vis
With UV-Vis, one major concern is stray light. Stray light can happen due to a multitude of factors: an imperfect diffraction grating, a broken cuvette, or even the broken seals on the UV-Vis spectrophotometer. When stray light occurs, it can interfere with the spectrophotometer’s ability to read the sample clearly. As a result, it could distort the readout and create inaccurate data.
Another common issue is when the sample itself has a contaminant, and thus the results would be inaccurate. In this case, it is important to use high-quality materials. Being extra cautious when preparing the samples can help prevent cross-contamination and keep your data intact.
In spite of the issues that can impact UV-Vis results, it remains a powerful tool with countless applications! Let’s review a few of the most prominent ones.
Real-World Applications of UV-Vis
We see UV-Vis in so many different fields, even where you wouldn’t expect it! In chemistry, one key use of UV-Vis happens in conjunction with the Beer-Lambert Law. The Beer-Lambert Law exploits a relationship between absorption and concentration, using proportions to calculate absorption from the UV-Vis data.
Air quality scientists can use UV-Vis in satellites to measure the absorption signatures of gas species! These measurements give them clues as they identify gases and learn about the substances that make up our air.
Back on the ground, in the lab, biologists and biochemists employ UV-Vis to identify the purity and concentrations of DNA and RNA. It’s also useful in food authentication, when it helps determine the purity of the food we eat every day. This is one of many ways science keeps us safe and protects our health. As we continue to improve UV-Vis spectroscopy and spectrophotometric technologies, there may be no limit to the applications benefiting from this invention.
Conclusion
UV-Vis spectrophotometry led to an incredible increase in the efficiency and accuracy of chemical analyses, highly sought-after during World War II. Since then, it has played a huge role in the rise of analytical chemistry, opening doors in so many different fields! We can use UV-Vis far and wide for researching nanoparticles, identifying macromolecules, analyzing the environment, and more. As technology continues to advance, so will its many applications.
