Seeing the Light: An Overview of Visible and UV-VIS Spectroscopy

Seeing the Light: An Overview of
Visible and UV-VIS Spectroscopy

Spectroscopy is the study of how materials react to radiated energy. Light is a radiated energy. Most of the spectrometers Cole-Parmer carries utilize light as the radiated energy source. “Light” exceeds the visible spectrum of color the human eye can see. As shown in the chart below, visible/white light breaks down into separate and unique colors: red, orange, yellow, green, blue, indigo and violet. These colors, and what the human eye can see, are a very small and limited range of the electromagnetic spectrum of radiated energies.

Breakdown of visible/white light chart

When visible light shines on a blue shirt, the dye in the shirt reflects the blue light and absorbs all the other colors we can see. This is a simple example of how a material, such as a shirt, reacts to light. Over time, the dye may wash out and its concentration in the fabric lessens. The loss in dye concentration can be seen as the color appears lighter and lighter. If looking at the shirt using a spectrometer set to the blue wavelength range, the transmittance (the transmission of radiant energy) would be increasing, and the absorbance (light-absorbing ability) decreasing. The law that surrounds spectroscopy is called “Beer’s Law” written commonly as

A = ε l c

“A” stands for absorbance, “ε;” for Molar absorptivity (also known as the extinction coefficient), “l” represents the pathlength, and “c” is concentration

All spectrometers are based on this law in one way or another. Given the equation, absorption is directly correlated to the extinction coefficient, pathlength, and concentration. If any of the variable on the right-hand side of the equation increases, absorbance will also increase.

Visible and Ultraviolet-Visible (UV-VIS) Spectrometers

As the name implies, the radiated energy source for these spectrometers is either visible light or both visible and ultraviolet light. Typically, ultraviolet (UV) covers the range of 190 to 400 nm and visible (VIS) covers the range of 400 to 800 nm wavelengths. Some molecules react to light within the UV range or will have bonds that react to light in the UV light range, thus needing the additional UV range on a spectrometer.

Below are a list of types of UV-VIS spectrometers and their advantages and disadvantages.

a: Single vs Double Beam

Spectrometers can have either a single beam or a split/double beam setup. In a single beam unit, the standard, or reference, sample is measured first in order to provide a baseline reading. The samples are then analyzed afterward. As the light source ages, even over a matter of minutes, there are slight changes to the output. Often, the standard will be reanalyzed between samples to ensure the readings are as accurate as possible. A double beam unit accounts for the drift over time by splitting the source lamp’s light into two light paths. One path goes toward the sample chamber, and the other is used to take reference sample measurements. Since the reference sample is always being monitored at the same time and to the same light, the user doesn’t have to recalibrate, or zero, the spectrometer repeatedly throughout many samples. For a simplified diagram of this process, look below and view how light travels through the sample and reference.

Light travelling through sample and reference diagram

Although a split/double beam setup makes for more accurate measurements, some drawbacks remain. There are two mirrors in a double beam unit; one mirror to direct light to the standard and another to direct light to the sample. Dust, dirt, and other debris may coat the mirrors unevenly causing erratic readings. Additionally, when changing the mirrors they must be done in pairs as the coating will need to be even in order to obtain similar readings. Since the replacement or repair must be done as a couple, the repair fee may also double as twice the work and parts are needed.

b. Scanning vs. Nonscanning

Scanning UV-VIS spectrophotometers are capable of quickly exposing the sample to the entire (or a set) range of wavelengths, and obtaining the absorbance measurements at all wavelengths across that range. A nonscanning UV-VIS unit does not have this capability and the user must manually change the wavelength to obtain a single absorbance measurement. Scanning spectrophotometers may also be used as single wavelength/nonscanning spectrophotometers, providing the user with greater flexibility. Scanning units may be used to determine what the greatest peak is for a sample throughout the UV-VIS range, so subsequent samples may be tested at a single wavelength. Or, they may be used to test quality. For example, the pure form of a sample should have similar scans. If the sample contains a contaminant, peaks may appear at other wavelengths indicating the presence of something else in the sample.

c. Colorimeters

Colorimeters are visible light spectrometers, typically single beam units, used to expose samples to specific wavelengths to obtain absorbance, but also programmed to output concentration values rather than just absorbance readings. These are commonly used for water quality testing when paired with specific reagents. The reagents will cause a color change based on the amount/concentration of the target dissolved ion. The colorimeter can measure the difference in color based on absorbance, and has preprogrammed equations that correlate the absorbance reading to a concentration for the target ion. Chlorine concentration testing is typically obtained using reagents and a colorimeter and is used in the water treatment industry. Specifically, this testing ensures that water piped into homes is within safe limits to keep the water clean from bacteria yet not too high as to harm consumers.

d. Nano-Volume Spectrometers

Nano-spectrometers also use visible and UV light as their radiated energy source. However, rather than using large volumes of sample (typically more than 1 mL is needed in cuvettes with a standard UV-VIS spectrometer), nano-volume spectrometers only need a small droplet. This small droplet (about 20 uL) of reference or sample can be placed on a platform. When the chamber is closed, the drop is suspended at a fixed distance, creating a path for the light to go through the sample. Fiber optic light, instead of a flashing bulb, is used in this type of spectrophotometer to more accurately focus the light through such a small sample. Revisiting Beer’s Law we can see if the pathlength is reduced, the absorbance would also be reduced, however this method is often used on highly concentrated samples. By using a small amount of concentrated sample, there is no need to dilute it (contaminate it) as if running on a standard spectrophotometer.

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Quartz Micro Cuvettes for UV Spectroscopy.