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UV-Vis Spectroscopy: How Does It Work?

You are a beverage manufacturer, creating your new drink that you will release soon. Imagine, for a moment, that you had two identical containers in front of you containing different substances. Even though they appear visually similar to your naked eye, these substances are very distinct. The problem is, how would you quickly, reliably, and safely determine what each chemical is?

In this case, the solution is absorption spectroscopy. This scientific approach measures how a substance consumes electromagnetic radiation to infer its chemical components.

Absorption spectroscopy can be performed using several distinct techniques depending on the specific wavelengths of light of significance. Below will describe UV-Vis Spectroscopy, often known as ultraviolet-visible spectroscopy, and its uses and purpose.

Introduction to UV-Vis Spectroscopy

UV-Visible spectrophotometry is a scientific approach that compares how much different wavelengths of UV or visible spectrum a sample absorbs or allows through to a reference or blank sample. The sample’s composition affects this attribute, which could reveal the types of substances present and their relative abundance.

The energy of light is equal to its frequency and inversely related to its wavelength. One must provide the energy to a substance to absorb it; this boosts the electron energy level of the substance to a detectable level.

Each bonding environment in a chemical has its unique set of conditions for the electrons present, and it takes a different amount of energy to boost those electrons to a higher level of energy. It explains why various compounds absorb light at varying wavelengths.

The range of wavelengths that can be detected as visible light ranges from roughly 380 nanometers (nm), which humans perceive as violet, to 780 nm, which humans perceive as red. The wavelength of ultraviolet light is about 100 nm shorter than visible light.

Because of this, UV-Vis spectroscopy can be used to evaluate or identify various compounds by identifying the exact wavelengths associated with their maximum absorbance, which light’s wavelength can characterize.

Use of Ultraviolet-Visible Spectroscopy

An instrument called a UV-vis spectrophotometer is used to take UV-vis spectroscopy. A spectrophotometer, in its best basic configuration, includes the following parts:

  1. Source of illumination producing radiation in the specified spectral range. For example, a tungsten or deuterium filament lamp.
  2. A monochromator, or wavelength selector, acts like a prism to split the light from a lamp into its component colors.
  3. Cuvette, a little cup used to hold the sample during analysis.
  4. A light detector, or photodetector, measures how much light is transmitted through a sample contained in a cuvette.

This type of spectrophotometer, commonly called a “single-beam spectrophotometer,” is the simplest. Yet, more intricate designs exist that allow for greater spectroscopic scope.

Other Complex Designs

Some of the complex spectrophotometer designs are listed below.

1. Double-Beam Spectrophotometer

This type uses two cuvettes for the measured sample and one as a reference. The mirrors are used to separate the light beam in half, allowing for a more thorough analysis of the chemical beneath inspection and a standard reference sample.

2. Simultaneous Spectrophotometer

All wavelengths are measured with the same photodetector in a simultaneous spectrophotometer; there is no wavelength selection. The light absorption at every wavelength is simultaneously measured as it passes across the sample using a diode array detector.

The Spectroscopy Procedure

UV-vis spectrophotometers can come in various shapes and sizes, but they all follow very standard spectroscopic procedures.

1. Adjustment of Spectrophotometer

The spectrophotometer is adjusted by analyzing the blank sample to ensure reliable results. This solvent-only blank sample is used to generate the real sample solution.

2. Making of Sample Solution

After the spectrophotometer is adjusted for accuracy, one can make the sample solution containing the test material and the solution. Because of the high UV light absorption cutoffs, dimethylformamide benzene and acetone are the most popular solvents in UV-vis spectroscopy. Below a certain wavelength, no light can pass through the solvent because it has reached its absorbance cutoff.

3. Transferring

After everything is set up, the solution is transferred to the cuvette, typically made out of fused quartz, in a UV-vis spectrophotometer. In contrast to materials like glass or plastic, quartz has a strong absorption cutoff of >380 nm and does not absorb UV light.

4. Analysis

When the sampling is ready, the analysis process can begin. This method directs a light beam from the illumination source via the sample and the wavelength selector. A one-xenon lamp is commonly used as the light source in UV-vis spectroscopy since it generates both ultraviolet (UV) and visible (VIS) light.

Several UV-vis spectrophotometers use a dual source illumination solution consisting of a visible-light-emitting halogen or tungsten lamp and a UV-emitting deuterium lamp.

5. Result Report

When the light beam emerges from the sample, it is obtained by the photodetector. The total light taken by the example is calculated by the photodetector and displayed graphically in the analysis results. The report’s absorption spectrum graph plots the amount of light taken up by the sampling across different wavelengths.

UV-Vis Spectroscopy Accurately Measures Chemical Content

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Regarding chemical analysis methods, UV-vis spectroscopy is among the quickest and most precise options. Due to UV-VIS spectroscopy’s precision and adaptability, UV-vis spectroscopy is employed in various fields, businesses and situations, such as DNA and RNA analysis, pharmaceutical analysis, bacterial culture, beverage analysis, and other applications.


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