The Agilent Uv-Vis Spectroscopy technique is a technological advancement widely used in various areas of Science. It’s used for bacterial culturing, nucleic acid purity checks, drug identification, quantitation, and quality control in chemical research and the beverage industries. This innovative technology makes the lives of researchers and scientists easier.
Let’s learn about Uv-Vis spectroscopy, how it works, and its advantages and limitations.
Table of Contents
Uv-Vis Spectroscopy is a quantitative and analytical technique that measures the amount of visible or UV light a chemical substance absorbs through a Uv-Vis spectrometer. The technique is done by measuring light’s intensity in wavelengths that passes through a particular sample and then comparing it with a blank or a reference sample.
Generally, Uv-Vis Spectroscopy is widely used in several sample types: liquids, glass, and thin films. It is an advanced technique that sees beyond the visible light spectrum our eyes can see, including ultraviolet and infrared light.
To give you a better understanding of how Uv-Vis spectroscopy works, let’s talk about its main components and the processes of how light is absorbed and measured by the spectrometer.
Even though Uv-Vis spectrometers come in various forms, all these machines can only function optimally when these components are complete and properly working:
For a Uv-Vis spectrometer to work, a light source is essential. One of the most common high-intensity light sources used for visible and UV ranges is a xenon lamp. However, it’s more expensive and less stable compared to halogen and tungsten lamps. If a spectrometer needs two lamps, a halogen or a tungsten lamp is typically used as visible light and a deuterium lamp as the UV light.
Because these are two different light sources, the spectrometer should switch when measuring the light’s intensity. Generally, the switchover occurs at 300 to 350 nanometers, when the light emission is the same for both the visible and UV light sources, allowing smoother transition.
Wavelength selection is done to determine which wavelength is suited to the type of analyte and sample to allow sample examination from the wavelengths the light source emits. The most widely used selector in Uv-Vis spectrometer is the monochromator, and this contains the following parts:
Polychromatic radiation or radiation of multiple wavelengths will enter the monochromator from the entrance slit. The beam will collimate and strike the dispersing element through a specific angle and split into several component wavelengths using the prism or the grating. Single radiation of a specific wavelength will leave the monochromator via the exit slit.
Some Uv-Vis spectrometers use monochromators and filters to narrow light wavelengths, allowing more precise measurements and improving the signal-to-noise ratio.
The sample container and reference solutions should be transparent compared to the radiation passing through. Fused silica or quartz cuvettes are commonly used for spectroscopy in both UV and visible regions.
The most common detector used in Uv-Vis spectroscopy is the photomultiplier tube, which contains a photoemissive cathode, dynodes, and an anode. A photoemissive cathode emits electrons when it’s struck by radiation photons, while the dynodes emit multiple electrons each time an electron strikes them.
When a radiation photon enters the tube, it strikes the cathode, which will emit several electrons. The emitted electrons will accelerate and strike the first dynode, which will emit more electrons as each incident electron strikes it. Again, the electrons emitted will accelerate to the second dynode, repeating the process until the electrons reach the anode.
Once the electrons reach the anode, the photon from the beginning of the process has already produced millions of electrons, and the resulting current will be measured and amplified.
Uv-Vis Spectroscopy has been widely used in various sample testing today. This technique has the following famous innovative applications:
One of the most widespread applications of the spectrometer is verifying the RNA or DNA purity and concentration. The Uv-Vis spectroscopy ensures that the DNA or RNA samples prepared for sequencing or other applications are not contaminated with any chemicals that could negatively affect the results.
The mathematical derivatives of a Uv-Vis spectrometer have been an effective machine to detect individual pharmaceutical compounds in overlapping absorbance peaks or different powder formulations.
Uv-Vis spectroscopy is also widely used in culturing bacteria, estimating their cell concentrations, and tracking their growth. The wavelength measurement commonly used is 600 nm to preserve the bacterial culture media properties and the cells when they are needed for consecutive and continuous experimentation.
Uv-Vis spectroscopy has also effectively identified the quantitative content of certain compounds in various beverages, such as caffeine content and colored substances like anthocyanin in wine.
Uv-Vis spectroscopy has proven its success and efficiency in various other applications, including the following:
The best advantage of utilizing Uv-Vis spectrometers is their optimal accuracy. These machines are guaranteed to give you accurate readings, which are essential when you need to prepare chemical solutions or record the movement of the celestial bodies.
Uv-Vis spectroscopy is also easy to understand with its simple analysis ability. The spectrometers are convenient and easy to operate, and there is only a rare chance that you will get the readings wrong.
The main disadvantage of Uv-Vis spectrometers is their challenging assembly, and it may take time to prepare using them. Ensure that the area where you’ll place the device is clear of any electronic noise, outside light, and other contaminants that could affect the measurements and readings of the spectrometer.
A Uv-Vis spectrometer is sensitive to external factors, so you must ensure your working area is clean and dust-free. Aside from that, the device’s stray light caused by a faulty equipment design could also influence the accuracy of the machine’s measurement. This is because stray light will likely reduce the linearity range and substance absorbency it’s measuring.
Even an advanced technique like Uv-Vis spectroscopy has limitations, too. You can grasp what these are below:
While this technique works well with liquids and other solutions, the readings may not be as accurate when the sample is a suspension of different solid particles. The sample will likely scatter the light rather than absorb it with solids, affecting the data. Generally, UV-vis spectrometers are more efficient in analyzing liquids and solutions.
When choosing what solvent to use, ensure that its absorbance cutoff is not close to the sample or compound to be tested. Aside from the solvent, the material of the container or cuvette is also critical because it also has a UV-vis absorbance cutoff. Generally, a quartz container or cuvette is more effective and practical because its absorbance cutoff is only around 160 nm.
Calibration references used to compare the sample readings should be accurately prepared to determine the sample’s concentration to be tested.
UV-vis spectroscopy provides researchers and scientists with more efficient methods to measure light wavelengths, providing accurate readings that are helpful in various biological and chemical analyses.
The UV-vis spectrometer device is precise and easy to operate, provided that you maintain a clean working area free from any external noise and dust that can affect the machine’s readings.
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