A Quick History and Underlying Principles
The UV-Vis Spectrophotometer wasinvented in the 1940s by Arnold Beckman as a means of detecting biochemical structures based on their ability to absorb light.
The original design used a glass prism and a vacuum tube photocell. The intention was to use it to test food quality, but the glass model lacked sophistication.
Because this design proved inadequate, additional models were developed using a quartz prism instead.
The quartz models were a success. These had the sensitivity to quantify specific compounds, and by the end of the 1940’s, the Beckman Spectrophotometer was being used to assess food quality and to study DNA.
Spectrophotometers use the Beer-Lambert law as their main principle, as you may remember from your freshman biology labs. Maybe it’s time for a quick refresher, if not!
The Use of Beer-Lambert’s Law in UV-Vis Spectrophotometry
The Beer-Lambert Equation, Simplified
A=ϵlc
Extended explanations of this equation are available, but for the sake of this article, we’ll keep it simple.
For this equation, ϵ is the molar extinction coefficient of the solute, l is the length of the light path, and c is the concentration.
The law states that the absorbance (A) of a compound is directly proportional to its concentration (c) in a solution.
This principle is used to positively identify compounds and to track their concentration, or infer reaction rates based on concentration changes.
Summary of Principles:
When you take a reading, you compare the peak absorbance of the sample to a blank reference in order to measure the absorbance of the molecule in question.
The value that you obtain is known as the optical density, or OD.
So, there it is in a nutshell! The bells and whistles behind the readout you get when you test your DNA concentrations, or the compound you arrived at this page thinking about.
Now, suppose you don’t have any experience with this tremendously undersung piece of hero lab equipment. We’ll cover some of the more common uses for it.
Key Applications of UV-Vis Technology
Because they measure the OD at specific wavelengths, they’re often used to detect molecular motifs. They can also be used to identify compounds that fit specific size ranges.
DNA and RNA
DNA and RNA absorb light at a specific wavelength, and for this reason the absorbance can be used to measure their purity and respective concentrations.
The absorbance ratio at 260 nm/280 nm can be used to determine the purity. Pure DNA has a ratio of 1.8, and pure RNA 2.0.
This method also allows the concentration of the sample to be calculated based upon a known constant for that nucleic acid.
The OD at 260 nm is multiplied by the dilution factor and the known concentration of pure nucleic acid at 1.0, as follows:
Nucleic acid concentration= Xug constant x dilution factor x OD260
Nanoparticle Testing
Nanoparticle solutions are commonly designed as antimicrobials, antifungals, and anticancer agents.
Different species have a strictly defined range of wavelengths that they absorb most highly at, so they can be identified and quantified with this method.
As an example, well-established methods for testing gold nanoparticles with UV-Vis are used in modern nanoengineering labs. Here is a simple protocol for characterizing gold:
A standard curve is prepared using known gold particles of 5, 20, 40, 60, and 100 nm. A sample of unknown size is measured, and the OD is used to place it within the curve.
This method is used regularly in food, cosmetic, medical, and household product industries for the characterization of nanoparticles.
Bradford Assay
The concentration of protein in a solution can be determined using the Beer-Lambert Principle as long as a coomassie stain is added to a sample of the protein beforehand.
The maximum absorbance at 595 nm is used to determine the concentration against a standard curve.
The strategy for doing this is simple. Using a Bradford kit a serial dilution curve is prepared in a microplate.
The standard curve is made with a known concentration of a protein like bovine serum albumin. A serial dilution is made from 20 ug/ul standard of protein, and diluted by half to as little as 0.125ug/ul.
The unknown samples are prepared alongside the standard curve in duplicate, and read at the required wavelength.
Optical density is plotted along the curve, and the concentrations are found using the slope.
A Summary of Points:
A standard curve showing the relationship between a known value for a reference and the optical density reading can be used to identify the qualities of an unknown.
Limitations and Use
To put it as simply as possible, the absorbance is a good test for the presence or absence of compounds that are well understood already.
Things that have unique motifs tend to be good candidates.
♪♪ This assay, what is good for?
It cannot identify peak absorbance for specific compounds unless you can purify them without knowing what they’re like.
What it CAN do is give you accurate concentrations of a known compound, and you can use it to determine purity and reaction rate within a solution for a known compound of interest.
Core Features of UV-Vis Spectrophotometers
Spectrophotometers are composed of a few basic parts:
- the lens
- the prism (or monochromator)
- the light source
- the photocell
- recording equipment
The sample is placed between the monochromator and the photocell, and light is focused onto it through the lens.
SmartDrop XF Nano Full Spectrum Absorbance Spectrophotometer
The photocell then delivers information about the photons that were transmitted to a computer that calculates the absorbance. Some machines offer printouts with the calculations.
The technology is pretty simple, but the options are unlimited!
Traditional machines required the use of a cuvette for holding about 1.5 ml of sample solution, but nanodrop machines that only require 1 ul of sample are common.
Although they’ve been around for quite some time, these machines haven’t lost relevance.
They are still used in the food industry, cosmetics, and even in academic laboratories to characterize new materials.
Recent improvements have led to portable spectrophotometer equipment for field testing of water quality, machines with automatic printouts for each reading, and even machines that interface with robotic handlers.
Maintenance and Safety Tips
In order to operate and maintain your instrument, you should always wipe the sensors clean with a lint-free wipe between samples and calibrate with a blank solution when changing sample type.
Basic Safety:
- Check the power cable and outlet to make sure there are no electrical breaches.
- If the machine shuts off or stops during readings, try replacing the power cable and changing the outlet. Contact the manufacturer if problems continue.
- If readings are erratic or consistently low and sample integrity cannot be blamed, contact the manufacturer for help. This may require a replacement detector.
Additional information for running and troubleshooting experiments with any of the Smartdrop models can be found on the Accuris website.
A World of UV-Vis Technology at Your Fingertips
Browse an entire online catalog of top-of-the-line spectrophotometry machines and accessories.
TheSmartdrop nanodrop equipment accepts microliter samples AND cuvettes. These are designed to instantly read your protein, DNA, and RNA samples in as little as 0.5 ul.
All of our machines have digital displays that provide high-sensitivity readings. Check out the Lu-mini if you really want to be wowed by how much you can do with something minimal.
The Stellar Scientific Catalog
See the rest of our online catalog of UV-Vis equipment and related accessories. We have cuvettes, pipettes, and even handheld spectrophotometers for on-the-go readings.
If you need help finding something, don’t hesitate to ask. Go to our contact us page and call us or chat online.
Footnotes:
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Barbas, CF., et al. “Quantitation of DNA and RNA.” Cold Spring Harbor Protocols, vol. 2007, no. 11, Nov. 2007, https://doi.org/10.1101/pdb.ip47.
“The Beer-Lambert Law.” Chemtalk, chemistrytalk.org/beer-lambert-law/. Accessed 15 May 2024.
“Bradford Assay Protocol.” Bradford Assay Protocol | Duke Department of Biostatistics and Bioinformatics, biostat.duke.edu/bradford-assay-protocol. Accessed 15 May 2024.Ganguli, Ishani.
“The First Commercial UV-Vis Spectrometer.” The Scientist, 1 Mar. 2006, www.the-scientist.com/the-first-commercial-uv-vis...
A Classic Instrument: The Beckman DU Spectrophotometer and Its Inventor, Arnold O. Beckman, Journal of Biological Chemistry, 5 Dec. 2003, www.the-scientist.com/the-first-commercial-uv-vis...
Quevedo, Ana, et al. “UV-Vis Spectroscopic Characterization of Nanomaterials in aqueous media.” Journal of Visualized Experiments, no. 176, 25 Oct. 2021, www.the-scientist.com/the-first-commercial-uv-vis...
