Combining UV-Vis and Fluorescence Spectroscopy for Optimal Results

Spectroscopic techniques play an important role in the preparation and assessment of biological samples. UV-Visible and fluorescence spectroscopy are two commonly used techniques that have their individual strengths in different applications. But while the approaches can successfully be used on their own, a combination of the two techniques can provide even greater insight. So next time, instead of choosing one technique or the other, why not choose both?

UV-Vis

UV-Visible (UV-Vis) spectroscopy relies on the measurement of absorbed UV or visible light wavelengths in comparison to a refence sample or blank. For many laboratories, the key strengths of the technique lie in its cost, speed, and accessibility. As Andrew Jones, Market Development Manager at DeNovix, explains, UV-Vis takes “direct measurements of the analyte so there isn’t a requirement to purchase an assay or set up standard curves, hence the method is quick and effectively zero cost per sample.” This coupled with the ease of use, and the instrument being commonplace in many laboratories, means that “absorbance is generally the go-to method for most sample quantification,” Jones states.

Another major advantage is that the technique is non-destructive. According to Ursula Tems, BioPharma Marketing Director, Molecular Spectroscopy at Agilent Technologies, “In many cases biological applications require working with nucleic acids, proteins, or samples that are in low volumes, are difficult to obtain, or expensive,” so a technique that allows the sample to be reused or advanced into downstream processing is a key benefit.

These advantages mean that two widespread applications of UV-Vis are taking routine optical density (OD) measurements in bacterial culturing, and quickly determining DNA and RNA purity—both of which are of critical importance to downstream experimental success. “The presence of residual contaminants carried over from extraction such as salts or phenol, or protein in DNA samples…can interfere with downstream processing, so easy identification of these saves researchers the time and expense of failed experiments.”

Fluorescence

Fluorescence spectroscopy, by contrast, works by one of two methods. It either takes advantage of the intrinsic fluorescence of many biological molecules, such as proteins and nucleic acids, to measure their emittance, or alternatively as an indirect measurement—measuring fluorescent light emitted from a fluorophore bound to your analyte of interest.

While the technique can be expensive and time-consuming, its strengths lie in its sensitivity and specificity. “When a UV-Vis absorption spectrum of a mixture is analyzed, all the molecules in the mixture that absorb light will contribute to the absorbance observed. With fluorescence spectroscopy, generally the excitation wavelength of a specific molecule can be used so that the emission profile that is observed is specific for that molecule,” Tems explains. This makes the technique the method of choice for specific quantification of a molecule in a mixture.

The sensitivity of the technique also means its use is favored in lower concentration samples or when your sample is scarce. The high extinction coefficient of fluorescence allows it to detect molecules at concentrations much lower than UV-Vis, meaning that less initial sample is required. So, if your concentration is low or you are running short on supply, bearing the extra cost of fluorescence spectroscopy may be your best bet.

The use of fluorescence is also a popular choice for examining how molecules interact. Variations of fluorescence spectroscopy include polarization and fluorescence resonance energy transfer (FRET) assays that are frequently used in oligomerization and high-throughput drug discovery screening studies respectively.

Use both, not either/or!

With both UV-Vis and fluorescence spectroscopy each having their respective advantages, it can be tempting to use only one technique in isolation. However, as Jones summarizes, “Absorbance and fluorescence should be considered complementary methods that can be combined to enable full sample quality control.” Put simply, using both techniques offers you the best of both worlds. It allows you to combine the respective advantages of each to create a complete analysis package—ensuring specific and sensitive quantification as well as information on potential contaminants.

And while the techniques can be used in unison, they offer particular advantages when used at different stages of the same workflow. As Gilbert Vial, Molecular Spectroscopy Product Manager, Shimadzu Scientific Instruments, explains “Often, UV-Vis will be used to identify the strongest wavelength of a fluorescent molecule” before it is analyzed by fluorescence spectroscopy. “Using the two techniques together [can help] prevent incorrect assumptions by researchers.”

Because of the benefits, UV-Vis and fluorescence spectrometers are often found beside one another in a laboratory. To ensure best results, Vial offers some tips for successfully using the two together. “UV-Vis and fluorescence spectroscopy are similar, and often use similar accessories,” Vial explains, so ensure “that the correct accessory is used for each technique as they are not always interchangeable.” A particular area to watch out for is the cuvettes—“Fluorescence cuvettes will have all four sides unfrosted to prevent any sample interference, whereas a UV-Vis cuvette will often have two sides frosted. If a UV-Vis cuvette is used in a fluorescence instrument, the frosted side will filter out some of the light from reaching the fluorescence detector” and affect your results.

The benefits of using both UV-Vis and fluorescence spectroscopy are clear, and this is now recognized by many companies. While in the past, using both techniques may have meant using two instruments, new instrumentation is being designed to incorporate both techniques—allowing users to measure both UV-Vis absorbance and fluorescence without the need to even change machine. So when using both techniques gives you greater insight, and with the added convenience of combined instruments now available—why wouldn’t you use both?