In the world of spectroscopy, where investigation into the presence, structure, and properties of molecules within a given sample involves the measurement and analysis of light spectra, instrumentation makes all the difference. Spectrophotometers are a unique set of instruments made to quantitatively measure absorption of visible and ultraviolet (UV-Vis) light or emission of fluorescence compounds. Absorbance approaches directly measure the light absorbed by a sample at a specific wavelength, where the absorbed light measurement is proportional to the concentration of the sample. Using fluorophores bound to compounds of interest enables sensitive measurement of the light emitted from the excited fluorophores.
UV-Vis and fluorescence spectroscopy each have their strengths for different applications, and advances in spectrophotometers are enhancing their usability and accuracy. Furthermore, instruments that offer both absorbance and fluorescence measurements allow definitive information for each approach as well as a thorough analysis of a single experiment in combination. By first acknowledging the strengths of and differences between the two approaches, an accurate assessment can be made as to which works best for a chosen experiment and how the two work together to provide the best of both worlds.
Choosing range or specificity
“Fluorescence and absorbance (UV-Vis-NIR) spectroscopy both study how light interacts with materials,” explains Kevin Grant, product manager and applications development manager at Agilent, “and both can be used to make qualitative, or what types of molecules are present, and quantitative, or how many of those molecules are present, measurements.” Spectrophotometers applying these techniques use a light source to illuminate a sample, where light is either reflected off the sample or passes through it (measuring UV-Vis spectra) or emitted from the sample (measuring fluorescence intensity).
Andrew Jones, market development manager at DeNovix, adds that while absorbance is quick and easy and doesn’t require the preparation or purchase of an assay, fluorescence assays are highly specific for the targeted analyte so that measurement accuracy is not affected by contaminants in a sample. Absorbance quantification methods are well suited to applications where samples are purified, where potential contaminants do not interfere with the measurement wavelength, or in contaminant detection. “Also, for microvolume measurements on instruments like our DS-11 FX series, the dynamic range is very large—0.015 to 750 absorbance units (1 cm equivalent). This equates to 0.75 to 37500 ng/µL dsDNA / 0.04 to 1500 mg/ml BSA, so samples never require dilution,” says Jones.
Absorbance is commonly used for determining purity ratios in nucleic acid and protein quantification. Typical approaches measure absorbance at wavelengths of 260 nm, 230 nm, and 280 nm in a UV-Vis spectrophotometer and calculate the 260/230 nm and 260/280 nm purity ratios. Cellular debris or residual reagent used during purification including proteins, salts, or phenols might be present in the solution and can generate a different absorbance spectrum from the nucleic acids in a sample. Core sequencing and array facilities routinely perform purity ratio analyses for sequencing preparation and other assays. Core facility managers note that UV-Vis spectroscopy is a fast and simple way to ensure proper nucleic acid concentrations and quality prior to sequencing runs.
A closer look at the sample surface for microvolume UV-Vis. Image courtesy of Denovix.
Fluorescence approaches still tend to be favored for lower sample concentrations, being more sensitive than absorbance, and delivering a specific concentration for the desired molecule to accurately set up downstream assays. However, fluorescence assays can be cost-prohibitive and time-consuming, require a standard curve to compare unknown samples, and have a defined range of concentration within which they are accurate. “If samples are outside that range then researchers will either need to dilute/concentrate the sample or use an alternative kit. For this reason, kits with a wider dynamic range are more convenient and save time and costs in the lab,” notes Jones.
Absorbance vs. fluorescence at a glance
- While absorbance is quick and easy and doesn’t require the preparation or purchase of an assay, fluorescence assays are highly specific for the targeted analyte so that measurement accuracy is not affected by contaminants in a sample.
- Absorbance quantification methods are well suited to applications where samples are purified, where potential contaminants do not interfere with the measurement wavelength, or in contaminant detection.
- Absorbance is commonly used for determining purity ratios in nucleic acid and protein quantification. It is a fast and simple way to ensure proper nucleic acid concentrations and quality prior to sequencing runs.
- Fluorescence approaches still tend to be favored for lower sample concentrations, being more sensitive than absorbance, and delivering a specific concentration for the desired molecule to accurately set up downstream assays.
- Fluorescence assays can be cost-prohibitive and time-consuming, require a standard curve to compare unknown samples, and have a defined range of concentration within which they are accurate.
Complementary techniques for comprehensive analysis
Applying both absorbance and fluorescence helps to garner more information about a sample and is especially important in applications such as next-generation sequencing, where both specific sample concentration and contamination data that may adversely affect the assay are needed. Jones offers that applications such as this may have different requirements for quantity and quality measurements at various stages of the workflow. Combining absorbance and fluorescence measurements delivers comprehensive sample quality control to deliver high-quality, reproducible results.
Yet, while UV-Vis and fluorescence are complementary techniques in many ways, it depends on what samples are being measured and what information is needed. “Both are quite simple experiments to do, both can measure liquid or solid samples without any prior preparation, and both are non-destructive,” explains Grant. Since not every sample fluoresces, the range of applications and samples that benefit from UV-Vis is much greater. And for samples that do fluoresce, the sensitivity of fluorescence spectrophotometry is significantly higher, so molecules can be more precisely characterized and smaller concentrations can be more accurately measured.
Novel technology
Both methods are benefitting from current technological advancements in instrumentation. UV-Vis is a very mature technology, having been preserved within new instruments over the decades. The new Agilent Cary 3500, however, is changing UV-Vis with a ‘supercharged’ lamp and new software and hardware purpose-designed for the instrument. Measurement modules do not contain any moving parts, improving usability and reducing instrument maintenance, nor do the thermal controlled systems rely on a water-cooler to control temperature. This provides an integrated air-cooled UV-Vis system with a temperature range of 0 to 110°C, enabling high-quality data to be collected quickly as the system is heated or cooled at faster temperature ramp rates than is currently possible.
The Cary 3500 enables laboratories to make significant gains in efficiency by running multiple experiments simultaneously (with programming for multiple temperature zones, allowing up to four different experiments at four different temperatures), performing individual experiments faster, and allowing custom-designed experiments in ways that have not before been possible. This eliminates key sources of experimental uncertainty and allows a greater level of accuracy in measurements.
Image: Modern design of the Cary 3500. Image courtesy of Agilent.
In addition to improvements in efficiency and accuracy in experiments, enhancements in flexibility and reliability are also front and center in new designs. DeNovix integrated microvolume and cuvette absorbance with a 4-channel fluorometer into a single, stand-alone instrument, so researchers can maximize the number and range of assays within a lab and ensure that future applications can be run on existing hardware. For microvolume absorbance, DeNovix developed SmartPath® technology to improve reliability and accuracy of 1 µL measurements, especially for protein samples. SmartPath compresses the sample, avoiding the danger of breaking the sample column from sample stretching, and also combines real-time absorbance feedback with high precision opto-mechanical hardware to select the optimum pathlength for each sample.
Technological advancements such as these significantly enhance current instrumentation to expand both performance and measurement capabilities. As instruments become easier to use and enable a broader scope of research, both absorbance and fluorescence spectroscopy will continue to be seen as preferred methods of quantitative analysis.