While the specificity of fluorescence is a benefit to assessing the concentration of the analyte of interest, the resulting figure gives no insight into any contamination within the sample. It is, therefore, necessary to prepare and measure a standard curve when performing fluorescence quantification assays. Samples must also be compared against similar samples of known concentration. Fluorescence analysis, by comparison, requires samples of interest to be bound with fluorescent reagents in an assay kit. Taking dsDNA as an example, the broadest range of fluorescence quantification kits detect to 4000 ng/μl, while the upper limit in microvolume spectrophotometers is currently 37500 ng/μl.Īn absorbance spectrophotometer directly measures the amount of a specific wavelength that is absorbed by a sample without dilution or assay preparation. Absorbance surpasses fluorescence analysis with respect to the width of the measuring range. The dynamic range of measurements is also a consideration in comparing methodologies. Fluorescence analysis using DeNovix Ultra High Sensitivity assay kits yields picogram (pg) levels of sensitivity enabling detection down to 0.005 ng/μl. The most sensitive microvolume spectrophotometer is capable of detection down to 0.75 ng/μl of double-stranded DNA in 1µL of the sample. In this blog post, DeNovix explores four of the key differences between absorbance and fluorescence analysis.īoth absorbance and fluorescence analysis are routinely used in life science laboratories for measurements of small sample volumes. Fluorescence analysis, meanwhile, has long been used to measure the emission spectra of fluorophores binding to samples excited by wavelengths for highly specific molecular quantification. Measuring the absorbance of ultraviolet and visible (UV-Vis) light at specific wavelengths is one of the most established methods of quantitating biomolecules. The good news is: (1) those later eluting compounds are easily separated chromatographically from each other, and (2) their spectra are unique for alkanes (paraffins, for petroleum people), which enables spectral deconvolution from other coeluting compounds, authoritative identification, and accurate quantification.Absorbance and fluorescence analysis are distinct yet complementary techniques used to detect and quantify analytes of interest in a sample. The situation changes as you get further out in carbon chain length, as illustrated in Figures 3 and 4, where the VUV spectra are less distinct from each other. As you can see from Figure 1, and from Figure 2 where I’ve zoomed in to the significant wavelength region, there are obvious differences in the spectra. Figure 1 is an overlay of VUV spectra for normal alkanes from propane (C 3) to heptane (C 7).
In contrast, small molecule spectral ion uniqueness can be rare for electron ionization mass spectrometry, given the limited acquisition “space” between 35 or 45 m/z and the molecular weight of the compound of interest.Īlthough it’s a bit of an intellectual exercise since they are well separated chromatographically, one of the ways to illustrate the advantage that VUV offers for small molecule analysis is to compare absorbance spectra for a homologous series of compounds, which often have very similar mass spectra. In a recent post I mentioned that VUV absorbance spectra are unique, especially for small molecules. VUV Absorbance Spectra for Small Molecules