Why is fluorescence more sensitive

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First, one or more standards of known concentrations are measured in order to generate the standard curve. Determination of sample concentration subsequently takes place in a second step, where the measured fluorescence intensities of the samples are evaluated in relation to the standard curve. Even if fluorometry offers advantages in certain cases, classic UV-Vis spectrophotometry continues to be the standard method when it comes to quantification of nucleic acids and proteins.

Spectrophotometry allows verification of sample purity, and highly concentrated solutions can be analyzed directly. Depending on the quality of the sample, as well as the requirements of downstream applications, it may be advisable to combine both techniques.

All rights reserved including graphics and images. Home Sample Handling Photometry Principles. Fluorescence — a Meaningful Addition to Absorbance? Share this article. Figure 1: A Principle of fluorescence measurements of nucleic acids left B The light that is utilized to excite the fluorophore EX has a shorter wavelength than the emitted light EM right. At pH 8. The intensity of each species is proportional to the concentration.

Representation of fluorescent emission spectrum of 2-naphthol as a function of pH under the conditions of slow left and fast right exchange. Now the emission is a weighted time average of the two forms. At the pKa value 9. What actually happens — is the exchange fast or slow? The observation is that the exchange of protons that occurs in the acid-base reaction is slow on the time scale of fluorescence spectroscopy. Remember that the lifetime of an excited state is about 10 -8 second. This means that the exchange rate of protons among the species in solution is slower than 10 -8 second and the fluorescence emission spectrum has peaks for both the 2-naphthol and 2-naphtholate species.

The pKa value of an acid is incorporated into an expression called the Henderson-Hasselbalch equation , which is shown below where HA represents the protonated form of any weak acid and A — is its conjugate base.

If a standard curve was prepared for 2-naphthol at a highly acidic pH and 2-naphtholate at a highly basic pH, the concentration of each species at different intermediate pH values when both are present could be determined. These concentrations, along with the known pH, can be substituted into the Henderson-Hasselbach equation to calculate pKa.

If you do this with the fluorescence spectra of 2-naphthol; however, you get a rather perplexing set of results in that slightly different pKa values are calculated at different pH values where appreciable amounts of the neutral and anionic form are present. This occurs because the pKa of excited state 2-naphthol is different from the pKa of the ground state. Since the fluorescence emission occurs from the excited state, this difference will influence the calculated pKa values.

A more complicated set of calculations can be done to determine the excited state pKa values. Because many compounds are weak acids or bases, and therefore the fluorescence spectra of the conjugate pairs might vary considerably, it is important to adjust the pH to insure all of either the protonated or deprotonated form.

Answering this question involves a consideration of the effect that collisions of the molecules will have in causing radiationless decay.

The advantages of the single beam design are low cost, high throughput, and hence high Sensitivity , because the optical system is simple. The disadvantage is that an appreciable amount of Time elapses between taking the reference I and Making the sample measurement Io so that there can be problems with drift.

Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold body radiation. The term ' luminescence ' was introduced in by Eilhard Wiedemann.

Source : Luminescence. Absorbance is related to the logarithm if internal transmittance. Why fluorescence is more sensitive than absorbance? Category: science chemistry. Why is fluorescence rather than absorption used for high- sensitivity detection?

Fluorescence is more sensitive because of the different ways of measuring absorbance and fluorescence. Light absorbance is measured as the difference in intensity between light passing through the reference and the sample. What factors affect fluorescence? Why does fluorescence occur?

Does pH affect fluorescence? Why is fluorescence spectroscopy used? How does fluorescence work? What is the unit for fluorescence intensity? What is the principle of fluorescence spectroscopy?