AAT Bioquest

What are the differences between absorbance and fluorescence?

Posted January 13, 2023


Basis of differentiation




Absorbance is a measure of the capacity of a substance to absorb light of a specific wavelength

Fluorescence is the emission of light from a substance that has absorbed energy previously 


Is less effective due to the technique not being sample-specific

Is more effective because the assay is highly specific 


Is a rapid and easy-to-use method

Is a more complex and time-consuming method

Sample preparation

Does not require sample preparation  (no dilution or assay preparation necessary)

Requires sample preparation (sample of interest must be bound with the fluorescent reagents in an assay kit)


The most sensitive spectrophotometer is able to detect as low as 0.75 nanogram per microliter of dsDNA in a 1 microliter sample 

Fluorescence analysis assays can detect 0.005 nanograms per microliter of sample

Dynamic Range

Has a higher broadcast range than fluorescence analysis (able to detect 37500 nanograms per microliter)

Has a lower dynamic range than absorbance (able to detect up to 4000 nanograms per microliter)

Contamination detection

Many contaminants can be identified using absorbance measurement across an array of wavelengths  typically 260/280 nm and 260/230 nm) 

Fluorescence is unable to identify contamination within a sample

Additional resources

Fluorescence-based monitoring of the pressure-induced aggregation microenvironment evolution for an AIEgen under multiple excitation channels

iFluor® Dye Selection Guide

iFluor® 488 succinimidyl ester

Fluorescence Imaging Assay Development Services