What are the differences between absorbance and fluorescence?

Question

Accepted Answer

Basis of differentiation

Absorbance

Fluorescence

Definition

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

Effectiveness

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

Is more effective because the assay is highly specific

Functionality

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)

Sensitivity

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

Basis of differentiation	Absorbance	Fluorescence
Definition	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
Effectiveness	Is less effective due to the technique not being sample-specific	Is more effective because the assay is highly specific
Functionality	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)
Sensitivity	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