Lab Quiz #5 - Fluorometry, Chemiluminescence, Reflectance Spectrophotometry

What is the measurement principle of Fluorometry?

Measures emission of light from fluorescent substances.

a type of luminescence in which electrons within a chemical are induced to absorb light and become excited using high-energy radiation.

The result is emission of low-energy light (longer wavelengths) very quickly after absorbing high-energy light (short wavelengths)

What is the measurement principle of Chemiluminescence?

measures light that is emitted by natural bioluminescent substances

What is the measurement principle of Reflectance Spectrophotometry?

measures light that is reflected from a solid surface.

Some light is absorbed by the surface; the light that is not absorbed is measured

Reflected light is not linear to concentration; concentration must be calculated by a computer

What are the light sources used in fluorometry?

a mercury or xenon arc lamp

What is the angle of the detector in fluorometry?

90 Degrees

What does fluorometry measure?

labels that have been placed on reagents

What is quenching?

A reduction in the overall output of emitted light owing to the presence of a chemical or colored interfering substance.

changes the structure of analyte, making it less fluorescent

What causes quenching?

Changes in pH (max. pH for analytes to fluoresce)

Changes in temperature of the reaction (⭣ in fluorescence with ⭡ temp.)

Changes in the solvent

Contamination of the reaction mixture.

What does quenching cause results to be?

Falsely decreased (less fluorescence)

What are the two most common chemical labels used in chemiluminescent measurements?

Acridinium esters

and

Luminol

Acridinium esters

A popular chemiluminescent label, acridinium ester, is a triple-ringed organic molecule linked by an ester bond to an organic chain.

In the presence of hydrogen peroxide

and under alkaline conditions, the ester

bond is broken and an unstable molecule (N-methylacridon) remains.

Light is emitted as the unstable molecule reverts to its more stable ground state.

Luminol

Simple, organic molecule.

When luminol is oxidized by H202 in the presence of a catalyst (peroxidase), it yields an unstable 3-aminophthalate

that emits light as it reverts to its ground state

What initiates the conversion process of those label molecules that allows the reaction to take place for Acridinium esters and Luminol?

H202 (hydrogen peroxide)

What is the substance measured in Chemiluminescence?

light that is emitted by natural bioluminescent substances

What are the advantages of Fluorometry?

1000x more sensitive than absorbance spectrophotometry

What are the advantages of Chemiluminescence?

no external light source is required

analytically sensitive and can be used as a label for immunoassay reactions to measure such substances found in low concentrations

What are the disadvantages of Fluorometry?

Can be susceptible to quenching

What are the disadvantages of Chemiluminescence?

Contamination of the reaction mixture may decrease chemiluminescence

hemolysis, lipemia, icterus, bacteria contamination, elevated protein levels