Module 3b: Luminiscence

Question 1

Define luminiscence

Answer 1

any emission of light when an electron returns from an excited state (by any other energy other than heat) to ground state

Question 2

There are four types of luminiscence which are

Answer 2

1. fluorescence
2. phosphorescence
3. chemiluminiscemce
4. electrochemiluminiscence

Question 3

Luminiscence should be observed at what direction and why?

Answer 3

Perpendicular to the incident light to avoid detection of scattered radiation

Question 4

In quantitative analysis, the intensity of luminiscence is proportional to

Answer 4

concentration of species (C)
incident radiant power (P0)
constant depending on emitting molecule (k)

Question 5

An atom or molecule that can fluoresce

Answer 5

fluorophore

Question 6

Makes Fluorescence measurements 100 to 1000 times more sensitive than absorbance measurements

Answer 6

1. intense light source
2. digital signal filtering
3. sensitive emission photometers

Question 7

Principle of Fluorometry

Answer 7

Molecule that has greater energy that the environment fluoresces where returning to ground state.

Question 8

Test based on the principle of fluorescence

Answer 8

Fluorometry

Question 9

Components of fluorometers

Answer 9

1. light source (excitation source)
2. excitation or primary monochromator (larger than absirbance spectrophotometers)
3. sample cell
4. emission or secondary monochromator
5. detector

Question 10

Difference between fluorometer and spectrofluormeter

Answer 10

Fluorometer - uses interference or glass filters

Spectrofluorometer - prism or grating monochromator

Question 11

Application of Fluorometry

Answer 11

• fluorescent tags or labels
• hematofluorometry of zinc protoporphyrin in whole blood
• flow cytometry

Question 12

Concentration effect that refers to the loss of excitation intensity across the cuvet path length as the fluorophor absorbs the excitation light

Answer 12

Inner filter effect

Question 13

Concentration effect where a macromolecule, like an antibody, is labeled heavily with a fluorophor, and radiationless energy transfer occurs.

Answer 13

concentration quenching

Question 14

The difference between excitation light and emitted fluorescence in fluorometry

Answer 14

Stoke’s Shift

Question 15

When the excitation and emission spectra overlap in fluorescence.

Answer 15

Light Scattering

Question 16

Limitation of fluorometer in which a serum or urine sample contain many compounds that fluoresce which contributes to unwanted background fluorescence.

Answer 16

sample matrix effects

Question 17

The most serious contributors to sample matrix effect in fluorometry

Answer 17

proteins and bilirubin

Question 18

The temperature in fluorometry must be regulated within

Answer 18

+/- 0.1 °C

Question 19

Fluorescence intensity ________ with increasing temperature by approximately

Answer 19

decreases

1-5 percent per degree Celsius

Question 20

Limitation of fluorrometry where weakly fluorescing or dilute solution are affected by intense light sources

Answer 20

photodecomposition

Question 21

Six limitation of Fluorometry

Answer 21

1. concentration effects
2. Light scattering
3. solvent and cuvette effects
4. sample matrix effects
5. temperature effects
6. photodecomposition

Question 22

Test that is similar to flurometry but is distinguished in that it continues to occur even
after the radiation causing it has ceased.

Answer 22

Phosphorescence

Question 23

Difference between the emitted light in Fluoremetry amd Phossphorescence

Answer 23

There is a larger shift in wavelength in phosphorescence

Question 24

Types of luminescence in which the excitation event is caused by a chemical or electrochemical reaction, not by photolumination

Answer 24

Chemiluminiscemce and bioluminiscence

Question 25

Principle of chemiluminiscence

Answer 25

Compounds react with an oxidizing agent, with a catalyst, It shifts into excited state. Light is emitted as the product returns to ground state.

Question 26

Common substrates in chemiluminiscence

Answer 26

luminol isoluminol acridinium estees luciferin

Question 27

Common oxidants in chemiluminiscence

Answer 27

hydrogen peroxide hypochlorite oxygen

Question 28

Commonly used catalysts in Chemiluminiscence

Answer 28

``` enzymes (alkaline phospahatase, horseradish peroxidase, microperoxidase) metal ions (copper, ferric phthalocyanine complex) ```

Question 29

substrate, oxidizer, and catalyst used in forensics as diagnostic tool for the detection of blood and other biological fluids.

Answer 29

luminol, hydrogen peroxide, peroxidase

Question 30

A special form of chemiluminescence occurring within biological systems.

Answer 30

bioluminiscence

Question 31

Biological substrate, oxidizer and catalyst in fireflies

Answer 31

Luciferin, oxygen, luciferase

Question 32

another example of biological caatalysts in bioluminiscemce

Answer 32

aequorin

Question 33

Type of luminescence where the reactive species that produce the chemiluminescent reaction are electrochemically generated.

Answer 33

Electrochemiluminiscence

Question 34

Advantages of Electrochemiluminiscence

Answer 34

reagent stability simple reagent prepartion enhanced sensitivity

Question 35

A physical phenomenon resulting from the interaction of light with particles in solution.

Answer 35

Light scattering

Question 36

Difference between fluorometry and light scattering techniques

Answer 36

scattered light has smiliar frequency as incident light

Question 37

Best application of Light scattering techniques

Answer 37

Immunoassay of specific serum proteins and haptens

Question 38

Two types of light scattering techniques

Answer 38

turbidimetry | nepehlometry

Question 39

Measures the decrease in intensity of the incident light at 180 degrees from the incident beam

Answer 39

turbidimetry

Question 40

It is the detection of light energy that js scattered or reflected toward a detector that is not in the direct path of the transmitted light, usually measured at a right angle.

Answer 40

nepehelometry.

Question 41

What is measured in turbidimetry?

Answer 41

blocked light

Question 42

what is measured in nephelometry?

Answer 42

scattered light

Question 43

Nephelometry that is commonly used in measuring macromolecules with size larger than the wavelength of incident light.

Answer 43

Forward scattering nephelometer