Fluorescence Spectrophotometry

Question 1

• A form of luminescence
• A substance emission of visible light after absorbing any form of electromagnet radiation
• examples: led lights, glow in the dark’s items, gemstones

Answer 1

Fluorescence

Question 2

It measure how much a chemical substance absorb light by measuring the lights intensity as a beam of light passes through a sample solution

Answer 2

Spectrophotometry

Question 3

Set of techniques that deal with the measurement of fluorescence that substances have emitted when they are exposed to uv visible or any electromagnetic radiation

Answer 3

Fluorescence spectrophotometry

Question 4

Basic principles

Answer 4

Excitation
Emission
Fluorophore
Jablonski diagram
Fluorescence lifetime

Question 5

• Begins when a molecule absorbs light causing it to promote electrons to a higher energy state
• From single ground state promoted to a single excited state

Answer 5

Excitation

Question 6

• Occurs when the excited state of a molecule returns to its ground state
•Involves emitting of photons that has a lower energy and longer wavelength than the absorbed

Answer 6

Emission

Question 7

• a molecule with fluorescence properties
• absorb light energy of a specific wavelength and re-emit it at a longer wavelength

Answer 7

Fluorophore

Question 8

• by physicist alexander joblonski
• shows the process involved in creation of an excited electronic single state by light absorption and subsequent emission of fluorescence

Answer 8

Jablonski diagram

Question 9

• The average time that the fluorophore spends in the excited state depending on the molecule type and its local environment
• varies from picoseconds to hundreds of nanoseconds depending on the molecule

Answer 9

Fluorescence lifetime

Question 10

General components

Answer 10

White light source
Excitation monochromator
Sample chamber
Emission monochromator
Detector

Question 11

• A light source that produce a wide range of wavelength from ultraviolet to infrared

Answer 11

White light source

Question 12

Selects the desired wavelength of excitation light which is focused at the sample position

Answer 12

Excitation monochromator

Question 13

How’s the cuvette (transparent tube) that is designed to hold samples that contain the analyte

Question 14

• Begins when a molecule absorbs light causing it to promote elections to higher energy state
• FROM SINGLE GROUND STATE PROMOTED TO A SINGLE EXCITED STATE

Answer 14

Emission monochromator

Question 15

• Occurs when excited state of a molecule return to its ground state
• involves emitting of photons that has a lower energy and longer wavelength than the absorbed photon

Answer 15

Emission

Question 16

Process of fluorescence spectrophotometry

Answer 16

1. The light passes through a lens that converts into a beam of light and strikes through the monochromator
2. The monochromator separates the beam of light into component wavelengths
3. The selected wavelength passes through and interacts with a sample.
4. The detector senses and measures the transmittance and absorbance of the sample
5. The detector will convert the information into a digital display

Question 17

Analytical steps (murag katong process naman ang analytical steps. Pero go)

Answer 17

1. Turn on the spectrophotometer. let it sit for 15 mins
2. Choose and set the wavelength of light to analyze the sample with
3. Calibrate the machine with the blank
4. Measure the absorbance of your experimental sample
5. Repeat the test with successive wavelengths of light