PHAR 204 - Spectrophotometry

What is a photomultipler

A device used to convert photons into an electrical signal

Describe the uses of a photomultipler

• Measures drugs + drug metabolites

• Important for endpoint quantitative measurements across scientific fields

Define absorbance

The weakening of radiation (ex: visible light, UV light) as it passes through a molecule

How is absorbance mathematically defined

As an inverse of transmittence

What are the common types of radiation absorbance assesses

UV (200nm - 400nm)

Visible (400nm - 800nm)

Define the Beer Lambert Law

Absorbance is directly proportional to molar absorptivity, path length, and solution concentration

What is a characteristic of molar absorptivity

Remains constant

Define path length

The distance light travels through a sample

What are the two limitations of Beer Lambert’s Law

• Saturation

• Insoluble substances

Why is saturation a limitation of Beer Lambert’s Law

• As concentration increases, absorbance can saturate and is no longer linear

Why is insoluble substances a limitation of Beer Lambert’s Law

cause light scattering

What are the two applications of absorbance

• Total DNA/RNA quantitation

• Total Protein Quantitation

What does a ratio of 260nm/280nm indicate

Purity of DNA/RNA in comparison to protein

What does a ratio of 260nm/230nm indicate

Purity of RNA/DNA compared to other contaminants

Explain how proteins can be quantified

Uses Bradford Assay

Define Bradford Assay

Uses Coomassie Blue to measure protein levels

Outline the outcomes of Bradford Assays

• Absence of protein —> 465 nm

• Presence of protein binding —> 595 nm

What is another factor that absorbance is dependent on

Conjugation

Define conjugation

The alternation of single and double bonds within a compound

How does absorbance influence conjugation

The longer the chain of alternating single + double bonds —> longer absorbance wavelength

Define monochromator

Filters light to one specific wavelength

Define photodiode array

Transmits the full spectrum of light into a sample.

Dispersed into individual wavelengths and detected

Define fluorescence

The absorbance of light at a particular wavelength and its subsequent emission at a longer wavelength

Define emission

Wavelength at which molecule releases light

Define excitation

Wavelength at which molecule absorbs light

Define stokes shift

Difference between maximum excitation and emission

Outline the procedure of fluorescene

1) Intense beam of light is directed towards sample

2) Emission is measured at 90

3) Emitted light is filtered and detected in visible spectrum

Why is light measured at 90 for fluoresence

To avoid interference from excitation beam

What is advantage of fluorescene

Lower LOD than absorbance

What is the disadvantage of fluorescence

Fewer compounds are capable of fluorescence

What is a factor that influence a compound’s ability to be fluorescent?

The presence of conjugated bonds (conjugation)

What is application of fluorescene

Fluorescence resonance energy transfer (FRET)

Define Fluorescence resonance energy transfer

Transfer of electrons between two light sensitive chromophores

What can FRET indicate?

Assesses if two proteins are in close proximity or interact with each other

Provide an example of how FRET can be measured

CFP-YFP tagged proteins

What wavelengths is emission and excitation for cyan fluorescent protein

Excitation = 440 nm

Emission = 480 nm

What wavelengths is emission and excitation for yellow fluorescent protein

Excitation = 480 nm

Emission = 530 nm

What is the procedure for CFP-YFP tagged proteins

1) Protein A is tagged with CFP

2) Protein B is tagged with YFP

3) 440 nm of light is directed at sample

Does the emission wavelength of 480 mean in CFP-YFP

No fret is occurring

What does the emission wavelength of 530 mean in CFP-YFP

Fret is occurring

Define luminescene

The emission of light

What are the two types of luminescene

Chemiluminescence + Bioluminescence

Define chemiluminescence

Emission of light due to a chemical reaction

Provide example of chemiluminescence

Used for Western Blotting

1) Uses HRP enzyme + luminol substrate which causes chemiluminescence that can be used for immunodetection

Define bioluminescence

Emission of light due to an enzymatic reaction

Provide an example of bioluminescence and how it may be used

Firefly luciferase

When modified, luciferase can be used to measure drug receptor activity

What is an application of bioluminescence

What are the advantages of luminescence

More sensitive than absorbance and fluorescence

What are the disadvantages of luminescence

• Requires a substrate

• Luminescence is short lived

What are the two types of luciferase

Flash = more sensitive but short acting

Glow = less sensitive but longer acting

What are the ways samples can be measured for photomultiplers

Cuvette + plate

Define cuvette

Small rectangular shaped tube to store sample for measuring

What is the disadvantage of cuvette

Low throughput; one sample can be measured

Define plate

Contains several samples depending on number of wells

Colour of plate is important for measurements

What are the number of wells that can be used

24

48

96

384

What colour of plate is used for absorbance

clear

What colour of plate is used for fluorescence

Black opaque

What colour of plate is used for luminescence

White opaque plate