Relationship between A(OD) and %T

Transmittance, T = P / P0%

Transmittance, %T = 100 T

Absorbance, A = log10 P0 / PA = log10 1 / T A = log10

100 / %TA = 2 - log10 %T 

Beer Lamert’s Law

Reflection

Light scattering

reflection

scattering

For Solution: Scattering 1/4

UV-Vis Spectrum of Milk

Prism

Diffraction grating

Spectrophotometer types -Single beam-Dual beam-Diode array

Single Beam - Spectrophotometer

Dual Beam - Spectrophotometer

Dual Beam – Single Detector

Diode Array - Spectrophotometer

NanoDrop

Bradford Assay

Substrate (S) and enzyme (E) combine to form the enzyme/substrate complex (ES). The complex then dissociates to yield enzyme (E) plus product (P).

Enzyme-Linked Immunosorbent Assay

ELISA

LDH Cytotoxicity Assay

Endpoint vs Kinetic

Endpoint vs Kinetic

Buffer Dilution

• V1 x C1 = Example: Need to make 1 L of 1mg/mL solution given 100mg/mL

stock

Example 2:Need to add component from 5.2x stock to 200mL of

sample

?V2 x C2

Fluorescence

is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength.

George Gabriel Stokes named the phenomenon fluorescence in 1852.

The name was derived from the mineral fluorite (calcium difluoride)

Molecular Orbital

Factors that influence on Fluorescence

pH

Solid state or Solution state

Solvent

Vibrational and rotational relaxation

Absorbance Fluorescence

Ene

rgy

The excitation and emission spectra of a fluorophore and the correlation between the excitation amplitude and the emission intensity. General diagram of the excitation and emission spectra for a fluorophore (left). The intensity of the emitted light (Em1 and Em2) is directly proportional to the energy required to excite a fluorophore at any excitation wavelength (Ex1 and Ex2, respectively; right).

The Stokes shift of the excitation and emission spectra of a fluorophore. Fluorophores with greater Stokes shifts (left) show clear distinction between excitation and emission light in a sample, while fluorophores with smaller Stokes shifts (right) exhibit greater background signal because of the smaller difference between excitation and emission wavelengths.

reflection

Emission

scattering

Exitation

Emission

Excitation

Spectrofluorometer

reflection

Emission

scattering

Exitation

reflection

Emission

scattering

Exitation

Detector

monochromator

EmissionExcitation

Dichroic Mirror

Microscope and Plate Reader

reflection

Emission

scattering

Exitation

reflection

Emission

scattering

Exitation

DetectorFilter

Optical Path Microplate Reader

reflection

Emission

scattering

Exitation

reflection

Emission

scattering

Exitation

http://www.chroma.com/products/catalog/11000_Series/11000v3

Filter and Dichroic Mirror

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html

https://www.omegafilters.com/curvo2/index.php