Relationship between A(OD) and
%T
Transmittance, T = P / P0%
Transmittance, %T = 100 T
Absorbance, A = log10 P0 / P
A = log10 1 / T
A = log10 100 / %T
A = 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).
ELISA
Enzyme-Linked Immunosorbent Assay
LDH Cytotoxicity Assay
Endpoint vs Kinetic
Endpoint vs Kinetic
Buffer Dilution
• V1 x C1 = ?
V2 x C2
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
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
Energy
Absorbance
Fluorescence
Vibrational and rotational relaxation
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.
scattering
Exitation
reflection
Emission
Spectrofluorometer
Detector
monochromator
Emission
Excitation
scattering
Exitation
reflection
Emission
Microscope and Plate Reader
Detector
Filter
Excitation
Emission
Dichroic Mirror
scattering
Exitation
reflection
Emission
Optical Path Microplate Reader
scattering
Exitation
reflection
Emission
Filter and Dichroic Mirror
http://www.chroma.com/products/catalog/11000_Series/11000v3
http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html
https://www.omegafilters.com/curvo2/index.php