Relationship between A(OD) and %T
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Transcript of Relationship between A(OD) and %T
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