Relationship between A(OD) and %T Transmittance, T = P / P 0 % Transmittance, %T = 100 T Absorbance, A = log 10 P 0 / P A = log 10 1 / T A = log / %T A = 2 - log 10 %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

Buffer Dilution V 1 x C 1 = 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 ?V 2 x C 2

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 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 AbsorbanceFluorescence Energy

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 Detector monochromator

Emission Excitation Dichroic Mirror Microscope and Plate Reader Detector Filter

Optical Path Microplate Reader

Filter and Dichroic Mirror