Experiment 3: Absorption, Beer Lambert law and Flourescene

Wavelength

Distance between 2 peaks, variable

Electromagnetic spectrum

Continuum of waves at different wavelengths,

Absorption

Interaction of electromagnetic field with mater, transfer of energy carried by photons to molecules that absorb a

Photon

Elementary particle, carries electromagnetic radiation

Energy of photons (E)

E=hc/lambda, h=planks constant, c=speed of light, lambda=wavelength of light carrying it, inversely proportional to wavelength

Absorption

Photon strikes molecule and is absorbed (energy photon=energy difference between energy levels), electron in ground state is excited into higher energy state, very quick (10^-15), remains for short time before return to ground (release energy)

Consider when detection of absorption

light source, select wavelength, direct and control shining, measuring the light, quantifying

Spectrophotometers key elements

Light source, Monochromator and slits, Cuvettes and cuvette holders, Dectectors

Light source depends

wavelength, consistency of intensity, durability and cost

Light source examples

Halogen (350-2500), Deuterium (<200-400), Xenon arc (<200-1100)

Monochromator

Separate light into different distinct wavelengths, primarily prisms or grating

Coloured filter

Mostly in fluorescence, selected for light using colorimetry

Slits

Select for particular wavelength, allows selective passage of light in wavelength

Cuvettes

1 or 3mL, path length typically 1cm, different materials for different wavelengths,

Cuvettes material

Polystyrene (325-750), PMMA (245-750), Quartz (170-2500)

Micro volume spectrophotometer

Small volumes (1-2.5 uL), use fiber optical cable to detect

Plate reader

Multiple samples, hold <150uL

Detectors

Gather and quantify intensity or wavelength reaching it

Blanks

Reference solution, same component as sample minus analyte

Dual beam spectrophotometer

Measure amount of light through blank and analyte simultaneously, split light into 2 beams, more expensive

Single beam spectrophotometer

Do not split light beam

Incident light (Io)

Intensity of light reaching detector during blank

I

Intensity of light reaching detector with analyte

Absorbance equation

A= log (Io/I)

Absorbance

Quantification of absorption process

Beer Lamber law considerations(3)

Wavelength used, thickness absorbing material, concentration absorbing material

Beer lambert law

A=Elc, A=absorbance, E=molar absorption coefficient(1/M*cm), l=path length(cm), c= concentration analyte(M)

Molar absorption coefficient

Constant of substance at particular wavelength, units 1/M*cm

Calibration curve

Graph that depicts response to some analyte under known conditions, relationship between [] and signal for instrument (absorbance proportional to concentration)

Absorption spectra

Graph, Absorption of light for molecules over broad range of wavelengths, peaks=elevated absorption

Lambda max

Wavelength at which highest absorption occurs

Chromophores

Structure in molecules that absorb light, tend to have conjugated double bonds,

Protein chromophores

Peptide bonds(lambda at 190), only aromatic side chains absorb (Trp/Tyr at 280), some have prose this groups or coenzymes (hemes)

Intrinsic absorption

Efficient absorption of Trp and Tyr for quantification

Hemes

In FAD and NADH, found oxygen transporting molecules or redox reactions

Nucleotides and nucleic acids

Aromatic rings absorbs at 260, quantitation of in solution, monitor change in secondary and tertiary structure

Light absorbing molecules

Proteins, nucleic acids, plants pigments (beta carotene)

Factors affecting absorption (4)

Solvents, Protonation, Redox state, Interaction effects

Solvents affects on absorption

Influence stability electronic states

Protonation affect on Absorbance

Affect absorption in certain regions (2nd peaks)

Redox states affect on Absorbance

Absorbance spectrum change depending redox state, increase sensitivity spectrophotometer to redox states

Interaction effects affect of Absorbance

Absorbance can change when interacting with other molecules (ex: dsDNA vs ssDNA), hyperchromicity

Fluorescence

Only when molecule is excited, measure photon release when falls back to ground state (less energy)

Fluorescence steps

Excitation- absorption of photons, internal relation in excited states (lose some energy), Emission-excited electron falls back to ground releasing photon

Stokes shift

Shift to higher wavelength when photon realized (less energy)

Sensitivity of fluorescence

Detect individual photons

Limitations of fluorescence

Scarcity of fluorescent. Molecules, Artefacts (probes attached), Photobleaching (lose fluorescence after continuous excitation), Toxic effects (kills cells), cost (more expensive)