Master 2 ème année : Sciences et Technologies Mention : Sciences de l’Univers, Environnement, Ecologie Spécialité : Océanographie et Environnement Marin Spécialité : Océanographie et Environnement Marin Unité d’Enseignement : Evolution du Phytoplancton Marin et Biogéochimie Methods of Photosynthesis Spectrometry Christophe Six
Définitions Spectrometry = spectroscopy : Methods of spectral analysis allowing to understand the composition the structure of matter and/or the study the composition, the structure of matter and/or the study of systems transferring energy � Qualitative and quantitative studies of spectra derived from the interaction � Qualitative and quantitative studies of spectra derived from the interaction between the matter and the wavy radiations of different frequences . Spectrophotometry is an analytic, quantitative method that consists in measuring the absorbance (= absorption = optical density) of a given chemical substance (or of a whole unicell organism) in solution, function of the light wavelength. (or of a whole unicell organism) in solution, function of the light wavelength. Spectrofluorimetry is an analytic, quantitative method that consists in measuring p y y , q g the emission and excitation levels of fluorescence of a given chemical substance (or of a whole unicell organism) in solution, function of the light wavelength.
Energy and wavelength E = (h . c) / λ 800 nm E : Photon energy E : Photon energy h : Plank constant factor Wavelength c : Light celerity in nanometers l : Photon wavelength 400 nm X-rays U.V. Visible Infrared Radio wavelengths
Absorbance of molecules and molecular complexes .Understanding photophysics and photobiology Very useful for assays .Very useful for assays � Using colorimetric assays � Using colorimetric assays
Photomultiplicator A = log (I 0 / I) Absorbance I Concept of absorbance and measurement A = -log T Sample > I 0 Transmittance T = I / I 0 Light Source
Spectrophotometers Components : Components : .One or several light source(s) Extended Visible (350-900 nm) : Tungsten, Halogen ( ) g g UV (<400 nm) : Deuterium . One monochromator : Selection of wavelengths . One sample compartment . One detector : photomultiplicator or photodiode detector . A result display system
Single beam spectrophotometers D.O. or monochromator or monochromator λ ( λ (nm) 400 400 500 500 )
Single beam spectrophotometers . The simplest system . A simple compartment for a single sample cuvette . The reference = blank is measured before the samples for zeroing the device Blank : all chemical components (buffer, solvant, etc) except the absorbing substance that you want to measure. It is actually rare to be able to use a perfect true blank but one should approach it as much as possible. approach it as much as possible. . Instrument useful for simple routine applications (single or few wavelengths) Various colorimetric assays (proteins, nucleic acids, pigments, etc.) V i l i t i ( t i l i id i t t ) . Main problems In single wavelength mode, one cannot check for artefacts The decrease of lamp intensity is not compensed I 0 I The making of these instruments is usually less careful (fix) (measured)
Double beam spectrophotometers M Monochromator h t Reference Cuvette I 0 Chopper Chopper Sample Cuvette Cuvette I
Double beam spectrophotometers . For each wavelength, one mesures the absorbance of the sample AND the absorbance of the reference (blank) . Good reliability of the measurements, ideal for absorption spectra (Eli (Elimination of solvent absorption) i ti f l t b ti ) . Correction of the variations of the light sources
Artefacts Refraction : déviation of a wave when its speed changes (interface between 2 media) Diopter (surface of the cuvette and surface of the sample) => A λ . Other optical phenomenons linked to diffusion, reflexion and diffraction . Other optical phenomenons linked to diffusion, reflexion and diffraction of light may also distort the measurement. of light may also distort the measurement.
Artefacts : Light diffusion . Turbid solutions, cell suspensions Turbid solutions cell suspensions => A λ Diffusion occurs when some light is deflected by particules and therefore does not reach the detector F( d , n ) σ S = = Diffusion of Rayleigh λ 4 λ d : Diameter of particules n : Refraction index Diffusion also depends on : λ : Wavelength - Particule concentration - Particule shape
Impact of diffusion on absorption spectra 5,0E-11 4,0E-11 y = 1 1 3,0E-11 Diffusion is λ -dependent x 4 2,0E-11 1 0E-11 1,0E-11 0,0E+00 350 450 550 650 750 850 Longueur d'onde (nm) g ( ) => A λ ok => A λ
Impact of diffusion on absorption spectra Example : absorption spectrum of a phycoerythrin I Fitting a correction curve Final spectrum Spectrum with diffusion ce sorbanc Abs Wavelength (nm)
Measuring absorbance in a diffusing sample => A λ Bringing the detector Bringing the detector nearer to the cuvette Increasing the surface g of the detector
Measuring absorbance in a diffusing sample Homogeneous Détecteur du Echantillon Light sample photomultiplicateur homogène detector A DO DO (nm) (nm) (nm) (nm) B DO DO Suspension Cell (nm) (nm) de cellules suspension C DO Light Rayon (nm) lumineux beam Source Light source and Light source and lumineuse et monochromator Sphère Integration monochromateur d’intégration sphere
If the absorbance of a sample is not stable… . Sample much colder than the atmosphere of the compartment � Condensation on the cuvette � Condensation on the cuvette � Gaz formation (diffusion) . Sample drops on the outside of the cuvette Sample drops on the outside of the cuvette . The sample contains absorbing particules that sink in the cuvette . There’s not enough sample in the cuvette and the beam passes through the meniscus . Cuvettes not adapted (micro-cuvettes)
The Beer-Lambert law At a given wavelength the absorbance of a solution is proportional to the At a given wavelength, the absorbance of a solution is proportional to the concentration of the absorbing chemical species that are present in this solution, and to the optical path A : Absorbance (no unit) A λ : Wavelength (nm) A λ = ε λ . l . C l : Optical path (cm) λ λ C : C C : Concentration (mol L -1 ) l L 1 ) t ti ( ε λ : E xtinction coefficient (L mol -1 cm -1 ) . The Beer-Lambert law is additive. Pour n chemical species : A λ = ε λ ,1 . l . C 1 + ε λ ,2 . l . C 2 + ε λ 3 . l . C 3 + … + ε λ ,n . l . C n = ε l C + ε l C + ε + ε A l C + l C A λ = ε λ . C => C = A / ε λ . For l = 1 cm : A λ = ε λ ,1 . C 1 + ε λ ,2 . C 2 + ε λ 3 . C 3 + … + ε λ ,n . C n = ε C + ε C + ε + ε A C + C
Stokes shift Fluorescence: what is it ?
Intensity of fluorescence emission . With fluorescence, there’s no general relation such as the absorbance Beer-lambert law The measurement depends strongly on : - The nature of the fluorescent system that is studied y - The device used to quantify fluorescence (light source intensity, optics configuration, etc.) � Need to use standard curves to quantify molecules by fluorescence � Need to use standard curves to quantify molecules by fluorescence . It is possible to quantify the fluorescence energy when a fluorescence quantum yield Q f : Energy of fluorescence emitted (I f ) = Absorbed energy ( I a ) x Q f Q f = f ( λ , T°C, pH, ions, etc.)
Spectrofluorimeters . None photon from the excitation light must be detected by the detector � excitation at 90° On average there is 10 6 times less photons that hit the detector of a spectrofluorimeter On average, there is 10 times less photons that hit the detector of a spectrofluorimeter than in a spectrophotometer Main components : - A light source : Mercury or xenon lamp A light source : Mercury or xenon lamp - Two monochromators selecting either the emission or excitation precise wavelengths - A dark compartment with the cuvette in a 90° excitation/emission cuvette holder - A photomultiplicator
Diagrammic representation of a spectrofluorimeter Photomultiplicator Photomultiplicator Xenon lamp Lens Entrance Slit Exit slit Monochromator Monochromator shutter Monochromator Slit Mirror Lens Lens Sample
Emission and Excitation spectra of fluorescence Emission spectrum Monochromator scanning Monochromator scanning all wavelengths ∼ 15 nm Quantification of the Fix monochromator : Fix monochromator : fluorescence emitted fluorescence emitted One given λ Emission by the excitation of a given λ Sample Excitation 400 500 600 700 At which λ is the maximum of fluorescence emission of the compound ? p Excitation spectrum Fix monochromator : One given λ Quantification of the fluorescence emitted many wavelengths λ Monochromateur scanning Emission all wavelengths Sample Excitation 400 400 500 500 600 600 700 700 Which λ gives rise to the fluorescence (Excitation spectra are often similar to absorption spectra) emission at a given λ ?
Fluorescence of marine picocyanobacteria : Synechococcus spp.
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