Absorption spectrometry summary

Transcription

1 Absorption spectrometry summary Rehearsal: Properties of light (electromagnetic radiation), dual nature light matter interactions (reflection, transmission, absorption, scattering) Absorption phenomena, change in light intensity: exponential and logarithmic function Lambert Beer law, absorbance, optical density, extinction coefficient, concentration, optical path (sample width) and their relationship Absorption (and emission): matter = sample = absorbent (synonyms) phenomena: excitation deexcitation, absorption emission, interaction of photon and electron atomic energy levels, energy difference of levels, photon energy, resonance condition atomic line type, molecular band type and highly heated matter s continuous (emission) spectra Interpretation of molecular BAND type spectra: origin, characteristics Born Oppenheimer approximation electronic, vibrational, rotational energy levels and their independence Jablonsky diagram (term scheme) interpretation of absorption phenomena on the Jablonsky diagram Absorption spectrometry fotometer: setup and arrangement: light source, monochromator, sample, detector (PMT), data aquisition and data handling unit (PC) principle of operation, one /two way fotometer, sample and reference

2 Absorption photometry Electron-spectroscopy Biophysics 2 nd semester Febr József Orbán University of Pécs, Department of Biophysics Rehearsal 1

3 Light electromagnetic radiation photon (quantum of radiation energy) : E = h f Dual nature of light: Electromagnetic wave (propagation) Maxwell Diffraction Interference Polarisation Particle (photon) (reaction) Einstein Photoeffect Compton-effect A b s o r p t i o n R e f l e c t i o n Propagation of electromagnetic wave If = 600 nm, then f = Hz x direction of propagation 2

4 E Propagation of electromagnetic wave electric field strength vector x magnetic field strength vector B x transversal wave wavelength c = f The electric- and the magnetic field strength vectors are perpendicular to each other and to the direction of propagation, as well! x Total spectra of electromagnetic radiations Energy, frequency (E=hf Wavelength ( =1/f) Gamma X-ray (Röntgen) E = hf c = f Visible range: light 3

5 Interaction of Light and Matter Radiation matter interaction electromagnetic (or radioactive) radiation matter = substance = absorber Radiation matter interaction I 0 (initial intensity) substance I I ~ n I: intensity n: photon number Reflection Transmission Absorption Scattering Rayleigh-type Which properties of the substance define the absorption? 4

6 light source Absorption matter I 0 I I 0 Exponential function!!! photon number: intensity: N I I ( x) ( x) N0 e I 0 e width Which function fits to the plot? x x Light absorption in a substance I 0 homogenous sample I 0 Always true: I 0 I 0 > I Intensity I reflected: R= I 0 -I 0 absorbed : A= I 0 -I transmitted : T= I width / optical path x In general reflection is negligible! Or it is eliminated by appropriate measurement setup. 5

7 light source Definition: ABSORBANCE I 0 general equation: Lambert-Beer equation (insolutions) matter I = I 0 e - x I DETECTOR I= I ( ) c x Why ( ) and not only? OD = A = - log 10 (I / I 0 ) = ( ) c x absorbance No dimension, additive quantity! optical density ( ): extinction coefficient (depends on constitution), c: concentration of solution, x: optical path (width) E x e r c i s e A substance transmits 10% of incoming light. Calculate the absorbance. A = OD = - log (I/I 0 ) = - log (0.1) = 1 If transmittance is 1%, the absorbance is? A = 2 Calculate the total absorbance if we use these together (10% and 1% transmittance)! A = 2+1 = 3, T = = = 0.1 % 6

8 S p e c t r a (line type emission spectra) Absorption and Emission of atoms 1. absorption state: ground state excited state e - e - + E abs = h + e - + phenomena: (photon) Absorption (electron) Excitation Absorption of the photon and the excitation of the electron occurs simultaneously! 7

9 Absorption and Emission of atoms 2. emission state: ground state excited state + e - +e - + E em = h e - phenomena: (electron) De-excitationexcitation (photon) Emission The de-excitation of electron and the emission of the photon occurs at the same time! The energy of the absorbed and emitted photon is equal and matches with the energy difference of the electron s ground and excited states energy! Electronic energy levels of atoms Bohr- and quantummechanic atommodel energy (level) energy difference Electrons have quantised (defined) energy energy levels! Figures are only for demonstration! 8

10 Spectrum Spectrum: (light) intensity or analogous quantity - plotted against wavelength or frequency. Types: line ( atoms ) band ( molecules ) continuous ( any material at high temperature: black body radiation ) Spectral types I emission absorption I Continuous, emission Line, emission Line, absorption See: continuous emission radiation, black body, Planck, Stefan-Boltzman Source of images: 9

11 Band type (absorption) spectrum of ACTIN molecule Absorption 2,5 2,0 1,5 1,0 0,5 How can you explain the shape? The line spectra of atoms and band spectra of molecules are characteristic! (depends on their chemical constitution) actin 0, Wavelength (nm) Absorption of proteins - aminoacids There are 3 aminoacids that absorbs in UV. (Measuring the absorption spectra of protein solution, the concentration can be determined.) tinction coefficient Ext wavelength 10

12 Spectra of molecules Born-Oppenheimer approximation: Atomic nuclei moves much slower than electrons, because the nucleus is much more heavy, therefore the simplified model of molecular motion considers the nucleus as not moving object. The electron- nucleus can be considered as two objects at two ends of a spring, with electric charge. Dynamic molecular structure Why band, and not line spectra? E total = E electronic + E vibrational + E rotational 11

13 Molecular energy-levels (terms) E total = E electronic + E vibrational + E rotational E total = E electronic + E vibration + E rotation E electronic ~ 1000 * E vibrational ~ * E rotational All the energy levels are below the zero level! More vibration level can appear at one electronic energy level. These vibrational energy levels are superimposed on each electronic level equidistantly separated from each other. One vibrational level can consist several rotational levels. As electron transitions can take place between several energy levels with not much different energy line spectra widens to band spectra! Jabłonsky-type term-scheme 0 Energy vibrational levels S 2 S 1 rotational levels S 0 All the energy levels are below the zero level! Each line represents a well defined (electronic, vibrational, rotational) energy state of the molecule. Ground state: the electrons stay at one vibrational level of the lowest electronic level (S 0 ). S stands for singlet state electronic levels: S 0, S 1, S 2 12

14 Jabłonsky-type term-scheme 0 S 2 1. Excitation & Absorption 2. De-excitation & Emission electron & photon Energy hf S 1 E = hf (resonance condition) Depending on the energy of the photon: Absorption electronic Emission hf vibrational rotational S mixed 0 transition happens. How do we determine absorbance? Setup of a (absorption) photometer light source monochromator (prism or grating) Sample Detector 1 Reference path Reference (blank) Detector 2 Light Electric signal Data aquisition (PC) To measure an absorbance spectra the absorbance values are determined from to, step by step) in a wide (spectral) range. 13

15 Basic concepts Light source PMT: Detection of light (photons): Converts electromagnetic radiation to electric signal. Photoelectron multiplier tube (PMT) photo on Photoeffect (photon force the cathode to eject an electron! photocathode dynodes electron If: 1e - 2 e - amplification: 2 n (n: number of dynodes) accelerating high voltage electron push other (1-6) electrons current/voltmeter Total amplification: 1 photon (photoelectron) 1-10 million electrons! 14

16 Two forensic investigator speaks regarding at a blood sample: - I am sure that he died of hampered respiration. - Cyanosis? How do you know it? - From the color of the blood! (How) is it possible? Oxy- and carboxy-hemoglobin The difference in absorption in the range nm explains the different appearent color. Two peaks of HbO 2 measured on practical: Spectroscopy and spectrometry 15

17 Interaction of Electromagnetic Radiation and Matter (absorbtion) Phenomenon to study Spectral Range Wavelength Inner electron shells, X-rays nm ionization Valency (outer) electrons Ultraviolet Visible nm nm Molecular vibrations (bond stretching), rotation Rotation & electron spin orientation in magnetic field Nuclear spin orientation in magnetic field Infrared 800 nm 0.8 mm Microwaves 0.8 mm - 30 cm Radio waves >100 cm Supplement /Spectroscopy /Spectrum White light can be split to several colours (with prism or optical grating) Red Orange Yellow Green Blue Violet R O Y G B V We may understand the colours of our colourful environment if we keep in mind that only photons reaching our eye can define the colour of the percepted objects. 16

18 Supplement Surface colours defined by reflection Red Orange Yellow Green Blue Violet What is the colour of this object? Search for the term: Additive / constructive colour mixing e.g.: colour of the furniture, wall, ink, paper, hair, iris Supplement body colours defined by absorption (transmission) Red Orange Yellow Green Blue Violet What is the colour of this object? e.g.: colour of the red blood cells, leaves, lead glass windows 17