The light Absorption photometry Szilvia Barkó University of Pécs, Faculty of Medicines, Dept. Biophysics February 2011 Transversal wave E Electromagnetic wave electric gradient vector wavelength The dual nature of the light: Wave (propagation) Particle (interaction) B x x magnetic field strength- vector The vectors of the electric the magnetic gradients are perpendicular to each other to the direction of the propagation of the wave. Diffraction nterference Polarization Photoeffect Compton-effect Types of the spectra line-type (atoms (gases in high temperature)) There is no intensity in wide spectral ranges The positions of the spectrum's lines are typical of the chemical element which emits (chemical ingredients, concentration) b type (molecules) Lots of lines near to each other, because of the rotation of the molecule, the vibrations of the atoms in the molecule Sir saac Newton (1642 1727) continuous (heated materials) All of the wavelength are observable The intensities of the neighbouring wavelengths connect to each other There is no big change in the intensities of the spectrum Classification of spectra based on the origin: emission type type 1
Continuous spectrum Heated fluid or solid materials Line type spectrum Emission spectrum of smouldering gases Joseph von Fraunhofer (1787 1826) Black lines in the spectrum of the sun, which are generated by the of some elements in the atmosphere of the sun at some wavelength. Line type spectrum Absorption spectrum of smouldering gases http://astro-canada.ca/_en/a3300.html The emission spectra of some elements He The spectrum Spallation of one wave e.g. electromagnetic wave to its component frequencies. One intensity-like quantity represented in the function of an energy-like quantity. Ne Ar intensity, count rate (e.g. measurement of radioactivity), number of photons, transmittancy, absorbancy (extinction, OD) energy energy-proportional quantities (e.g. frequency, wavelength, wavenumber) (nm) nteraction of the light with matter Quanted energy uptaking (photon) nteraction of electromagnetic wave with atomic system (matter): reflection transmmission The assumption of the biological effect s formation! The of the light The electric field of the incident lightbeam obliges to oscillate the charges of the small particles tiny oscillators or radios They emit electromagnetic waves f it is the resonance frequence: the amplitudes of the oscillations are going to grow (inner friction ) The oscillator s energy decreases The matter are going to particularly sink the radiation 2
Transmittance Exciting light source f resonance Atomic/molecular system Light intensity change Light source detector T = / Usually given in %. Absorption The - should be clear - should be easily measurable - additive E OD A = - log ( / ) = ( ). c. x =. 10 - ( ) x c Lambert-Beer law ( ): the extinction coefficient (depends on the material), c: the concentration of the, x: the optical length Note: the transmittance (T=/ ) is not additive: if 30% of the incoming light go through one substance 60% go through the other substance, then they are mixed together, it (the mixture) will not ever let go through 90% of the incoming light! Why ( ) not only? How to measure? photometry = spectroscopy The simple scheme of a photometer light source monochromator Prism or optical grating + slit detector (nm) The depends on the, that is why the must be depend on it too. Gives continuous light, e.g.: halogen, deuterium, xenon, etc. lamps n plastic, glass or quartz cuvettes 3
Single dual channel photometers Directly we can measure only the intensity! Emission measurements The linearity of the spectrophotometer; the stray light effect emission 3D direction! linear arrangement! The linearity of the spectrophotometer; the stray light effect n the case of low The origin of the problem: monochromators are not perfect! the second-third harmonics!!!! 99% chosen 89% chosen Optical grating 4
n the case of high Light scattering Can be an other problem in highconcentration s. 99% chosen 1% chosen The detector detects 2%!!! The application of the photometry Absorption by proteins To determine the concentration of different solutions (e.g. protein solutions!!!) Following time-dependent changes The evaluation of electrophoresis s the interpretation Measuring the of proteins The importance of the baseline Protein BUFFER!!! 5
Measuring the of proteins Measuring the of proteins Measuring the of proteins Additivity Application: to determine the protein-concentration protein fluorescent label (nm) Time-dependent measurements 0,2 Following material, qualitative or conformational changes (e.g. chemical reactions) Absorption 0,0-0,2-0,4 0 1000 2000 3000 4000 Time (s) 6