SUBJECT

Spectroscopy of Plant Materials

practical

master

Faculty of Science

Biology MSc

3

Semesters 1-4

Autumn/Spring semester

1) The theory of absorption spectroscopy. The structure of spectrophotometers, the function of the optical elements. Common methods of absorption spectrocopy in biochemical laboratories. Room-temperature and 77 K measurements. Derivative, difference and kinetical measurements. Error sources and the possibilities to avoid them. False signals of the spectrophotometer, light scattering (Éva Sárvári).

2) Commercial spectrophotometers, their elements, spearparts and accessories.. How to control the wavelength-correctness and -accuracy? Calibration procedures (Éva Sárvári)

3) Concentration determination. Determination of calibration curves. Protein concentration measurements. Chlorophyll extraction with different solvents, and the measurent of their concentrations (Éva Sárvári).

4) Basic rules of absorption spectrum measurements. The effect of the slit broadness on the shape of the absorption spectrum. Difference spectroscopy: measurement of cytochrome-f and P700. Measurement of scattering samples (chloroplasts suspension with "opal glass technics" (Éva Sárvári).

5) Measurement of enzyme kinetics (Éva Sárvári).

6) The theoretical ground of fluorescence measurements. The electronic structure of fluorophores. The Jablonski diagram. Electronic transitions and absorption/fluorescence bands. The role of the molecular environment of fluorophores in forming the spectral properties. The effect of dielectric constant of solvents on the electron structure of the chromophores. Chromophore-cvhromophore interactions and exciton splitting (Béla Böddi).

7) The structure of spectrofluorometers. How to measure emission and excitation spectra, steady-state and kinetical measurements. Error sources and possibilities for error corrections (Béla Böddi).

8) Concentration determination with fluorescence. Sensitivity and error sources. The concentration caused fluorescence quenching: measurement. How to avoid ghost peaks? How to use polar filters: advantages and dangers (Béla Böddi).

9) Selective excitation: measurement of chlorophyll-a and chlorophyll-b mixtures at different excitation wavelengths. Slit broadness and spectral resolution (Béla Böddi).

10) Measurement of solid samples: measurement of leaf pieces at room temperature. The shape of the spectrum and sample geometry. How to detect, identify and avoid interference ghost signals. The effect of sample orientation: false bands, false amplitude ratios (Béla Böddi).

11) Measurement of light scattering spectra. The role of the surface geometry. Interference signals and slit problems (Béla Böddi)

12) 77 K fluorescence spectroscopy. The rules for working with liquid nitrogen. Advantages and error sources. Measurement and comparison of 77K and 293 K spectra of leaf pieces, suspensions and solutions (Béla Böddi).

13) Data tranfer in ASCII code. Data analysis with the software SPSERV (copyrights: C. Bagyinka): Computerized analysis of the spectra I.: smoothing and its danger, baseline correction, calculation of difference spectra (Béla Böddi).

14) Computerized analysis of spectra II.: derivative spectroscopy. The role and importance of the derivation side-bands in the 2nd and 4th derivative spectra. Conversion of spectra into wave number function. The decomposition of spectra into Gaussian components and other decomposition peocedures. Errors and results (Béla Böddi)

15) How to publish the spectra in the papers: figure editing softwares (Béla Böddi)

• Jan Greve et al.: Spectroscopy of Biological Molecules, Springer Science & Business Media, 2012

• Handouts from the teachers