Lecture 4 -BMS2043 - Spectroscopy final.pdf

BMS2043- ANALYTICAL AND CLINICAL BIOCHEMISTRY SPECTROSCOPY DR PENNY LYMPANY [email protected] 28AY04 OBJECTIVES At the end of this series of lectures you will be able to: Identify key features of different methods of spectroscopy Identify uses of these applications Links to prior learning – BMS...

BMS2043- ANALYTICAL AND CLINICAL BIOCHEMISTRY SPECTROSCOPY DR PENNY LYMPANY [email protected] 28AY04 OBJECTIVES At the end of this series of lectures you will be able to: Identify key features of different methods of spectroscopy Identify uses of these applications Links to prior learning – BMS1041/BMS1030 http://bareket-astro.com/en/project/spectra.htm WHAT ARE WE GOING TO BE LOOKING AT? UV/vis absorbance spectroscopy Photoluminescence (Fluorescence) Bio- & Chemi-luminescence WHY? Clinical diagnosis Basic science Forensics SPECTROSCOPY - DEFINITION Latin - ghost or spirit Greek - to see Studying the properties of matter through its interaction with different frequency components of the electromagnetic spectrum. With light – interaction not with the matter but the “ghost.” Each type of spectroscopy—different light frequency—gives a different picture → the spectrum THE QUESTIONS What does light do to sample? How do you produce a spectrum? What EXACTLY is a spectrum a measurement of? This Photo by Unknown Author is licensed under CC BY-SA WHAT DOES A SPECTRUM MEASURE? SPECTRUM OF VISIBLE LIGHT http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/10_Spectroscopic_Methods/10A_Overview_of_Spectroscopy ENERGY LEVELS – THE PHYSICS Energy levels Absorption : Ground state to Excited state (10-15 sec) Relaxation: Excited state to Ground state – Internal Conversion (IC) nonradiative relaxation through vibrational states (10-12 - 10-14 s) – Emission Fluorescence (spontaneous emission: 10-10 - 10-8 sec) Phosphorescence (10-3 - 102 sec) – BASICS - ABSORPTION BASICS - EMISSION PHYSICS – THE SPECTRUM ABSORPTION SPECTROSCOPY ABSORPTION PROCESS Transfer of light energy to molecule What happens next depends on the characteristics of the molecule http://www.analyticalspectroscopy.net/ap5-6.htm Absorption Spectroscopy excited states Energy change Photon - ground state http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/10_Spectroscopic_Methods/10A_Overview_of_Spectroscopy ABSORPTION: THE BEER-LAMBERT LAW Concentration can be determined by UV-visible spectrophotometry A = ·c·l  = Extinction coefficient (M-1 cm-1) C = concentration (Molar, M) L = pathlength (cm) ABSORPTION PROCESS – THE REALITY Primary deduction from Beer-Lambert law is Light intensity exponential attenuation with : 1 Absorber molecule conc. 2 Sample cross section (Optical path length). This is not the complete picture [What if Sample is Turbid?] Scattering effects http://www.waterontheweb.org/under/waterquality/turbidity.html ABSORPTION PROCESS - CHARACTERISTICS Main parameters controlling optical characteristics POLARISATION Change of phase of light on a sample – rotation of polarisation HOW DO WE MEASURE ABSORPTION? Measure change in light intensity as it passes through a sample http://en.wikipedia.org/wiki/Fluorescence_microscope USES OF ABSORPTION SPECTROSCOPY - KINETICS Extinction coefficient () of NADH at 340 nm: 6,220 M-1 cm−1 OXY AND DEOXY HAEMOGLOBIN Soret peak https://openi.nlm.nih.gov/detailedresult?img=PMC3629043_pone.0061767.g002&req=4 USES OF ABSORPTION SPECTROSCOPY – DNA QUANTIFICATION AND PURITY 260 nm – quantification 260/280 - purity https://tipbiosystems.com/wpcontent/uploads/2020/05/AN-LS-001-DNAMeasurement.pdf PHOTOLUMINESCENCE Photoluminescence is light emission from any form of matter after the absorption of photons EMISSION SPECTROSCOPY – TYPES OF EMISSION Luminescence – emission of light – no heat Phosphorescence Fluorescence Chemiluminescence This Photo by Unknown Author is licensed under CC BY FLUORESCENCE 1. Emission of a photon from the singlet excited state to the singlet ground state. 2. Average lifetime of an electron in the excited state is only 105 – 10-8 s. 3. Fluorescence decays rapidly once the source of excitation is removed FLUORESCENCE – KEY FEATURES http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/10_Spectroscopic_Methods/10A_Overview_of_Spectroscopy FLUORESCENCE: EXCITED STATES Relaxation of excited state Collisions with other species in the sample Photochemical reactions Emission of photons Quantum yield – fraction of excited state molecules returning to the ground state by fluorescence range from 1, when every molecule in an excited state undergoes fluorescence, to 0 when fluorescence does not occur. FLUORIMETRY https://www.chromedia.org/chromedia?waxtrapp=mkqjtbEsHonOvmOlIEcCArB&subNav=cczbdbEsHonOvmOlIEcC ArBP USES OF FLUORESCENCE SPECTROSCOPY 1. Qualitative analysis shapes and peak positions of the excitation spectra and emission spectra of fluorescent substances can be compared with the spectra of standard solutions to achieve the purpose of qualitative analysis. 2. Quantitative analysis At low concentrations, the fluorescence intensity of the solution is directly proportional to the concentration of the fluorescent substance: USES OF FLUORESCENCE SPECTROSCOPY EXAMPLES Quantification - DNA, antibodies, antigens Drug analysis, pharmacokinetics, efficacy analysis – e.g. quinine, antimicrobial drugs Food – detecting minerals, metallic elements, aas, vitamins, fungal contamination etc Environmental analysis – water, soils Currently not for treatment or diagnosis ADVANTAGES OF FLUORESCENCE SPECTROSCOPY Fluorescence analysis qualitatively and quantitatively analyse substances based on characteristics and intensity of fluorescence produced widely used to characterise physical and chemical properties of the system and its changes – e.g. conformation and properties of biological macromolecules. FLUORESCENCE ANALYSIS suitable for analytes that can be dissolved in solvents like water, ethanol and hexane analytes need to absorb UV or visible light analytes need to emit visible or near infra red radiation quantitative measurements of a single analyte in solution (Or more than one analytes in solution provided they do not interfere with each other.) Not Analytes that have a photochemical reaction at (or above) the wavelength range of interest Intransparent, not clear or colloidal samples Compounds that do not show fluorescence EXCITATION VS EMISSION SPECTRA excitation spectrum is obtained bymonitoring emission at a fixed wavelength, variable excitation wavelength After correction for source intensity & detector ~ absorbance spectrum Emission spectrum – Fixed wavelength to excite sample Monitor emitted wavelength intensity Molecules have single excitation spectrum but two emission spectra – fluorescence, phosphorescence FLUORESCENCE MICROSCOPY http://en.wikipedia.org/wiki/Fluorescence_microscope QUENCHING Quenching and dequenching is the basis for activatable optical contrast agents for molecular imaging. Many dyes undergo self-quenching, which can decrease the brightness of protein-dye conjugates for fluorescence microscopy, or can be harnessed in sensors of proteolysis Quenching is the basis for Förster resonance energy transfer (FRET) assays energy from excited molecular fluorphore (donor) is transferred to another fluorophore (acceptor) FRET 1-10 nm (10-100 Ă) ~ 3-30 nucleotides in DNA double helix Key information – Energy level of donor –returns to ground state without own fluorescence Limited distance between two molecules Emission spectrum of donor overlaps absorption spectrum of acceptor If acceptor is fluorophore – transferred energy emitted as fluorescence If acceptor not fluorophores – energy lost as heat and not light FRET IN MICROSCOPY FLUORESCENCE: FURTHER CONCEPTS Fluorescence Recovery After Photobleaching (FRAP): a method of determining the kinetics of diffusion in living cells (usually) using fluorescence microscopy A. The bilayer is uniformly labelled with a fluorescent tag B. label is selectively photobleached by a small (~30 micrometre) fast light pulse C. The intensity within this bleached area is monitored as the bleached dye diffuses out and new dye diffuses in D. Eventually uniform intensity is restored FLUORESCENCE: FURTHER CONCEPTS BIOLUMINESCENCE: FIREFLY FLASHES In bioluminescence, a photon is released after excitation in a biochemical reaction, e.g. luciferase BIOLUMINESCENCE: FIREFLY FLASHES CONT. LUCIFERASE ASSAYS To determine if a protein is able to activate (or suppress) transcription of a gene of interest, recombinant DNA technology to produce a construct in which the gene's promoter is placed adjacent to a luciferase reporter gene CHEMILUMINESCENCE In chemiluminescence, a photon is released after excitation in a chemical reaction, e.g. luminol http://en.wikipedia.org/wiki/Luminescence CHEMILUMINESCENCE - LUMINOL Activation – e.g. H2O2 in water Catalyst e.g. Fe – H2O2 oxidised to H20 + O2 Luminol reacts with OH – dianion Dianion reacts with O2 to produce peroxide – unstable – made by loss of N2, and change of electrons from excited to ground state – emission of photons (blue glow) Organic peroxides contain peroxide functional group ROOR MICROSCOPY: EPIFLUORESCENCE Nucleus: blue (DAPI) Microtubules: green Actin: red http://en.wikipedia.org/wiki/Fluorescence_microscope FLUORESCENT PROTEINS http://en.wikipedia.org/wiki/Green_fluorescent_protein Confocal microscopy http://en.wikipedia.org/wiki/Confocal_microscopy NIR- ABSORPTION SPECTROSCOPY NIR- absorption spectroscopy – The interesting wavelength range for this type of spectroscopy is 800-2500 nm (invisible). – The related wavenumber (1/λ) range is 12500 – 4000 cm-1. Examples: Water owes its blueness due to selective absorption in the red portion of its visible spectrum. "heavy" water (chemically identical to regular water, but with the two hydrogen atoms replaced with deuterium atoms) has a similar absorption curve, shifted to higher wavelengths outside of the visible spectrum of light. Heavy water is thus colourless. http://www.webexhibits.org/causesofcolor/5B.html ? ANY QUESTIONS? We made it! This Photo by Unknown Author is licensed under CC BY-SA Please use the discussion board for general questions and queries