Types of luminescence Types of emission processes Luminescence Photoluminescence Chemiluminescence Other examples Fluorescence Phosphorescence Biolumi

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1 Instrumentation of Spectrofluorometers 里 Interactions between electromagnetic waves and your samples E = hν The internal energy of a molecule: E total =E electronic + E vibrational + E rotational UV-Vis, Fluorescence & Phosphorescence spectra of most molecules consists of broad bands Related to combination of ( Electronic + Vibrational + Rotational ) transitions Energy levels of molecular Fine Structure Considered Organic Compounds Energy and molecular transition of UV-Vis σ to σ*, n to σ* n to π* π to π*. 3 4 What is Luminescence? Luminescence an emission of light occurring at a temperature below that of incandescent bodies. It is distinct from incandescence, in which materials emit light as a result of their high temperature. 'Luminescence comes about through the ability of certain substances to absorb light of relatively high frequency and re-emit it in installments of discrete lower frequencies (longer wavelength) ' Luminescence ( Emit ) Excited State ) Photon ) 量 Ground State ) 來說列 Molecular fluorescence spectroscopy Molecular phosphorescence spectroscopy Chemiluminescence spectroscopy 5 6 1

2 Types of luminescence Types of emission processes Luminescence Photoluminescence Chemiluminescence Other examples Fluorescence Phosphorescence Bioluminescence For photoluminescence, the energy is provided by the absorption of IR, visible or ultraviolet light. Bioluminescence is the name given to chemiluminescent reactions that occur in living organisms. Electroluminescence Thermoluminescence Sonoluminescence,etc Photoluminescence excitation by absorption of light fluorescence, lifetime to 10-7 secs phosphorescence, lifetime 10-3 to 10 2 secs Chemiluminescence excitation by chemical reaction bioluminescence is a chemiluminescent reaction in a biological system 7 8 類 1.Vibrational Relaxation ( in the same electronic state level ) 2. External Conversion : 量 3. Internal Conversion : 輻量 ( to lower electronic level ) 4. Intersystem Crossing : Radiative Transitions ground singlet state Electronic states excited singlet state excited triplet state Singlet state: All electrons in the molecule are spin-paired Triplet state: One set of electron spins is unpaired 9 10 Possible physical process following absorption of a photon by a molecule? Fairly large conjugate sysytem Delocalized electron Rigid structure Reduce interaction with medium Planar

Essential component of fluorometer or spectrofluorometer Cary Eclipse 濾濾理 13 14 (UV-Vis) Absorption spectrophotometer I 0 I Fluorescence spectrophotometer excitation I 0 I f I 0 Φ (1-10 -Abs )

3 Essential component of fluorometer or spectrofluorometer Cary Eclipse 濾濾理 (UV-Vis) Absorption spectrophotometer I 0 I Fluorescence spectrophotometer excitation I 0 I f I 0 Φ (1-10 -Abs ) Spectrum Abs I emission Measurement of: Fluorescence Phosphorescence Bioluminescence / 冷 Wide range of accessories Cary Eclipse Cary Eclipse Main components Lamp Module Excitation Monochromator 參 Reference Detector Sample compartment Emission Monochromator Emission Detector 零 Electronics

4 Schwarzschild collection optics Xe Lamp 濾 on both mono 19 參 Beam Splitter 20 靈度 Excellent sensitivity and low signal noise The power of Xenon! 不 Unique room light immunity for Fluorescence samples provides greater flexibility in measuring a wider range of samples. Dark current correction ( before sample measurement ) Ratio result =(sample PMT-dark)/(ref PMT-dark) x 1000 This is not applicable to PHOSPHORESCENCE BIOLUMINESCENCE Cary Eclipse Anthracene room light immunity sample chamber open sample chamber closed Monochromator Feature Monochromators Czerny- Turner Design minimizes aberrations in critical applications Entrance Slits Grating Exit Slits Czerny Turner design 六 1.5, 2.5, 5, 10, 20, 10 round Horizontal slits Benefit 1cm cuvette, sample <0.5 ml Increased S/N (region of viewing) 5~30 0.5mL 4

5 Cary Eclipse design Detectors: 2 R928 photomultiplier tubes large dynamic range ( Volts) red-sensitive - Included as standard Revolutionary new electronics Capture a phosphorescence data point every microsecond (10-6 s) Cary Eclipse Cary Eclipse optics tour 27 5

6 Instrument Function Modes Scan 里兩 Excitation spectrum: wavelength and amount of light absorbed Emission spectrum : wavelength and amount of light emitted Fluorescence Signature or Fingerprint 兩 Fluorescence Signature Eclipse 兩 often just referred to as the emission spectrum or the fluorescence spectrum 兩兩異 Synchronous scan Absorption, fluorescence,and phosphorescence spectra Stokes Shift nm 離 The difference in nanometers between the peak excitation and emission wavelengths of a fluorescent species. 量 This indicated the energy dissipated during the lifetime of the excited state before fluorescence. Stokes Shift

7 vs. Is characteristic of molecular structure and environment of material The shape of the fluorescence spectrum is independent of the excitation wavelength because the emission always originates from the lowest excited state. The fluorescence spectrum is often a mirror image of the absorption spectrum Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does not change the emission profile but does produce variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum. 例 Absorption and Emission of Anthracene 不度 Is a plot of intensity at a particular emission wavelength against the wavelengths of the exciting light. When collecting an excitation spectrum you are asking the question where do I have to excite this system to see fluorescence at my chosen emission wavelength? Excitation and Emission spectra Synchronous scans Light source Monochromator Sample Light source Monochromator Sample excitation excitation emission emission Generally plot intensity vs ex. mono. WL Monochromator Monochromator 離 Anthracene Detector Detector Permit separation of complex mixtures such as crude oil (after appropriate dilution) Int Excitation scan Int Emission scan Spectra have unusual shape

synchronous scan 例 Synchronous scans Source Monochromator Sample (Fluorescein + RhB mixture) Fluorescein Synch scan 25nm Delta excitation Emission scan of mixture RhB Synch scan 25nm Delta emission

8 synchronous scan 例 Synchronous scans Source Monochromator Sample (Fluorescein + RhB mixture) Fluorescein Synch scan 25nm Delta excitation Emission scan of mixture RhB Synch scan 25nm Delta emission Monochromator Synch scan of mixture 25nm Delta Detector Artifacts in a fluorescence spectrum Scattering 雷禮 Rayleigh scatter scatter of the excitation light peak at the excitation wavelength 拉 Raman scatter scatter due to solvent 量 peak at a fixed energy from the excitation wavelength (solvent dependent) 2nd Order Scatter of higher order excitation light 數 peak at multiple of excitation wavelength Software detects scattering artifacts.. Intensity (a.u.) Scattering. Rayleigh scatter Real luminescence 2nd Order Wavelength (nm) 拉 Raman Scattering During the Rayleigh scattering process, some of the incident energy can be abstracted and converted into vibrational and rotational energy. The resulting energy scattered is therefore of lower energy and longer wavelength than the incident radiation. The amount of energy abstracted is always constant,raman bands appear separated from the incident radiation by the same frequency difference

9 拉 Question! 若拉 band What will you do if the Raman band of the solvent coincides with the fluorescence emission of the solute? Types of collection modes Fluorescence (lifetime to 10-7 sec) Types of collection modes Generate emission with light source on Measure the emission with the light source on Types of collection modes Phosphorescence (lifetime 10-3 to 10 2 sec) Generate emission with light source on Measure emission with light source off Types of collection modes Chemi-/Bio-luminescence Generate emission with light source off Measure emission with light source off Delay time Gate time

10 Types of collection modes How many flashes should I perform? Example #1 Usefulness of the No. of flashes parameter Example # Phosphorescence data mode Single Reads example Simple Reads, Advanced Reads, Concentration applications Total Decay Time = 20ms Delay Time =10ms Ave time = Gate time = 5ms Ave. time = Gate time Intensity = I Ave. time = 5 Gate time Total Decay Time = 20ms Delay Time =10ms Gate Time = 5ms Phosphorescence scanning Ave time = Gate time Total decay time = 40ms Delay Time = 10ms Ave time = Gate time = 20ms Ave Time = 25ms Ave Time/Gate Time = 5 Intensity = A 1 + A 2 + A 3 + A 4 + A

11 3 pulse width Delay time Gate time 3 pulse width Delay time 3 pulse width Delay time Gate time Gate time Gate time Phosphorescence scanning Ave time = 3 x Gate time Bio-Chemi/luminescence data mode Scanning examples Total decay time = 40ms Delay Time = 10ms Gate time = 20ms Ave time = 60ms Bio/Chemiluminescence scanning Time dependent measurements Time dependent measurements Intensity Kinetics Vs Lifetimes 63 量參數度 Measure the intensity as a function of time 力 Kinetics 度 change in intensity usually due to chemical reaction time scale of seconds to minutes to hours Lifetimes (phosphorescence) 度 change in intensity usually due to uni-molecular processes time scale of time scale of ms to sec Intensity Intensity 64 High [A] Intensity Time (s) Time (ms) High [B] Time (min) Intensity Intensity Time (ms) Time (ms) Kinetics A + B C A + Excitation A* A* A + Emission Lifetimes A + Excitation A* A* A + Emission Time (ms) 6

12 Kinetics application Measure the increase or decrease in emission intensity as a function of time. Fluorescence Phosphorescence Chemi/Bio-luminescence Obtain an Intensity vs time plot Fit the data with either a Zero, First or Second order equation Total Decay Time = 20ms Delay Time =10ms Gate Time = 5ms Ave Time = 25ms Cycle Time = 1sec Stop Time = 3sec Ave Time/Gate Time = 5 I 2 = A 1 + A 2 + A 3 + A 4 + A Lifetimes application Time scales Phosphorescence (µs to sec) 1 µs data interval capabilities Advanced Cary Eclipse electronics enables data to be collected real time in most cases Fluorescence (nanoseconds) High end Fluorometers ONLY, not Cary Eclipse Laser-based systems Lifetimes Definition of Lifetime (Decay time) the average amount of time a molecule remains in the excited state following excitation Lifetimes (time resolved) A + Excitation A* A* A + Emission Time based measurement like Kinetics change in intensity usually due to uni-molecular processes Why is this important? Characteristic of a particular molecule under certain conditions Solvent and temp effects Applications Protein labelling e.g. Eu labelling paired long fluorescence electron lifetime spins Lifetimes Jablonski Diagram unpaired electron spins Phosphorescence lifetimes

13 度 Concentration Effects fluorescence intensity is proportional to input light 量 Quantitative fluorescence intensity at low concentrations at high concentrations other effects come into play self (concentration) quenching energy transfer formation of new species after absorption inner filter effect collection geometry - e.g. front face illumination (triangular cells) Inner Filter Effect Inner filter effect I=I o exp(-εlc) Coenzyme NADH in D.I. water concentrated solution I=I o exp(-εlc) weak fluorescence signal dilute solution stronger relative fluorescence signal detected Linear range from 10-8 to 10-4 M 量 Other factors affecting quantitative accuracy 度量 Working with dilute solutions 度 Temperature effect 數 Change number of collisions 度 Change viscosity Adsorption Cuvette material and quality e.g. solvents should not stored in plastic containers Photo-decomposition 75 度 ph effects Change intensity and spectral characteristics of fluorescence Quenching( ) 76 e.g. the presence of O 2 or traces of peroxide Stopcock grease The growth of micro-organisms in buffer. Filter ( phenols present from the original wood) 8

Quantitation summary 度量 Measuring concentrated solutions 度度度. Typically C max = 0.05/εl where ε is the molar extinction coefficient at the excitation wavelength, and l is the pathlength.

14 Quantitation summary 度量 Measuring concentrated solutions 度度度. Typically C max = 0.05/εl where ε is the molar extinction coefficient at the excitation wavelength, and l is the pathlength. L-format (usual) front surface (for concentrated solutions and films) Thermal Analysis Temperature dependent measurements BioMelt TM Package DNA work being investigated using fluorescence Higher sensitivity Use Fluorescence Resonance Energy Transfer (FRET) Characterization of DNA melting curves Drug diagnostics Thermodynamic properties of DNA attach various groups to DNA molecule Accessories 4 cell peltier accessory Temperature controller Temperature probe Example Tm value using 9mer DABCYL labelled PNA probe with 5 6- carboxyfluorescein labelled DNA. 81 Details refer to Varian Fluorescence At Work No

Microsoft PowerPoint _代工實例-1

Microsoft PowerPoint _代工實例-1 4302 動態光散射儀 (Dynamic Light Scattering) 代工實例與結果解析生醫暨非破壞性分析團隊 2016.10 updated Which Size to Measure? Diameter Many techniques make the useful and convenient assumption that every particle is a sphere. The