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  1. 1. It's type of spectrophotometry deals with fluorescence , the phenomenon in which light incident on amaterial at agiven wavelenth . It's type of electromagnetic spectroscopy which analyze fluorescence from asample, it involves using abeam of light , usually ultraviolet light that excites the electrone in molecule of certain compound and causes them to emit light of alower energy , typically . Device that measure fluorescence called fluorometer . INTRODUCTION
  2. 2. <ul><li>Molecules have various states referred to an energy leveles fluorescence spectroscopy is primarily concerned with electronic and vibrational states . </li></ul><ul><li>Generally the species being examined has aground electronic state (alow energy state) of interest and an excited (electronic state of higher energy ) , within each of these electronic states are various vibrational states . </li></ul>Theory
  3. 4. <ul><li>The species is first excited by absorbing aphoton from its ground electronic state to one of the various vibrational states into the excited state collisions with other molecules cause the excited molecules to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state . the molecule then drops down to one of the various vibrational level of ground state again emission of aphoton which will have different energies and thus frequencies </li></ul><ul><li>By analysing the different frequency of light emitted in F.S , along with their relative intensis the structure of different vibrational level canbe determine </li></ul>
  4. 5. 1- The NanoBiophysics Core Facility offers a Spectrofluorimetric System from PTI. The System includes QuantaMaster spectrofluoromete r, which is a sophisticated multifunctional instrument. It has dual-emission detection in the UV-VIS spectral range, and with the included calcite polarizing prisms, accurate fluorescence anisotropy measurements can be made, along with the capability of monitoring anisotropy changes as a function of time .  It also has a sample holder where the temperature is controlled and changed using the Peltier effect .  The range of set point temperatures on the Peltier controller is – 20º C to + 100º C .
  5. 6. The instrument also includes the ability to perform fluorescence lifetime measurements . Three LED excitation sources (at 280, 295, and 370 nm) are included; these wavelengths are appropriate for the excitation of tyrosine and/or tryptophan in intrinsic protein fluorescence work (280,295), and, for example, exogenous probes such as acrylodan (370). The spectrofluorometer has double excitation monochromator for enhancing the spectral purity of the excitation band.  PTI double monochromators have a unique ability, and that is that with the appropriate options, it can also function as a single-pass monochromator.  The instrument will make use of this capability to its maximum, because it will be supplied with two arc lamp housings, so that users will have the choice (for excitation wavelengths in the 250-600 nm range) of using either single-pass or double-pass excitation (depending on the needs dictated by the sample) without having to move the lamp housing from one double monochromator entrance port to another. A programmable excitation shutter is part of the spectrofluorometer.  This device is useful for minimizing photobleaching between data acquisitions or even between the taking of data points in a long time based scan.
  6. 7. A solid sample holder is also included in the package.  With this holder, the user has both translational and rotational control over the positioning of the sample relative to the incoming exciting radiation.  Thin films, samples on slides, paper, irregularly-shaped solid samples, as well as cuvettes can be accommodated by the holder.  For the study of very turbid solutions, this holder does an excellent job of redirecting scattered exciting light away from the emission axis; this scattered radiation, if not efficiently removed, would contaminate the fluorescence spectrum of the sample with a high, non-constant background. The spectrofluorometer includes a near-infrared fluorescence option .  The main components of this option are the following: a)  a 1200 gr/mm, 600 nm blaze excitation grating (placed in a turret with the second            300 nm blaze grating in the double excitation monochromator; b)  a 600 gr/mm, 1000 nm blaze emission grating (placed in a turret with the 400 nm      blaze grating in one of the emission monochromators; c)  a thermoelectrically-cooled InGaAs detector (spectral range 500-1700 nm); d)  a lock-in amplifier and chopper for noise suppression; Two common applications where this option finds applicability is in the study of novel materials such as semiconductor nanoparticles (quantum dots) as well as singlet oxygen detection. The software that comes with the spectrofluorometer also includes a FRET calculator , for the calculation of Rο , the D-A distance, as well as kDA . Basic QuantaMaster QM-4SE Spectrofluorometer Specifications Signal-to-noise Ratio :  10,000:1 (based on the Raman spectrum of water, excitation wavelength 350 nm, exc. and em. spectral bandwidth 5 nm, 1 s integration time, no smoothing)
  7. 8. Excitation Source : Type:  continuous Xe arc lamp, 75W Spectral range :  200-2000 nm (output dependent on monochromator grating) Monochromators : Type:  Czerny-Turner Focal Length: 200 mm f number: 4 Spectral bandwidth: 0 to 25 nm Resolution: +/- 0.5 nm Gratings: ruled, 1200 gr/mm, blazed at 300 nm (excitation) and 400 nm (emission) Detectors : Standard: R1527 PMT (200-680 nm) Optional: R928 PMT (200-870 nm), InGaAs photodetector (500-1700 nm) PMT Detection modes: photon counting, analog (user-switchable) Reference channel photodiode (calibrated against a standard photodiode), variable-speed magnetic stirrer, filter holders, and lid-activated emission shutter are all standard. Time-resolved Fluorescence Accessory Specifications Excitation wavelengths: 280, 295, and 370 nm (others available) Detection: PTI-patented stroboscopic detector Timescale protocols: linear, arithmetic, logarithmic (menu-selectable) Pulse width: 1.5 ns (for 280 and 295 nm LED’s) Range of Measurable Lifetimes: ~100 ps to >1 μs (LED-dependent) Acquisition mode: sequential or random Lifetime Analysis: 1-to-4 exponentials, global analysis, non-exponential, micellar kinetics, lifetime distribution analysis by ESM, lifetime distribution analysis by MEM, anisotropy decays FRET Calculator
  8. 9. 2- The SPEX FluoroMax3 spectrofluorometer from Horiba Scientific is located in the Michael Swann building, room 3.5. Booking, costs and rules for the use of the spectrofluorometer Features of the FluoroMax3 spectrofluorometer: ideal for high-resolution, sensitive fluorometric work (ca five times the sensitivity of the SpectraMax M5 Multimode plate reader). ideal for intrinsic tryptophan and tyrosine fluorescence. temperature of the sample block is controlled using a circulating water bath. accommodates standard fluorescence cuvettes. proprietary software, Fmax3, is very easy to use; it controls the experimental set-up and data acquisition. Note: this software is very old and has limited functionality with regard to data processing, we normally use this software only for data acquisition and viewing, and routinely copy the data collected directly into an Excel spreadsheet for processing/analysis. Xenon arc lamp wavelength selection is achieved by the optical gratings of the excitation and emission monochromators. adjustable slits (adjustable in the software) further resolve light wavelength s.. .
  9. 10. Samples: 0.2 μm filtering is recommended as light scatter can be a problem. sample solutions should be buffer-matched to any buffer controls. for titrations, corresponding buffer control titrations should be performed to take into account any fluorescence originating from the titrant. cuvette must be thoroughly clean before and after use. Use lens tissue to clean the outer surface (remember dust particles scatter light!). recommended volume for the standard cuvette is 0.5 ml (minimum volume is 350 μl although recommend using at least 400 μl). typical protein concentrations are 1-10 μM (obviously this is molecule-dependent and is related to the environment, and proportion, of tryptophans and other aromatics/fluorophores
  10. 11. APPLICATIONS : 1-. Determination of polyaromatic hydrocarbons Benzo[a]pyrene i s a product of incomplete combustion and found in coal tar.   - Benzo[a]pyrene , is a 5-ring polycyclic aromatic hydrocarbon that is mutagenic and highly carcinogenic It is found in tobacco smoke and tar The epoxide of this molecule intercalates in DNA, covalently bonding to the guanine base nucleotide   - Excitation and fluorescence spectra for benzo(a)pyrene in H 2 SO 4 . In the diagram the solid line is the excitation spectrum (the fluorescence signal is measured at 545 nm as the exciting wavelength is varied). The dashed line is the fluorescence spectrum (the exciting wavelength is fixed at 520 nm while the wavelength of collected fluorescence is varied).
  11. 13. 2- B. Fluorimetric Drug Analysis Many drugs possess high quantum efficiency for fluorescence. For example, quinine can be detected at levels below 1 ppb - In addition to ethical drugs such as quinine, many drugs of abuse fluoresce directly. For example lysergic acid diethylamide (LSD) whose structure is: - Because LSD is active in minute quantities (as little as 50 g taken orally) an extremely sensitive methods of analysis is required. Fluorimetricaly LSD is usually determined in urine from a sample of about 5mL in volume. The sample is made alkaline and the LSD is extracted into an organic phase consisting of n -heptane and amyl alcohol. This is a &quot;clean-up&quot; procedure that removes potential interferents and increases sensitivity. The LSD is then back-extracted into an acid - --- - solution and measured directly using and excitation wavelength of 335 nm and a fluorescence wavelength of 435 nm. The limit of detection is approximately 1 ppb:
  12. 14. CALCULATION CONCENTRATION AND FLUORESCENCE INTENSITY : The power of fluorescent radiation, F , is proportional to the radiant power of the excitation beam absorbed by the species able to undergo fluorescence: F = K '( P 0 - P ) where P 0 is the power incident on the sample, P is the power after it traverses a length b of the solution and K ' is a constant which depends upon experimental factors and the quantum efficiency of fluorescence.   Beer's law can be rearranged to give: P/ P 0 = 10 -  bc where A =  bc is the absorbance. Substitution gives: F = K ' P 0 (1 - 10 -  bc ) This is the fluorescence law Unlike Beer’s Law fluorescence isn’t in general linear with concentration   This expression can be expanded (Taylor series): To a good approximation if  bc is small (< 0.05) the higher-order terms are nearly zero, we have: F = 2.3 K '  bcP 0
  13. 15. EQUATION
  14. 16. EQUATION which demonstrates two important points: that at low concentrations fluorescence intensity is proportional to concentration; that fluorescence is proportional to the incident power in the incident radiation at the absorption frequency     For a concentration above c 1 the calibration curve is no longer linear

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