Chapter 17: Fundamentals of Spectrophotometry

Transcription

1 Chapter 17: Fundamentals of Spectrophotometry Spectroscopy: the science that deals with interactions of matter with electromagnetic radiation or other forms energy acoustic waves, beams of particles such as ions and electrons Spectrophotometry: a more restrictive term, - any procedure that uses light to measure chemical concentrations. - the quantitative measurement of the intensity of electromagnetic radiation at one or more wavelengths with photoelectric detector.

2 17-1. Properties of Light Electromagnetic radiation ; EM wave ; radiation ; radient ray ; ray ; light ne linearly (or plane) polarized and consists of a single frequency, that is, is monochromatic.

3 Dual properties: 1) wave: perpendicular, oscillating electric and magnetic fields explains diffraction, reflection = C/ ( : frequency, C; speed of light, ; wavelength) 2) particle (photon): absorption, emission E = h =h C/ =hc where E is the energy in joules (J) h; plank constant ( 6x10-34 J s) ; wavenumber (1/, cm -1 reciprocal centimeter ; Kayser)

4 17-2 Absorption of light Absorption of light: increases the energy of molecule (the molecule is promoted to an excited state) Emission of light: decreases the energy of molecule Ground state: lowest energy state of a molecule Excitation Relaxation M + h υ M* (life time: 10-6 ~10-9 S) M* M + light (fluorescence, phosphorescence) or M* M + heat

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6 Absorption Single-beam spectrophotometric experiment Light source Wavelength selector (monochromator) P o Sample P Light Detector b When light is absorbed by a sample the radiant power of the beam of light is decreased Radiant power (P): the energy per second per unit area of the light beam Transmittance (T): T = P/P o (T = 0 ~ 1) Absorbance (A), or optical density: A = log (P o /P) = -log T (if 90% light is absorbed, 10% transmitted: T = 0.1P o /P o = 0.1, A= - log T=1) Absorption spectrum: absorbance vs wavelength

7 Absorption: Beer s Law The part of molecule responsible for light absorption: chromophore Absorbance is directly proportional to the concentration Beer-Lambert law: A = εbc ε : molar absorptivity (extinction coefficient) characteristic of a substance that tells how much light is absorbed at a particular wavelength b: path length c: concentration Beer s law works for monochromatic radiation passing through a dilute solution < 10 mm Colorimetry: a procedure based on absorption of visible light

8 17-3 Measuring Absorbance For visible and UV spectroscopy, a liquid sample is contained in a cell called cuvet: - fused silica: UV and VIS - glass; suitable for VIS but not for UV spectroscopy (it absorbs UV) For IR spectroscopy: - cells of NaCl or KBr

9 17-4 Beer s Law in Chemical Analysis Determination of serum iron (Fe 3+, from transferrin) Fe ferrozine 2- (ferrozine) 3 Fe 4- purple complex ( max = 562 nm) Colorimetry: based on visible radiation

10 17-6 What happens when a molecule absorbs light? Ground state (planar) electronic transition ( n π* (S 1 )) e movement from one M to another M Excited state (pyramidal) Molecular orbitals (M) describe the distribution of electrons in molecules

11 Infra Red (IR) Absorption of IR radiation: vibration Absorption of microwave radiation: rotation Nonlinear molecule with n atom: 3N-6 Linear molecule with n atom: 3N-5 (Formaldehyde: 3N-6 = 3x4-6 = 6) Electronic transitions involve simultaneous vibrational and rotational transition IR spectroscopy: good for structural information

12 What happens to absorbed energy? IC: raditionless transition between states with the same quantum state (S 2 S 1, S 1 S 0 ) ISC: raditionless transition between states with different quantum state (S 1 T 1 ) S 1 : singlet excited state T 1 : triplet excited state Life time: s Life time: s S 0 : singlet ground state

13 What happens to absorbed energy? R: vibrational relaxation

14 Fluorescence vs Phosphorescence Fluorescence: S 1 S 0 Phosphorescence: T 1 S 0 (very rare) ISC (T1 S0)can occur before phosphorescence: cooling required The relative rate of internal conversion, intersystem crossing, fluorescence, and phosphorescence depend on the molecule, the solvent, and conditions such as temperature and pressure.

15 17-7. Luminescence Luminescence: 1) photoluminescence (fluorescence, phosphorescence) 2) chemiluminescence 3) bioluminescence 0 (abs) < 0 (emission) The absorption and emission spectra will have an approximate mirror image if the spacings between vibrational levels are roughly equal and if the transition probabilities are similar

16 Relationship between absorption and emission spectra The o does not exactly overlap: In the emission spectrum, o comes at slightly lower energy than in the absorption spectrum Frank-Condon Principle: o (absorption) < o (emission) Electronic transition are so fast, relative to nuclear motion

17 Fluorescence Spectrometer Luminescence is observed at 90 o to the incident light Emission spectrum: constant ex and variable em Excitation spectrum: constant em and variable ex

18 Excitation and emission spectra Emission spectrum: constant ex and variable em Excitation spectrum: constant em and variable ex An excitation spectrum looks very much like an absorption spectrum

19 Luminescence in Analytical Chemistry Relation of emission intensity to concentration: I = kp o C I: emission intensity P o : radiant power of incident light C: concentration of emitting species In FL: Higher radiation power higher intensity better detection In Absorbance: Higher radiation power no change in absorbance (Laser-induced fluorescence; LIF) good for the detection of trace amount Emission intensity is not proportional to analyte concentration at high concentration, or in the presence of significant amount of absorbing species Self-absorption

20 Luminescence in Analytical Chemistry Incident Photons Absorbed Photons Transmitted Photons Transmittance (P/Po) /10 = /100= Fluorescence (arb.unit.) Higher radiation power no change in absorbance Higher radiation power higher intensity better detection

21 Luminescence in Analytical Chemistry Self-absorption

22 Luminescence in Analytical Chemistry Most compounds are not fluorescent: Fluorescent moiety (fluorophore) should be coupled to the compound. H 3 C N CH 3 H H 3 C N CH 3 S Cl + H 2 N C R CH R S N C H H Dansyl Chloride Amino Acid CH Dansyl Amino Acid Fluorophore Chromophore No FL, no absorbance Derivatization: the chemical alteration of analyte so that it can be detected easily or separated easily from other species

23 (a) Luminol Chemiluminescence NH NH H -, H 2 2, catalyst * APA + ligh (425 nm blue emission) NH 2 NH 2 (b) 3-aminophtalate* (3-APA*) Catalysts: Heme proteins (HRP, hemoglobin..) transition metal ions (Co 2+, Cu 2+, Fe 2+,...) H 2 N C 2 H 2 N (CH 2 ) 4 aminobutylethylisoluminol (ABEI) NH NH Application: -H 2 2 detection at submicromolar concentration: H 2 2 producing substrates using oxidase enzymes - Immunoassay using HRP, isoluminol, or ABEI as labels - Amino acid detection using isoluminol or ABEI as labels - Transition metal detection - BD sensor (HRP used) - Forensic science (blood trace)

24 (a) 루미놀화학발광 NH NH H -, H 2 2, catalyst * APA + ligh NH 2 NH 2 3-aminophtalate* (3-APA*) 촉매 : 금속이온 (Co 2+, Cu 2+, Fe 2+,...) (b) 헴단백질 (HRP, 헤모글로빈 ): 혈액이화학발광의촉매로작용 NH C 2 H 2 N NH H 2 N (CH 2 ) 4 aminobutylethylisoluminol (ABEI)

25 혈흔검사 : 루미놀반응

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