Modern Optical Spectroscopy Introduction of AES, NMR, and Laser Shu-Ping Lin, Ph.D. Institute of Biomedical Engineering E-mail: [email protected] Website: http://web.nchu.edu.tw/pweb/users/splin/ Nuclear Magnetic Resonance (NMR) Spectroscopy Molecular Spectroscopy Nuclear magnetic resonance (NMR) spectroscopy: A spectroscopic technique that gives us information about the number and types of atoms in a molecule, for example, about the number and types of 1 hydrogen atoms using H-NMR spectroscopy. 13 carbon atoms using C-NMR spectroscopy. 31 phosphorus atoms using P-NMR spectroscopy. Nuclear Spin States An electron has a spin quantum number of 1/2 with allowed values of +1/2 and -1/2. This spinning charge has an associated magnetic field. In effect, an electron behaves as if it is a tiny bar magnet and has what is called a magnetic moment. The same effect holds for certain atomic nuclei. Any atomic nucleus that has an odd mass number, an odd atomic number, or both, also has a spin and a resulting nuclear magnetic moment. The allowed nuclear spin states are determined by the spin quantum number, I, of the nucleus. The Chemistry of Life - Atoms The basic unit of each chemical element is the atom. Atoms have a large nucleus, composed of protons and neutrons held together by the Strong Force. The electrons "orbit" the nucleus, attracted by the Electrical Force. The number of protons determines the chemical element, and the number of neutrons determines the isotope of the element. No net electrical charge Ions -- when atoms gain or lose electrons http://cass.ucsd.edu/public/tutorial/scale.html Carbon Orbitals around a nucleus Figure1.1 Periodic Table Periodic table indicating the atomic properties of all elements found on earth. Periodic Table Explorer is a simple Periodic Table software. http://www.technosamrat.com/freewares/periodic-table-explorer/ Nuclear Spin States A nucleus with spin quantum number I has 2I + 1 spin states; if I = 1/2, there are two allowed spin states. Spin quantum numbers and allowed nuclear spin states for atoms common to organic compounds. Element 1H 2H 12C 13C 14N 15N 16O 19F 31P 32S Nuclear spin quantum 1/2 1 0 1/2 1 1/2 0 1/2 1/2 0 number (I ) Number of 2 3 1 2 3 2 1 2 2 1 spin states Nuclear Spins in H0 1 13 Within a collection of H and C atoms, nuclear spins are completely random in orientation. When placed in a strong external magnetic field of strength H0, however, interaction between nuclear spins and the applied magnetic field is quantized. The result is that only certain orientations of nuclear magnetic moments are allowed. Nuclear Spins in H0 1 13 for H and C, only two orientations are allowed. Nuclear Spins in H0 In an applied field strength of 7.05T the difference in energy between nuclear spin states for 1 H is approximately 0.120 J (0.0286 cal)/mol, which corresponds to a frequency of 300 MHz (300,000,000 Hz). 13 C is approximately 0.030 J (0.00715 cal)/mol, which corresponds to a frequency of 75MHz (75,000,000 Hz). Nuclear Spin in H0 The energy difference between allowed spin states increases linearly with applied field strength. 1 Values shown here are for H nuclei. 在較強磁場中，二自旋狀態之能量差△E，比在較 弱磁場中之能量差更大。其能量差△E與磁場強度Ho成 正比，即 h EH (12-1) 2 o 其中 △E=α及β自旋狀態之能量差 h=蒲郎克常數 Ho=外磁場強度，高斯 (gauss) γ=迴轉磁係數 (gyromagnetic ratio)，每一質子為 26,753-1高斯-1 當一質子在合適磁場接受一光子時，可由α-自旋 狀態跳至β-自旋狀態，稱此原子核是共振 (resonance)。 一光子的能量以E=hν表示，此式與式 (12-1) 合併可得 h E h H (12-2) 2 o 解出為ν 1 H 2 o (12-3) 一質子之26,753-1高斯-1 1 (26,753)H 4257.8 s11高斯 H (高斯 ) (12-4) 2 oo 質子共振頻率發生於光譜的無線電頻率區。對 於共振所需無線電頻率可由磁場Ho求出。 最常用的NMR操作頻率為60~300MHz(60~300百 萬赫 ) 相當於14,092 高斯的磁場。對於較高解析度的 NMR，則使用300~600MHz之頻率操作。 Nuclear Magnetic Resonance When nuclei with a spin quantum number of 1/2 are placed in an applied field, a small majority of nuclear spins are aligned with the applied field in the lower energy state. The nucleus begins to precess and traces out a cone-shaped surface, in much the same way a spinning top or gyroscope traces out a cone- shaped surface as it precesses in the earth’s gravitational field. Nuclear Magnetic Resonance If the precessing nucleus is irradiated with electromagnetic radiation of the same frequency as the rate of precession, the two frequencies couple energy is absorbed the nuclear spin is flipped from spin state +1/2 (with the applied field) to -1/2 (against the applied field). Nuclear Magnetic Resonance (a) Precession and (b) after absorption of electromagnetic radiation. Bo=Ho Nuclear Magnetic Resonance Resonance: In NMR spectroscopy, resonance is the absorption of energy by a precessing nucleus and the resulting “flip” of its nuclear spin from a lower energy state to a higher energy state. The precessing spins induce an oscillating magnetic field that is recorded as a signal by the instrument. Signal: A recording in an NMR spectrum of a nuclear magnetic resonance. Nuclear Magnetic Resonance 1 If we were dealing with H nuclei isolated from all other atoms and electrons, any combination of applied field and radiation that produces a signal for one 1H would produce a signal for all 1H. The same is true of 13C nuclei. Hydrogens in organic molecules, however, are not isolated from all other atoms. They are surrounded by electrons, which are caused to circulate by the presence of the applied field. The circulation of electrons around a nucleus in an applied field is called diamagnetic current and the nuclear shielding resulting from it is called diamagnetic shielding. Nuclear Magnetic Resonance The difference in resonance frequencies among the various hydrogen nuclei within a molecule due to shielding/deshielding is generally very small. The difference in resonance frequencies for hydrogens in CH3Cl compared to CH3F under an applied field of 7.05T is only 360 Hz, which is 1.2 parts per million (ppm) compared with the irradiating frequency. 360 Hz 1.2 = 6 = 1.2 ppm 300 x 106 Hz 10 Nuclear Magnetic Resonance Signals are measured relative to the signal of the reference compound tetramethylsilane (TMS). CH3 CH3 Si CH3 CH3 Tetramethylsilane (TMS) 1 For a H-NMR spectrum, signals are reported by their shift from the 12 H signal in TMS. 13 For a C-NMR spectrum, signals are reported by their shift from the 4 C signal in TMS. Chemical shift (): The shift in ppm of an NMR signal from the signal of TMS. Chemical Shift - 1H-NMR Chemical shift δ is usually expressed in parts per million (ppm) by frequency, because it is calculated from: Since the numerator is usually in hertz, and the denominator in megahertz, delta is expressed in ppm. NMR signal that absorbs at 300 Hz lower than does TMS at an applied frequency of 300 MHz has a chemical shift of: Although the frequency depends on the applied field the chemical shift is independent of it. On the other hand the resolution of NMR will increase with applied magnetic field resulting in ever increasing chemical shift changes NMR Spectrometer Schematic diagram of a nuclear magnetic resonance spectrometer. NMR Spectrometer Essentials of an NMR spectrometer are a powerful magnet, a radio-frequency generator, and a radio-frequency detector. The sample is dissolved in a solvent, most commonly CDCl3 or D2O, and placed in a sample tube which is then suspended in the magnetic field and set spinning. Using a Fourier transform NMR (FT-NMR) spectrometer, a spectrum can be recorded in about 2 seconds. NMR Spectrum 1 H-NMR spectrum of methyl acetate. High frequency: The shift of an NMR signal to the left on the chart paper. Low frequency: The shift of an NMR signal to the right on the chart paper. Equivalent Hydrogens Equivalent hydrogens: Hydrogens that have the same chemical environment. A molecule with 1 set of equivalent hydrogens gives 1 NMR signal. O H3 C CH3 CH3 CCH3 ClCH 2 CH2 Cl C C H3 C CH3 Propanone 1,2-Dichloro- Cyclopentane 2,3-Dimethyl- (Acetone) ethane 2-butene Equivalent Hydrogens A molecule with 2 or more sets of equivalent hydrogens gives a different NMR signal for each set. Cl Cl CH3 CH3 CHCl O C C H H 1,1-Dichloro- Cyclopent- (Z)-1-Chloro- Cyclohexene ethane anone propene (3 signals) (2 signals) (2 signals) (3 signals) Signal Areas Relative areas of signals are proportional to the number of H giving rise to each signal, Modern NMR spectrometers electronically integrate and record the relative area of each signal. Chemical Shift - 1H-NMR Type of Chemical Type of Chemical Hydrogen Shift () Hydrogen Shift () ( CH3 ) 4 Si 0 (by definition) O RCH 0.8-1.0 3 RCOCH3 3.7-3.9 Chemical RCH2 R 1.2-1.4 O Shifts R3 CH 1.4-1.7 RCOCH2 R 4.1-4.7 1H-NMR R C= CRCHR 2 2 1.6-2.6 RCH2 I 3.1-3.3 RC CH 2.0-3.0 RCH2 Br 3.4-3.6 ArCH 3 2.2-2.5 RCH2 Cl 3.6-3.8 ArCH R 2 2.3-2.8 RCH2 F 4.4-4.5 ROH 0.5-6.0 ArOH 4.5-4.7 RCH2 OH 3.4-4.0 R2 C= CH2 4.6-5.0 RCH2 OR 3.3-4.0 R2 C= CHR 5.0-5.7 R2 NH 0.5-5.0 ArH 6.5-8.5 O O RCCH3 2.1-2.3 RCH 9.5-10.1 O O RCCH2 R 2.2-2.6 RCOH 10-13 Chemical Shift Chemical shift depends on the (1) electronegativity of nearby atoms, (2) hybridization of adjacent atoms, and (3) diamagnetic effects from adjacent pi bonds.