Analysis & Structure of Molecules 문명희 화학과 분석화학전공 CHEMISTRY 물질의 특성 및 변화 물질의 합성 유기화학 무기화학 분석 분석법개발 분석화학 에너지변화 물리화학 Analytical Chemistry & Life Drunken driver Athletes River contamination Explosives by terrorists Food Environments QC in Manufacturing Clinical Examinations Etc. Detection of Chemicals Accuracy Precision Development of Analytical Technologies Classical Technologies manual Time taking Labor oriented poor detection limit •Titration acid-base, redox, complex •Precipitation-Weighing Group Analysis •Colorimetry litmus paper, Computer Robotics Instrumental Analysis instrumental reproducible Ultra low detection (10억분의 1영역) •Spectrophotometric methods X-ray, Atomic Spectroscopy UV-VIS, IR, microwave •Separation (chromatography) GC, HPLC, SFC •Electrochemical Classical Analytical Chemistry • Quantitative Analysis – gravimetric analysis (중량분석) Ni Ni-dimethylglyoxime salt (침전) 무게측정 지시약첨가 – Volumetric analysis (부피분석) titration (적정) 색변화관찰 What is Titration ? Final Goal : Amount ? 농도를 아는 용액 (산, 또는 염기) H-Acid + M-Base BH + MA types 분석대상용액 (염기 또는 산) Acid-Base titration Redox titration Precipitation Spectrophotometric Physical Methods in AC • A Great Advance 19th century - Atomic Spectroscopy Chemical compound Ex) Na ---- 589.0 nm Light (bright yellow) What if light source is plasma ? ICP (Inductively Coupled Plasma) Colored light Inductively Coupled Plasma “torch” atomic emission 4000 ~ 8000 K plasma load coil (~27MHz, 2kW) Ar 의 이온화 – coil 의 spark Ar sample solution (11~17L/min) Ce emission ICP/ optical emission spectrum of 100 ppm Cerium solution plasma continuum 140Ce+ ICPMS spectrum of 10 ppm Cerium Analysis of Complicated Mixtures ? Needs Simplification of Matrix Separation (or isolation) Emergence of Chromatography Chromatography In Greek Word, Color + To Write 1903 Tswett (Russian Botanist) First Observed Separation of Plant Pigments as bands on chalk column (CaCO3) with ether Chromatographic Separation M.P Samples Sample A&B Solid materials Glass wools A Sample B Modern Instruments of Chromatography injector detector pump column Injector Sample loop column PC Data record Separation Process in Chromatography Sample Components are carried by a mobile phase through a bed of stationary phase Mobile Phase : Gas Liquid Stationary Phase : Solid (silica, alumina, etc.) Liquid Classifications by Phase Type Mobile Liquid Stationary Solid Liquid Gas Solid Liquid Supercritical Fluid LSC LLC GSC GLC SFC Retention of sample molecules ! Where ? -- Stationary Phase (S.P.) By how ? -- Various Mechansims 1. Adsorption 2. Partition 3. Ion-Exchange 4. Size Exclusion 5. Affinity 1. Adsorption Stationary Phase - Solid : silica, alumina LSC, GSC Separation is due to a series of Adsorption/Desorption Steps Solid (particle) M.P. Adsorption Common S.P. : Silica & alumina Both Solutes and Solvent are attracted to the Polar Sites on the S.P. For Separation, Solutes need to have different degrees of ATTRACTION to the phase 2. Partition (distribution) Solute Partitioning between TWO PHASES S.P.: Lig. M.P.: Gas --- GLC S.P.: Lig. M.P.: Liq --- LLC How does liq. S.P. exist ? Liq. Immobilized to solid (C18-silica) Separation is Based on RELATIVE SOLUBILITY Phase A Phase B Partition Basic Principle - similar to EXTRACTION High Affinity (Solubility) for S.P. Retain Longer One phase - polar The other phase - nonpolar Separation of Solutes is based on differences in this relative solubility Example i.e. S.P. : Nonpolar (C18) M.P.: Water or Methanol * separation order - order of interaction between C18-sample 3. Ion Exchange Stationary Phase has ionically charged surface interaction between Surface-ions: counterions S.P. : Exchange Resins (Cation, Anion Types) sample M.P. + + + Solid (particle) Ion Chromatography (IC) Chromatographic Process to separate Ions and some polar molecules Stationary Phase attract ionic species by the following principles. Typical Mechansims 1. Ion Exchange - IEC 2. Ion Exclusion 3. Complexation Effect Ion Exchange Chromatography Stationary Phases Anion Exchange Resins Cation Exchange Resin + resin + (analyte) Ion exchange + R resin resin NH+ ClR H NH+ ClH + + resin strong resin SO3-H+ weak resin COO-H+ Ion Exchange What Affects the Separation Order ? Ionic Charge : Larger the stronger attraction Li+, Na+, K+ < Ca+2, Mg+2 F-, Cl-, Br-, H2PO4-, < SO42Atomic Number : Higher the larger Electron Cloud -- stronger van der Waals Force Li+ < Na+ < K+ , Ca+2 < Mg+2 F- < Cl- < Br- < H2PO4- Example Ion Exchange Separation of Common Anions Eluent: 1.8mM Na2CO3, 1.7mM NaHCO3 Example Ion Exchange Separation of Common Cations Eluent: 20mM HCl or Methansulfonic acid Example Ion Exchange Separation of Organic Acids Eluent: 1.6mM Heptafluorobutyric acid 4. Size Exclusion Stationary Phase : Porous Gel Solute passes pores or is excluded Gel Permeation Chromatography or Gel Filtration Chromatography polymer M.P. Porous particle Schematics of Porous Particles being used in SEC Size Exclusion Large Species Elute First since they can pass through as many pores so they spend little time in S.P. Various Columns needed to separate SAMPLES of SPECIFIC SIZE RANGES Useful for Determining Size & Size Distribution for Polymers, Proteins, ... Example Application by SEC Gas Chromatography (GC) First Commercial Instrumental Chromatographic System Separation in GC Sample should be converted to Vapor State Mobile Phase : Inert Gas (H2, He, N2, Ar) Types of Stationary Phases GC Solid (GSC) : silica gel, alumina, charcoal, etc Liquid(GLC): nonvolatile liq. Coated on firebrick (Chromosorb..), diatomaceous earth thickness : ~ 0.25 mm i.e.: Poly(dimethyl siloxane) (Dipheny)0.05(dimethyl)0.95polysiloxane Separation is achieved by order of Sample’s Polarity. Nonpolar Solute - attracted to Nonpolar S.P. Types of Columns GC Types of Columns GC Conventional 1/8-1/4 inch OD, SS or glass tube 6-20 feet in length Capillary 0.1 - 0.5 mm ID 10-100 meters in length OTC WCOT GC Operation Isothermal : Constant Temperature Temperature Programming : Temp. varies during the analysis By Increasing T, Increase the activity (diffusion, ad/desorption,etc.) Increase speed recovery For Temperature Programming, Solubility Variations, Volatility of Solutes should be considered Example Applications Example Applications Example Applications Chemical Structure ? By How • Electromagnetic - Radiation • Electron Beam Molecule • Neutron Most Typical Probe ! Electromagnetic Radiation Measure Physical & Chemical changes What is electromagnetic Radiation ? Light, microwaves, x-rays, and TV and radio transmissions are all kinds of electromagnetic radiation. They are all the same kind of wavy disturbance that repeats itself over a distance called the wavelength. The different names refer to different wavelengths. frequency c E h h Planck const. Light speed wavelength What can happen when light shines on a material ? heat Visible light Atomic level Inner shell Electron transition X-ray Diffraction Outer shell Electron transition UV-VIS Spectroscopy Atomic Absorption Atomic Emission Rotational Motion NMR, MRI, ESR Molecular vibration IR Spectroscopy Raman Spectroscopy Molecular level X-ray Diffraction or X-ray Crystallography X-rays are diffracted by crystals scattered by the electron cloud of an atom of comparable size. Diffraction Pattern Crystal lattice Molecular structure X-Ray Diffraction (X-Ray Crystallography) • The most straightforward way of examining the structure of a compound Bragg’s Law Bright X-ray: Synchrotron radiation High-resolution X-ray crystallography Conditions for constructive interference of the beam at angle θ AP + PC = n AP = PC = d sin θ n = 2d sin θ n sin θ = 2d 1. Crystals are rotated through all angles to obtain diffraction pattern 2. The conversion of diffraction pattern to structure If we know, and θ, We can get d (inter-atomic distance) X-ray Diffraction or X-ray Crystallography Light Spectroscopy II UV-VIS Absorption Spectroscopy • Interaction of UV-VIS light with electronic energy levels in molecules • Good means of identification for transition metal coordination compound or some organic compounds (double bond 포함 시료의 경우) • Good for quantitation (정량분석, HPLC의 검출기) • Spectral band: provides a great deal of information about the electronic structures of molecules S2 excited electronic state S1 rotational levels ho- h1 ho virtual state overtones v=3 Energy Ground state h1 UV-Vis absorption Raman Scattering Fluorescence IR Absorption v=2 v=1 NIR Absorption Light Spectroscopy III IR Spectroscopy • Information on chemical groups in molecules • Stretching and shaking of molecules: IR region (4000 cm-1 – 200 cm-1) • Provides ‘fingerprint’ of the various chemical groups in the molecule (예: carbonyl group: 1700 – 1800 cm-1) • FT-IR (fast, better resolution and S/N) see box 2 CO stretching band Vibrational Modes of Molecules c E h h Light speed wavelength Vibrations in polyatomic molecules: linear molecule: 3N-5 (N = number of atoms) nonlinear molecule: 3N-6 for water: 3N-6 = 3 symmetric stretch 3652 cm-1 asymmetric stretch 3756 cm-1 bend 1595 cm-1 2000 cm-1 1500 929 1000 1000 671 1500 Galactic Library 1994 [Chloroform w/ 0.75% Ethanol; liquid] 2500 1045 1522 2000 1216 2500 1425 OSU FTIR (between NaCl plates) 2401 3000 772 1216 3021 3000 3022 absorbance CHCl3 CHCl3 normal modes 364 Cl 667 cm-1 (sym. stretch) Cl 4 C cm-1 3019 cm-1 (C-H stretch) 3 9 1,2 7,8 H Cl 5,6 760 cm-1 260 cm-1 1215 cm-1 (C-H bend) mode labels are from Hyperchem, e vibrations are doubly degenerate Fourier Transform IR (FT-IR) Interferogram: x=0 FT x=0 Mirror travel Single beam spectrum of air: 100% H2O 4000 CO2 H2O 400 Frequency, (cm-1) Perkin Elmer Galaxy 2000 FTIR top of beamsplitter He-Ne laser Nuclear Magnetic Resonance (NMR) 시료주입 magnet Nuclear Magnetic Resonance • Depends on magnetic properties of certain nuclei (H, C, F, ..) • Certain nuclei rotates about an axis and thus have a property of spin • A spinning charged nucleus creates a magnetic field, thus magnetic moment. Energy ~ radio wave 영역 Spin - spin coupling Nuclear Magnetic Resonance I 초기 NMR: radiowave파 : 고정된 frequency 사용 - magnetic field: 시간에 따라 변화시킴 Signal vs magnetic field 최근 NMR (Pulse FT NMR) - 강한 radiofrequency (다양한 frequency 함유)의 pulse를 가함 - Free induction decay(FID) decayed - Time domain의 신호를 Fourier Transformation으로 frequency의 영역으로 변화시킴 Radio frequency 영역의 빛 흡수 (megahertz) : depends on magnetic field and nucleus being studied (chemical structure에 의존) Magnetic Resonance Imaging (MRI) Imaging tool for clear and detailed pictures of internal organs and tissues using magnet and radio frequency MRI is particularly good for some types of brain tumour, for primary bone tumours and soft tissue sarcomas and for tumours affecting the spinal cord.