儀 器 分 析 李茂榮 國立中興大學化學系
分析化學乃研究探討、開發新的技術或方法，解決各領域所遇到的問題。
• 分析化學是一門工具科學，在科學研究上可以幫助我們擴大和加深對於自然界的認識，起著「眼睛」的作用。 •在現代的化學研究中，分析手段尤其是不可缺少的，而且對分析化學的要求也越來越高。在其他科學領域中，例如在礦物學，地質學，海洋學，生物學，醫藥學，農業科學，天文學，甚至考古學中，也都要用到分析化學。 •國民經濟的許多部門，分析化學具有很大的實用意義。例如，工業上，資源的勘探，生產的控制，產品的檢驗，三廢(廢水，廢氣，廢渣)處理和環境的監測;農業上，土壤的調查，作物營養診斷，農產品質量檢驗等等，都廣泛地應用分析化學。
從對分析化學的要求，分析手段必須越來越靈敏、準確、快速、簡便和自動化。例如，半導體技術中的原子級加工，要求測出單個原子的數目;環境保護工作要求測定ppb級甚至ppt級的污染物;純氧氣吹煉鋼每爐只用幾十分鐘，他要求快速的爐前分析;在地質調查、勘探工作中，需要獲得上百萬、上千萬個數據，不但要求快速和自動化，而且要求發展偵測技術。不僅如此，分析化學的任務也不再限於測定物質的成分和含量，而是往往還要知道結構、價態、狀態等等，因而它活動的領域也由宏觀發展到微觀，從總體進入到微區、表面或薄層，由表觀深入到內部從靜態擴展到動態。
元素缺乏症及過量累積的毒性
QITMS: Low Cost permits unique applications
4 Ss of Trace Analysis Sensitivity (靈敏度) Selectivity (選擇性) Speed (速度) $ (分析費用)
化學分析和儀器分析 分析方法的建立，是以被測物質在某種變化中或某種條件下所顯示的性質為依據的。以物質的化學反應為基礎的分析方法稱為化學分析法。在定性分析中，許多分離和鑑定反應，就是根據成分在化學反應中生成沉澱、氣體或有色物質而進行的。在定量分析中，主要有重量分析、滴定分析等方法。這些方法歷史悠久，是分析化學的基礎，所以又稱為古典化學分析法。
儀器分析特性 準確度 (Accuracy) 靈敏度 (Sensitivity) 再現性 (Reproducibility) 4. 解析力 (Resolution)
以被測物質的某種物理性質為基礎的分析方法稱為物理分析法。這類方法不需要進行化學反應，可以直接進行鑑定或測定，例如X-射線分析法就屬於這一類。但物質的某些物理性質往往要通過化學反應才能顯示出來，這種性質我們稱之為物理化學性質。以被測物質的物理化學性質為基礎的分析方法稱為物理化學分析法。這兩類方法都需要特殊的儀器， 所以又稱為儀器分析法。儀器分析法主要有光學分析法、電化學分析法、色譜分析法、質譜分析法和放射化學分析法等，種類很多，而且新的方法正在不斷地出現。
放射光譜法[(X光,UV,可見光,電子,歐皆(Auger)]; 火焰光度法;螢光法(X光,UV,可見光)放射化學法 分析信號 輻射線的放射 放射光譜法[(X光,UV,可見光,電子,歐皆(Auger)]; 火焰光度法;螢光法(X光,UV,可見光)放射化學法 輻射線的吸收 光譜光度法(X光,UV,可見光,IR);比色法;原子吸收;核磁共振以及電子自轉共振光譜法 輻射線的散射 濁度分析法;懸液法;拉曼光譜 輻射線的折射 折射法;干涉法 輻射線的繞射 X光與電子繞射法 輻射線的旋轉 旋光法;旋光色散;圓形二向色性測量法 電位 電位法;瞬時電位法 電流 極譜法;電流滴定法;電量法 電阻 導電度測量法 質量電荷比 質譜法 反應速率 濃度-時間解析法 熱性 導熱度與焓測量法 質量 重量分析 體積 容積分析
/Raman Regions of The Electromagnetic Spectrum
Ultraviolet/Visible spectroscopy The component colors of the visible portion can be separated by passing sunlight through a prism, which acts to bend the light in differing degrees according to wavelength.
Transmittance (T%) = Psolution/Psolvent × 100% = P/P0 × 100% Absorbance (Abs) = log(Psolvent/Psolution)= log(P0/P)= -log(P/P0) Reflection and scattering losses with a solution contained in a typical glass cell.
Beer’s Law Radiation of initial radiant power P0 is attenuated to transmitted power P by a solution containing c moles per liter of absorbing solution with a path length of b centimeters.
Application of Beer’s Law to Mixture Limitation to Beer’s Law Mismatched Cells Real Limitations to Beer’s Law Absorbance is not proportional to concentration at high concentrations (usually > 0.01 M) due to solute-solvent interactions, solute-solute interactions, and hydrogen bondings,… Beer-Lambert Limiting Law
Types of instruments Single Beam Double Beam
UV spectra of phenylalanine, tyrosine and tryptophan at pH 6.0
UV spectra in water of the four bases commonly found in nucleic acids: thymine (T), cytosine (C), adenine (A), guanine (G), and uracil (U).
Application of Ultraviolet/Visible spectroscopy The Determination of Food Colours in Mixtures Manufacturers often mix dyes to obtain an acceptable colouring in foodstuffs. UV/Vis spectrometer can be used for the determination of the concentrations of dyes in food products.
Application in drug analysis Different drugs create a different pattern
Infrared spectral regions Non-destructive technique Required only a small amount of sample for analysis Widely used for analysis of organic and inorganic materials
Theory of IR Absorption Spectrometry Dipole Moment Changes During Vibrations and Rotations Net Change in Dipole Moment Adsorb IR Radiation Ex: HCl, NO, CO,… No Net Change in Dipole Moment No IR Absorption Ex: O2, N2, Cl2 IR absorption spectrum of a thin polystyrene film. Wavenumber (in cm-1): 10 mm = 1/(10 × 10-4) = 1000 cm-1
Types of Molecular Vibrations Stretching (Changes in Bond Length) Symmetric Asymmetric Bending (Changes in Bond Angle) Scissoring Twisting Wagging Rocking
Near-IR Absorption Spectrometry For the routine quantitative determination of species, such as water, proteins, products of agricultural, food, petroleum, and chemical industries. MPA Multi Purpose FT-NIR Analyzer Analysis of liquids, solids, powders and tablets Automated 30-position sample wheel Used for vials and tablets of all shapes and sizes Can be connected to laptop via Plug can even be placed on a utility cart to improve its mobility. Fiber probes
Regions of the Infrared Spectrum 4000-2500 cm-1 N-H, C-H, O-H (stretching) 3300-3600 N-H, O-H 3000 C-H 2500-2000 cm-1 CºC and C º N (stretching) 2000-1500 cm-1 double bonds (stretching) C=O 1680-1750 C=C 1640-1680 cm-1 Below 1500 cm-1 “fingerprint” region
Infrared Spectra of Hydrocarbons C-H, C-C, C=C, C C have characteristic peaks absence helps rule out C=C or C C
Cyclohexane (top), cyclohexene (bottom)
IR Spectra - Examples sp3 C-H str no O-H str no N-H str no sp or sp2 C-H str no C=O str no C=C str C-H bend This is an alkane.
IR Spectra - Examples C=C str sp2 C-H str This is an alkene.
Applications of Infrared spectroscopy
Application in Crime Scene Investigation (CSI) IR spectra of individual glitter particles in three different commercial cosmetic products
Overlay of IR spectra from two different glitters shows reproducible differences
Library search of top spectrum (Whiteopal-top)
Application of Infrared spectroscopy Infrared thermal imaging Human temperature screening Internal building inspection saves on energy bills External building inspection identifies insulation problems Thermal imaging used for surveillance at the port Fugitive Searches Tactical Entry Teams Search and Rescue
Sir Chandrasekhara Venkata Raman Raman spectroscopy Sir Chandrasekhara Venkata Raman (1888-1970) Discovered Raman Effect in 1928 while studying the scattering of sunlight, made quasi –monochromatic by the use of a filter. Won the 1930 Nobel Prize in Physics
What is Raman scattering? Raman is a light scattering technique, whereby a molecule scatters irradiant light from a source laser. Most of the scattered light is at the same wavelength as the laser source and does not provide useful information, but a small amount of light is scattered at different wavelengths.
Energy diagram for IR absorption/emission, Rayleigh scattering, and Raman scattering. Excited electronic state Ground electronic state Virtual state IR absorption and emission Rayleigh scattering Stokes Raman Anti-Stokes Raman Energy hvM 1 hv0 h(v0 – vM) h(v0 + vM)
488 nm 476.3 nm 500.2 nm weak; no fluorescence strong; fluorescence Raman spectrum of CCl4, excited by laser radiation of λex = 488 nm (νex=20,492 cm–1).
Asymmetric stretching Information of Raman Scattering and Infrared Spectroscopy are Complementary Infrared Active: Net Change in Dipole Moment Ex: CO2 Symmetric stretching is IR Inactive Asymmetric stretching is IR Active Raman Active: Polarizability of The Molecule Must Change with The Vibrational Motion is Raman Active
Raman Spectra: Fingerprinting a Molecule Raman spectra are molecule specific Spectra contain information about vibrational modes of the molecule Spectra have sharp features, allowing identification of the molecule by its spectrum Examples of analytes found in blood which are quantifiable with Raman spectroscopy
Raman Spectrum of Cholesterol
The most common applications for Raman analysis 1. Pharmaceutical and cosmetic 5. Geology/Mineralogy/Gemology Characterization of active pharmaceutical ingredients and excipients in a tablet by Raman spectrometer. Geological Materials Analysis 2. Semiconductors 6. Arts/Museums Strain measurements of a Si cap layer deposited on a SiGe substrate determination of Ge content. The non-destructive and in-situ analysis of pigments. 3. Nanotubes and nano-material 7. Forensic science The non-destructive and in-situ identification of controlled drugs and narcotics 4. Biology 8. Polymer In vivo Raman measurements of human skin. Raman analysis of single bacteria cells. Characterization of polymers in industrial applications. HORIBA Scientific
Raman Spectral Pathology of Atherosclerosis (動脈粥狀硬化 ) normal artery lipid-rich plaque calcified plaque collagen elastin actin cholesterol -carotene proteins Ca hydroxyapatite
Nuclear Magnetic Resonance Spectroscopy What is NMR? N (nuclei): 原子核 自旋角動量不為零的原子核 M (magnetic field): 磁場 外加磁場，B0 R (resonance): 共振 同調頻率下的能量/磁矩交互作用 In contrast to ultraviolet, visible, and infrared absorption, nuclei of atoms rather than outer electrons are involved in the absorption process. The energy radiated back out is at a specific resonance frequency which depends on the strength of the magnetic field and other factors. Two general types of NMR spectrometers: continuous-wave (CW) and pulsed or Fourier transform (FT-NMR) spectrometers
Continuous wave (CW) vs. Fourier Transform (FT) NMR In CW-NMR the resonance condition ν = γB0/2π is matched for one frequency at a time, by sweeping the field B0.
Continuous wave (CW) vs. Fourier Transform (FT) NMR Richard R. Ernst The Nobel Prize in Chemistry 1991 Fourier transform
Advantages of FT over CW Faster – no sweeping, all the frequencies at once The possibility f multiple scanning, to improve the signal to noise ratio. The possibility of spin-engineering- manipulation of the spins through pulses and delays reveals more information (2D NMR)
a c b Ha Hc Hb
What can NMR do? Structural determination and dynamics Small molecules to macromolecules Liquids, semisolids, solids Compounds, complexes, mixtures Noninvasive observation of internal structure of almost everything (Imaging) Magnetic Resonance Imaging (MRI)
Molecular structure of small molecule
3D structure of proteins NMR can deal with proteins ~30,000 Da (300 residues)
Solid state NMR Superconductivity phase transition Polymers Advanced materials Nanoparticles Phys. Rev. Lett., 1997, 78, 72
Application of NMR Magnetic Resonance Imaging (MRI) – The Noble Prize in Physilogy and Medicine 2003 MRI image of brain MRI image of plant MRI is an imaging technique that utilizes the nuclear magnetic resonance of the hydrogen atom.
Magnetic Resonance Imaging: MRI An MR image is (usually) a map of water protons, with intensity determined by local physical environment Contrast and image quality are determined by Pulse sequence Field strength Shim quality Acquisition time
Mass Spectrometry
What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! 12 units
What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! Number of counts 12 units 8 9 10 11 12 13 14 15 16 mass/charge
What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! Number of counts 14 units 8 9 10 11 12 13 14 15 16 mass/charge
What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! Number of counts 12 units 8 9 10 11 12 13 14 15 16 mass/charge
1906 Physics Nobel Laureate Joseph John Thomson 1906 Physics Nobel Laureate JJ Thomson’s cathode ray tube, 1897
Gas Phase/ Ionize Separate Based on Mass/Charge Sample Why Ionize ? Difficult to manipulate neutral particles on molecular scale. If they are charged, then we can use electric fields to move them around. Detector
Mass spectrometry studies ions Mass spectrometers determine mass-to-charge ratio of ions The analysis is performed in the gas phase not in solution Structure can be elucidated by determining the mass of fragments
Ion source 目的: 將分析物離子化 Electron impact ionization (EI) Chemical ionization (CI) Fast atom bombardment (FAB) Atmospheric pressure ionization (API) ＊electrospray ionization (ESI) ＊atmospheric pressure chemical ionization (APCI) ＊atmospheric pressure photoionization (APPI) Matrix-assisted laser desorption ionization (MALDI) Inductively coupled plasma (ICP)
M M(g) + e- M+(g) + 2e- M+(g) A+Fragment 1 (g) + BFragment 2 (g) Electron energy is chosen by compromise. Fragment Information is useful. It can help structural determination. However, many ions produce only fragments with no molecular ion remaining. Molecular ion often very unstable. 70 eV “Classical Spectra” to be used for comparisons
Electrospray Ionization (ESI) Source:http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/index.html
ElectroSpray Ionization – sample is sprayed out of a narrow nozzle in a high potential field. Generates positive (M+nH)n+ and negative (M - nH)n- ions and almost no fragmentation. Generates multiple charged ions.
5KV 特別容易形成 Multi-charged Ions [M+H]+ [M+2H]2+ [M+3H]3+ N2 Sheath Gas And Aux Gas [M+2H]2+ [M+3H]3+
Multiple Charged Spectrum of ESI 16+ 18+ 17+ Myoglobin 15+ 19+ M≈16965Da 20+ 14+ 21+ 13+ 22+ 23+ 12+ 24+ Multiple charging means heavier ions can be analyzed by analyzers with a lower range; also, charge states can be deconvoluted to get a rather good mass for the uncharged species. 11+ 25+ 10+
MALDI Source:http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/index.html
Mass Analyzer 目的 : 分離不同質荷比 (m/z) 之離子 Magnetic sector Quadrupole Quadrupole ion trap Time-of-flight (TOF) Fourier transform mass spectrometry (FT-MS)
Full Scan V.S. SIM Full Scan Mode SIM mode
Tandem Mass Spectrometry Usually referred to as MS/MS. Here one can take a mass spectrum of a mass spectrum. A particular ion fragment isolated in the first stage is directed to the second phase for further fragmentation and dispersion. For large biomolecules, this is essential for spectral analysis. There are two types: Tandem in space EBEB: Double Double Focusing. Two double focusing magnetic sector stages. QQQ Q-ToF TOF-TOF B. Tandem in Time Ion Trap FT-MS
Four scan modes in tandem mass spectrometry
MS, MS/MS, and MS/MS/MS of TBZ Matrix peak No matrix peak MS (500 pg) MS/MS (10 pg) MS/MS/MS (10 pg) 20180-220 20117465-220 Note MS/MS might be satisfactory on some columns but the small amount of 174 from the interfering compound will interfere as the column ages.
Applications of Mass Spectrometry in Trace Analysis
毒奶粉中三聚氰胺
三聚氰胺(Melamine)的性質 化學式： 俗稱： 特性： C3H6N6 蜜胺、蛋白精、美耐皿，是一種含氮雜環有機化合物。 白色結晶體。幾乎無味，微溶於水(3.1 g/L常溫)，微毒。 Melamine
為何被濫用? 凱氏定氮法常於食品工業中被使用—經由測定氮原子的含量來間接推算食品中蛋白質的含量。 由於由於三聚氰胺(含氮量66%)比蛋白質(平均含氮量16%)含有更高比例的氮原子，因此被偷加在奶粉中，可產生蛋白質含量較高的假象。 水 + 三聚氰胺 含氮量 奶粉 β-乳球蛋白、γ-乳白蛋白 、乳清蛋白、酪蛋白
形成結石的真正元兇 三聚氰胺(Melamine)與三聚氰酸(Cyanuric acid) 兩者結合之晶體，易在腎臟中兩者結合沉積，從而形成腎結石，堵塞腎小管，嚴重將導致腎衰竭。
前處理方法: 液液萃取法 (Liquid-Liquid Extraction) 取出1 g 檢體於15 mL 離心管， 加入 5 mL 萃取溶液 (ACN/H2O/1N HCl, 12:12:1) 振盪(1 min) 後，離心 (4000 rpm,5 min) 取出上層液1 mL至離心管，加入 10 mL Dichloromethane 振盪(2min) 後，離心 (4000 rpm,5 min) 取出上層液 0.5 mL 於eppendorf ，加入H2O 0.5 mL 振盪(1 min) 後，離心 (4000 rpm,5 min) 取出上層液體於eppendorf中,振盪5 sec 後,進行SPE
前處理方法: 固相萃取步驟 (SCX, Cation Ion Exchange) Condition Loading Washing 甲醇 5 mL 去離子水 5 mL Condition 注入上述所有萃取液 Loading 0.1 N HCl 5 mL 甲醇 1 mL Washing 5% 氨水甲醇溶液 5 mL Elution 吹乾，以ACN:Ammonium acetate (9:1)溶劑 0.5 mL 定容, 注入LC/MS/MS分析 N2 91
三聚氰胺質譜層析圖 (a) (b) (c) 50 ppb標準品 空白真實樣品 添加1 ppm RT: 0.00 - 12.00 1 2 3 4 5 6 7 8 9 10 11 Time (min) 10000 20000 30000 5000 15000 Intensity 500000 1000000 1500000 2000000 液相層析串聯質譜術所得到的三聚氰胺(50 ppb)質譜層析圖(a) 50 ppb 之標準品(b) 空白真實樣品 (c) 添加1 ppm之三聚氰胺於空白樣品中。
真實樣品之質譜層析圖 (a) 三聚氰胺標準品 (b) 罐裝咖啡真實樣品 RT: 0.00 - 12.00 1 2 3 4 5 6 7 8 9 10 11 Time (min) 500000 1000000 1500000 2000000 2500000 3000000 3500000 Intensity (a) 三聚氰胺標準品 (b) 罐裝咖啡真實樣品 液相層析串聯質譜術所得到的質譜層析圖 (a) 50 ppb 之三聚氰胺標準品 (b) 罐裝咖啡真實樣品。
結論 目前各領域所面對的困難即要解決的問題，均是複雜基質上微量的成分分析，欲達成此目的，非有更精密的分析儀器，無法竟其功，但了解及如何應用儀器的特性，則更形重要。