Liquid Chromatography/ Mass Spectrometry Fundamental Jimmy Chan WW Seminar Tour Spring 2006

The Advantage of LC-MS A general detector It can connect with HPLC to separate and get more structure informations It can use in quantitative and qualitative analysis WW Seminar Tour Spring 2006

The Limit of LC-MS Sample must be ionization Ionic compound can motion、transfer and analysis MS must vacuum 离子飞行的平均自由程需最大化 理论上，真空度越高越好 MS must analysis with M/Z WW Seminar Tour Spring 2006

The Solvent of API-MS Suitable for both ES and APCI Methanol Acetonitrile Water Ethanol Propanol Isopropanol Butanol DMF(1) DMSO(1) Acetone CH2Cl2 CHCl3 Suitable for only APCI Toluene Benzene（2） Hydrocarbons (e.g., Hexane) Styrene （2） CCl4 CS2 Cyclic Hydrocarbons (e.g., Cyclohexane) At lower solvent percentages (~10 % or less) under ES conditions These solvents not often used due to environmental health and safety issues WW Seminar Tour Spring 2006

The Selection of API Addtive pH 乙酸，甲酸，TFA，氢氧化铵 一般的缓冲液/离子配对试剂 乙酸铵/甲酸铵 三氟乙酸 (TFA) 七氟丁酸 (HFBA) 四乙基或四丁基氢氧化铵 (TBAH) 阳离子化试剂 20-50 mM的乙酸钠或乙酸钾 一般考虑 挥发性（污染喷雾室，堵塞喷嘴） 导电性（对离子气化过程减少小液滴的形成） 离子配对（进入气相时中和预带电离子） 不能使用在HPLC中常使用的磷酸盐，硫酸盐或硼酸盐! WW Seminar Tour Spring 2006

The Application of wide-range Small Molecular: Medical drug Analysis Drug abuse Analysis Antibacterial drug Analysis Steroid Analysis Structure comformation Proteomics: Peptide and Protein molecular Weight Peptide Sequence Peptide and Protein Purity Analysis Protein identification WW Seminar Tour Spring 2006

The LC method transfer to LC/API-MS method 非挥发性的缓冲盐 挥发性缓冲盐 浓度 <10 mM (对于 ES )或<100 mM (对于 APCI) 用醋酸铵、或甲酸、三氟乙酸（TFA）、七氟丁酸（TFBA）、羟基四丁基胺取代磷酸盐、硫酸盐和硼酸盐。 如果必须使用非挥发性盐，, 使用仅仅阳离子或阴离子部分不挥发的缓冲盐，例如，用醋酸钠而不用磷酸钠。 Formic acid, acetic acid, TFA, ammonium hydroxide 挥发性离子对试剂可以使用 WW Seminar Tour Spring 2006

API-Electronspray Ionization + 加热的氮气干燥气 双电子毛细管入口 雾化气 (气体用红色显示) 溶剂喷雾 电喷雾离子 -4000 V  蒸发 Rayleigh 极限 Coulomb 爆裂 被分析离子 WW Seminar Tour Spring 2006

API-Chemical Ionization 蒸汽 通过电晕针放电形成带电荷的反应剂离子 溶剂在蒸发器中蒸发 + 含有气溶胶的分析物 分析物离子 电荷转移至 分析物分子 流动相 分析物 溶剂--------->[溶剂+H]+ [溶剂+H]++M--------->溶剂+[M]+ WW Seminar Tour Spring 2006

The Characteristic Of API ESI Compound with volitility Compound with thermo unstabilite Ionization form in liquid Besides singal charge, it can form mutiple charges APCI Compound with volitility Compound polarity lower than ESI Compound with thermo stabilite Ionization form in gas Only singal charge WW Seminar Tour Spring 2006

The Relative Application of LC/MS WW Seminar Tour Spring 2006

What is LC/MS?? WW Seminar Tour Spring 2006

Which mode do you get the singal charge?? [M+H]+ 基峰: 谱图中最强的离子 苯基丁氮酮（Phenylbutazone）的API-ES 谱图 WW Seminar Tour Spring 2006

Which mode do you get the singal charge?? Ginsenoside WW Seminar Tour Spring 2006

Which mode do you get the mutiple-charge?? 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 Abundance 5000 10000 15000 20000 25000 30000 35000 40000 m/z--> A+25 679.45 A+24 707.65 A+23 738.45 A+22 771.90 A+21 808.60 A+20 848.95 A+19 893.55 A+18 943.15 A+17 998.55 A+16 1060.85 A+15 1131.50 A+14 1212.30 Abundance 16400 16600 16800 17000 17200 17400 17600 m/z--> 280000 A Assume adduct is a 240000 16959.09 proton: 200000 M1=(A+n)/n 160000 M2=(A+n+1)/(n+1) 120000 80000 Solve for A 40000 肌红蛋白(Myoglobin)的API-ES 谱图 WW Seminar Tour Spring 2006

3D-Data（R.T、m/z、Intensity） Mass Spectrum Concept 3D-Data（R.T、m/z、Intensity） TIC 反应产物的联机分析结果 a—色谱图；b—总离子流图；d—质谱棒图 WW Seminar Tour Spring 2006

Raw, Filtered Full, and Condensed Data Abund Raw Data 原始数据 Filtered Full Data 过滤后的数据 Profile Condensed Data 精简数据 line spectra m/z Abund threshold m/z Abund m/z WW Seminar Tour Spring 2006

Mass Spectrum WW Seminar Tour Spring 2006

Total Ion Chromatography (TIC) UV TIC WW Seminar Tour Spring 2006

Extract Ion Chromatography (EIC) Increase S/N Subtract background Separate the co-elution What the purpose of EIC?? Quantitative !! EIC 285 TIC WW Seminar Tour Spring 2006

The Explain of API Mass Spectrum!! ESI and APCI can supply the compound messgae The ion depend on additive and condition Negative [M-H]- 碱性条件 [M+X]- X =溶剂或缓冲溶液中的阴离子 [M-H+S]-溶剂添加剂 Positive [M+H]+ 酸性条件 [M+Na]+, [M+K]+ (有盐时) [M+NH4]+ 有铵盐缓冲溶液 [M+X]+ X = 溶剂或缓冲溶液中的阳离子 [2M+H]+ 在高浓度时形成的二聚体 [M+H+S]+ 溶剂添加剂 WW Seminar Tour Spring 2006

What is the Collision-Induced Dissonciation(CID)? 通过与中性分子碰撞将能量传递给离子的过程。能量传递足以导致键的开裂和所选离子的重排。 WW Seminar Tour Spring 2006

The CID in API Transition region 这是什么? 利用 API-MS 的传输区的电场在中压区(~0.01 - 1 torr)碰撞激活离子来产生CID质谱图。 硬件: ION TRAP WW Seminar Tour Spring 2006

The Process of HED and EM 正离子采样 负离子采样 Lens Dynode 7 kV EM 0 to -3000 e- SQ WW Seminar Tour Spring 2006

Introducing the Agilent 6130 Singal Quadrupole Mass Spectrometer WW Seminar Tour Spring 2006

6410 Triple Quadrupole LC/MS 6510 Quadrupole Time-of-Flight New Agilent 6000 LC/MS Series A new and expanded portfolio for applications from life science to chemical analysis, combining exceptional performance and legendary Agilent reliability Updated Ion Trap Family 6300 Series Ion Trap LC/MS New QQQ 6410 Triple Quadrupole LC/MS New QTOF 6510 Quadrupole Time-of-Flight LC/MS The Triple Quadruple (QQQ) is a benchtop instrument for MS/MS quantitation. It combines unsurpassed reliability with best-of-industry sensitivity at best price value. It includes a working Autotune function that dramatically enhances reliability and ease-of use and will be available at a price significantly lower than comparable competitor instruments. . Updated TOF 6210 Time-of-Flight LC/MS 6100 Series Single Quad LC/MS New Single Quads WW Seminar Tour Spring 2006

Lassen LC/MS Singal Quadrupole system introduction WW Seminar Tour Spring 2006

Atmosphere Pressure Ionization -Mass Spectrometry (API-MS) API-MS is usde for analysis liquid samples that introduce by HPLC、 FIA and Infusion. Application in wide mass range. 1pg sensitiveity limit. WW Seminar Tour Spring 2006

6100 Series SQ: 6110、6120、6130 and 6140 1. Entry Level (10 to 1500amu)    p/n G6110AA 2. Mid-range (POS/NEG, 10 to 1500amu)  p/n G6120AA 3. Research (2 to 3000amu)  p/n G6130AA 4. Research (fast scan, 10 to 1350amu)  p/n G6140AA 6120 SIM Scan ( 10pg; S/N>20:1 ) Mass range: 10~1500 m/z Pos/Neg Switching and Muti Signal Acquisition WW Seminar Tour Spring 2006

Function Block Diagram Inlets Ion Source Ion Optics Mass Filter Detector Vacuum System Electronics WW Seminar Tour Spring 2006

Agilent LC-6320 Ion Trap/MS Shield hole Focus Len HED/EM Liquid introduce Nubalizer gas Quadrupole capillary Octopole Nubalizer + + Neutral Molecules Analyte Ions Clusters Salts + + + + + + + + + + + + + + + + Heat N2 碎片碰撞电压 (CID) WW Seminar Tour Spring 2006

Mutimod Source ( ESI+APCI ) Capillary HPLC inlet Nebulizer Drying gas Corona needle ESI Zone APCI Zone Thermal container IR emitters Charging electrode Reversing electrode Sensor for vapor temperature APCI counterelectrode WW Seminar Tour Spring 2006

The G1978A Multimode Source APCI Counterelectrode Nebulizer IR emitters Field-shaping electrodes Sensor for vapor temperature APCI corona needle WW Seminar Tour Spring 2006

Desolvation (Dry Gas with Heat) 冷却的毛细管 加热的干燥气直接吹向喷雾的液滴 WW Seminar Tour Spring 2006

Desolvation and Capillary WW Seminar Tour Spring 2006

Skimmer、Octopole and Lens WW Seminar Tour Spring 2006

Octopole RF Ion Introduction 更高的灵敏度 更强的分辨率 减少离子能量的色散 Skimmer assembly Lenses WW Seminar Tour Spring 2006

Octopole RF Ion Guide Octopole WW Seminar Tour Spring 2006

Agilent Singal Quadrupole Mass Spectrometry WW Seminar Tour Spring 2006

WW Seminar Tour Spring 2006

Discussion and Question?? WW Seminar Tour Spring 2006