LCMS Technology Connects to Your Application Dr.Rittichai - PowerPoint PPT Presentation

LCMS Technology Connects to Your Application Dr.Rittichai Charoensapyanan June, 2018 (LCMS Product Specialist) 1

Topics  Fundamental of Liquid Chromatography (LC)  Fundamental of Mass Spectrometer (MS)  LCMS Applications 2

Fundamental of Liquid Chromatography 3

Liquid Chromatography (LC) Retention time  Identification 4.7 Mobile phase (continuous) Peak area  Quantification 1.9 6.3 Stationary Phase • Liquid Chromatography (LC) : Separation technique which liquid is used as mobile phase • Separation : Between two phases (Stationary phase and Mobile phase) • Compounds are separated from each other based on their difference in affinity for the stationary or mobile phase. 4

HPLC System Degasser : Remove air bubble in solvents • Pump : - Mix solvents • - Control the flow rate of mobile phase and analytes Autosampler : Inject the sample into a running system • Column : Separate each components • Column Compartment : Control a column temperature • Detector : Detect signal from analytes after separation • DAD (UV, VIS) Fluorescence Reflective Index Mass Spectrometer 5

HPLC System Range RSLCnano x2 Dual RSLC RSLC x2 Dual LC  UHPLC system for Nano/Cap/Micro Standard  x2 Dual UHPLC System  20 nL/min – 50 µL/min up to 800 bar  Two systems in one  Continuous direct flow  1000 bar up to 5 mL/min  New standard in retention time  800 bar up to 8 mL/min precision Basic Automated  Binary and Quaternary UHPLCs  Oven temp. 5 – 110 º C  Snap-in valves  1000 bar up to 5 mL/min  200 Hz DAD, MWD, VWD, FLD  nanoViper fitting system for easy  800 bar up to 8 mL/min  Parallel and Tandem LC operation  Oven temp. 5 – 110 º C  Online SPE-LC  Two systems in one  200 Hz DAD, MWD, VWD, FLD  Automated method scouting  Improved sub 2-µm particle column  620 bar UHPLC compatible  Offline 2D-UHPLC compatibility  Flow rates up to 10 mL/min  Turn key Viper kits for ease of use  Ultrafast/ultra resolution system  Oven temp. 5 – 80 º C  3 rd Generation Modules  Automated Application Switching  620 bar UHPLC compatible  Parallel and Tandem LC  Flow rates up to 10 mL/min  Online SPE-LC  Highly economic & reliable  Oven temp. 5 – 80 º C  Automated method scouting  620 bar UHPLC compatible  100 Hz DAD, MWD, VWD, FLD, CAD  Turn key Viper kits for ease of use  Flow rates up to 10 mL/min  Highest flexibility  100 Hz detector range  Modular flexibility Basic Standard x2 Dual LC RSLC x2 Dual RSLC RSLCnano 6

The Highest Pressure HPLC Vanquish TM Max Pressure 1517 bar 7

Advantage of Small Particle Higher efficiency, independent of flow rate means… Faster runs without loss of performance 15 Increasing Column Efficiency 5 µ m u opt 10 3 µ m H E T P ( µ m) u opt 5 u opt 1.9 µ m 0 1 2 3 4 5 6 Linear Velocity (mm/s) Increasing Flowrate 8

Advantage of Small Particle N = 142,000 plates/m 1.9 µ m (189% higher) N = 75,000 plates /m 5 µ m ( ) α − 1 1 k = R s N Efficiency is the key!!! α + 4 1 k Selectivity Efficiency Retention Higher resolution – narrower peaks Higher sensitivity – taller peaks Higher peak capacity (more peaks / unit time) – narrower peaks 9

Advantage of Small Particle Increase Speed, Maintain Resolution 200x2.1mm Speeding up analysis with 1.9 µ m Hypersil GOLD 600µl/min 655 bar 1.9 µ m 400µl/min 190 bar 3 µ m Speed 250µl/min 102 bar 5 µ m 150µl/min 68 bar 8 µ m 100µl/min 56bar 12 µ m 0 2 4 6 8 10 12 14 16 18 Time (min) 10

The UltiMate ™ 3000 HPLC Systems Isocratic Binary Quaternary Dual-Gradient Pump Standard Thermostatted + Fractionation Basic Automated Autosampler Standard With Valves Column Compartment VWD MWD/DAD Fluorescence Corona Coulochem Detector 11

HPLC Applications • Built-in column compartment with 2-position, 6-port switching valve Switching Valve (2-position, 6-port) 12

HPLC Applications • Online SPE: Extraction and separation at the same time !!! Load Position Injection Position 13

HPLC Applications • Parallel LC: Analysis of two distinct methods !!! 14

Fundamental of Mass Spectrometer 15

What is Mass Spectrometer? “The basis in mass spectrometry (MS) is the production of ions, that are subsequently separated or filtered according to their mass-to-charge (m/z) ratio , and detected. The resulting mass spectrum is a plot of the (relative) abundance of the produced ions as a function of the m/z ratio.” • Measure gas-phase ions • Operate at very low pressure (10 -5 to 10 -7 torr) • Mass spectrometer work with IONS • Determine the mass are separated according to their mass-to-charge (m/z) ratio 16 Niessen et al ., LC-MS: Principles and Applications , 1992, Marcel Dekker, Inc., New York, p. 29.

Information Rich Data 17

Mass Spectrum Mass to charge (m/z) = ( molecular weight + charge ) / charge (512.287 x 2) - 2 = 1022.6 (1023.566 x 1) - 1 = 1022.6 18

Mass Spectrometry: Block Diagram Liquid Ionization Mass Analysis Chromatography 19

Ionization • Ion source : Converts sample molecules (neutral) into charged molecules or molecular ions. • Type of ionization techniques o Matrix Assisted Laser Desorption Ionization (MALDI) o Atmospheric Pressure Ionization (API) - Electrospray Ionization (ESI) - Atmospheric Pressure Chemical Ionization (APCI) Ion Source No one ionization technique is applicable to all classes of chemical species ! 20

Atmospheric Pressure Ionization (API) ESI APCI 21

Atmospheric Pressure Ionization (API) ESI APCI • Ions formed by gas phase chemistry • Ions formed by solution chemistry • Good for volatile / thermally stable • Good for thermally labile analytes • Good for polar analytes • Good for non-polar analytes • Good for large molecules (protein/peptide) • Good for small molecules (steroids) 22

Mass Spectrometry: Block Diagram Mass Analysis 23

Mass Analyzer • Triple Quadrupole (QqQ) • Orbitrap 24

Mass Analyzer: Triple Quadrupoles (QqQ) HyperQuad quadrupole mass Active collision cell (Q2) filter (Q3) Ion beam guide with neutral HyperQuad quadrupole mass blocker Dual-mode discrete-dynode filter (Q1) detector Asymetric RF drive Electrodynamic ion funnel (EDIF) High-capacity transfer tube (HCTT) 25

Mass Analyzer: Triple Quadrupoles (QqQ) Q1 and Q3 are “Mass filter” where • ions are scanned by varying the DC/AC & RF voltages across the quadrupole set 26

Mass Analyzer: Triple Quadrupoles (QqQ) Q2 is “Collision Cell” where precursor • ions are fragmented and pass through Q3 for ion sorting again Precursor Ion Product Ions Fragmentation (Collision gas: Ar) 27

Scan Modes in QqQ Purpose: Survey scan of a chromatographic peak m/z 200-400 Purpose: Quantitation on a specific m/z range of ions m/z 250 28

Scan Modes in QqQ Purpose: Targeted quantitation 29

Scan Modes in QqQ Fixed m/z: 400 Fixed m/z: 500 m/z 400 m/z 400 m/z 500 m/z 500 m/z 300 Fixed m/z: 400 Fixed m/z: 400 m/z 400 m/z 400 m/z 400 m/z 300 m/z 400 30

SRM m/z 400 m/z 250 m/z 400 m/z 150 m/z 250 m/z 400 m/z 300 m/z 400 m/z 100 Q3: Product Ion Q1: Precursor Ion Q2: Fragmentation Fixed m/z: 400 Fragment Fixed m/z: 250 m/z 400 m/z 250 m/z 400 m/z 300 31

Scan Modes in QqQ 32

Full Scan VS SIM RT: 0.00 - 75.04 SM: 7G 52.33 NL: 2.91E8 100 Base Peak F: + c NSI Full ms [ 90 400.00-1800.00] MS data14 80 70 Relative Abundance 60 Full Scan 50 47.88 40 30 31.30 55.14 20 34.47 50.24 10 39.42 1.00 18.87 23.56 6.50 8.09 11.51 17.22 24.15 63.65 65.28 70.26 72.63 42.17 44.24 56.03 0 31.30 NL: 7.97E7 100 Base Peak m/z= 1030.90-1031.90 F: 90 + c NSI Full ms [ 400.00-1800.00] 80 MS data14 70 Relative Abundance 60 SIM 50 40 30 20 10 39.85 38.39 3.23 30.99 47.88 3.45 40.53 52.44 55.53 59.41 64.64 67.24 73.57 10.36 14.03 19.66 21.90 27.26 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) 34

SIM VS SRM 0 NL: 2.67E4 25.37 100 m/z= 271.50-272.50 F: + c SIM Relative Abundance ms [236.50-237.50, 80 271.50-272.50, 306.50-307.50] MS probe20f_sim 23.53 60 RT: 23.76 27.35 22.93 40 26.62 20.19 23.43 24.55 21.25 22.41 24.67 25.48 26.26 24.02 20 20.57 22.17 19.61 SIM 0 RT: 23.76 NL: 9.94E3 100 m/z= 306.50-307.50 F: + c SIM Relative Abundance ms [236.50-237.50, 80 271.50-272.50, 306.50-307.50] 24.59 MS probe20f_sim 60 23.13 24.17 25.18 25.34 22.93 26.31 26.46 27.09 40 23.37 21.89 22.52 21.67 20 21.15 20.57 19.65 20.15 0 RT: 23.77 NL: 6.10E5 100 m/z= 207.50-208.50 F: + c EI Relative Abundance SRM ms2 237.000 80 [207.999-208.001] MS Genesis Probe20F 60 40 20 SRM 0 NL: 1.06E6 RT: 23.77 100 m/z= 236.50-237.50 F: + c EI Relative Abundance SRM ms2 272.000 80 [236.999-237.001] MS Genesis Probe20F 60 40 Superior Selectivity 20 0 Free from sample matrix 18 19 20 21 22 23 24 25 26 27 Time (min) 35

Mass Analyzer • Orbitrap 36

Mass Analyzer: Orbitrap 37

Orbitrap Mass Analyzer: Principle of Operation (r,z) (r, φ ) Image Current k ω = z / m q 38 Makarov A. Anal. Chem. 2000, 72 , 1156-1162.