UHPLC-MS Technology and Applications Rittichai Charoensapyanan March, 2018 Product Specialist LC/MS
Topics � Fundamental of Liquid Chromatography � Fundamental of Mass Spectrometer � Applications in Food Safety, Halal Food and Pharmaceutical
Fundamental of Liquid Chromatography
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 • Pump : - Mix two or more solvents - Control the flow of mobile phase and analytes • Degasser : Remove air bubble in solvents • 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
HPLC System Range RSLCnano x2 Dual RSLC RSLC x2 Dual LC § UHPLC system for Nano/Cap/Micro § x2 Dual UHPLC System Standard § 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 § Binary and Quaternary UHPLCs § Oven temp. 5 – 110 º C Basic Automated § 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 § Flow rates up to 10 mL/min § Turn key Viper kits for ease of use compatibility § Oven temp. 5 – 80 º C § Ultrafast/ultra resolution system § 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
The Highest Pressure UHPLC Vanquish TM Max Pressure 1517 bar
Advantage of Small Particle Higher efficiency, independent of flow rate means… Faster runs without loss of performance 15 Increasing Column Efficiency 5 µ m 10 u opt 3 µ m H E T P ( µ m) u opt 5 1.9 µ m u opt 0 1 2 3 4 5 6 Linear Velocity (mm/s) Increasing Flowrate
Advantage of Small Particle 1.9 µ m N = 142,000 plates/m (189% higher) 5 µ m N = 75,000 plates /m ( ) α − 1 1 k = R s N α + 4 1 k Efficiency is the key!!! Selectivity Efficiency Retention Higher resolution – narrower peaks Higher sensitivity – taller peaks Higher peak capacity (more peaks / unit time) – narrower peaks
Advantage of Small Particle Increase Speed, Maintain Resolution 200x2.1mm Speeding up analysis with 1.9 µ m Hypersil GOLD 600 µ l/min 655 bar 1.9 µ m 400 µ l/min 190 bar 3 µ m Speed 250 µ l/min 102 bar 5 µ m 150 µ l/min 68 bar 8 µ m 100 µ l/min 56bar 12 µ m 0 2 4 6 8 10 12 14 16 18 Time (min)
The UltiMate ™ 3000 LC Systems Isocratic Binary Quaternary Dual-Gradient Pumps Standard Thermostatted + Fractionation Basic Automated Autosampler Standard With Valves Column Compartments VWD MWD/DAD Fluorescence Corona Coulochem Detectors
UHPLC + Applications • Built-in column switching valve • 2-position, 6-port column switching valve Switching Valve
UHPLC + Applications Online SPE Parallel LC
Fundamental of Mass Spectrometer
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.” Operate at very low pressure (10 -5 to 10 -7 torr) (Atmosphere = 760 torr) • • Mass spectrometer work with IONS • Measure gas-phase ions • Determine the mass are separated according to their mass-to-charge (m/z) ratio Niessen et al., LC-MS: Principles and Applications , 1992, Marcel Dekker, Inc., New York, p. 29.
Information Rich Data
Mass Spectrum mass to charge = ( molecular weight + charge ) / charge (512.287 x 2) - 2 = 1022.5 (1023.566 x 1) - 1 = 1022.5
Mass Spectrometry: Block Diagram Liquid Ionization Mass Analysis Chromatography
Ionization • Ion source : converts sample molecules (neutral) into charged molecules or molecular ions. • Type of ionization techniques Electron Impact (EI) o Chemical Ionization (CI) o Matrix Assisted Laser Desorption Ionization (MALDI) o Atmospheric Pressure Ionization (API) o - Electrospray Ionization (ESI) Ion Source - Atmospheric Pressure Chemical Ionization (APCI)
Atmospheric Pressure Ionization (API) ESI APCI
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 non-polar analytes • Good for polar analytes • Good for small molecules (steroids) • Good for large molecules (protein/peptide)
Mass Spectrometry: Block Diagram Liquid Ionization Mass Analysis Chromatography
Mass Analyzer • Triple Quadrupole (QqQ) • Orbitrap
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)
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
Mass Analyzer: Triple Quadrupoles (QqQ) • Q2 is “Collision Cell” where precursor ions are fragmented and pass through Q3 for ion sorting again Precursor Ions Product Ions Fragmentation (Collision gas: AR)
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
Scan Modes in QqQ Purpose: Targeted quantitation
Scan Modes in QqQ Fixed m/z: 400 Fixed m/z: 400 Fixed m/z: 500 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
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 Q1: Precursor Ion Q2: Fragmentation Q3: Product Ion Fixed m/z: 400 Fixed m/z: 250 m/z 400 m/z 250 m/z 400 m/z 300
Scan Modes in QqQ
TSQ Triple Quadrupole MS http://www.youtube.com/watch?v=LFB14D8pkoc
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 50 Full Scan 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)
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 18 19 20 21 22 23 24 25 26 27 Free from sample matrix Time (min)
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