UHPLC-MS Basic Principles and Applications Jitnapa Voranitikul February, 2018 Product Specialist LC/MS
Fundamental of Liquid Chromatography https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ
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 3
Thermo Analytical LC Systems Vanquish TM Max Pressure 1517 bar
What is the Small Particle Advantage ? 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
Efficiency is the key!!! Small Particle Advantage 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 Selectivity Efficiency Retention Higher resolution – narrower peaks Higher sensitivity – taller peaks Higher peak capacity (more peaks / unit time) – narrower peaks
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 Application Switching Tandem LC Automated Method Scouting Online SPE Parallel LC
Fundamental of Mass Spectrometry https://www.thermofisher.com/order/catalog/product/TSQ02-10001?SID=srch-srp-TSQ02-10001
What is Mass Spectrometry? “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.” Niessen, W. M. A.; Van der Greef, J., Liquid Chromatography–Mass Spectrometry: Principles and Applications , 1992, Marcel Dekker, Inc., New York, p. 29.
Information Rich Data
Applications of Mass Spectrometry • Pharmaceutical analysis • Biomolecule – Bioavailability studies characterization – Drug metabolism studies, – Proteins and peptides pharmacokinetics – Oligonucleotides – Characterization of potential drugs • Environmental analysis – Drug degradation product analysis – Pesticides on foods – Screening of drug candidates – Soil and groundwater contamination – Identifying drug targets • Forensic analysis/clinical
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 Characteristics • 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
Mass Spectrometry – Block Diagram Liquid Chromatography Very important! - Many columns - Many solvent systems • ESI • APCI • APPI
ION SOURCE IONIZATION TECHNIQUES
Type of Ionization techniques • Electron impact (EI) • Chemical Ionization (CI) • Atmospheric Pressure Ionization (API) • Electrospray Ionization (ESI) • Atmospheric Pressure Chemical Ionization (APCI) • Atmospheric Pressure Photo-Ionization (APPI) • Matrix Assisted Laser Desorption/Ionization (MALDI)
Electrospray Ionization (ESI) Three Fundamental Processes: 1. Production of charged droplets. 2. Droplet size reduction , and fission. 3. Gas phase ion formation.
Ion Evaporation Theory Droplet As droplet evaporates, field Raleigh Limit – Droplet Capillary increases and ions move to Instability - releases containing ions +4 kV surface smaller droplets ions
ESI - Ion Max Source
Atmospheric Pressure Ionization Atmospheric Pressure Electrospray Ionization Chemical ionization
Chemistry Considerations ESI or APCI ESI: APCI: Ions formed by solution Ions formed by gas phase chemistry chemistry Good for thermally labile analytes Good for volatile / thermally stable Good for polar analytes Good for non-polar analytes Good for large molecules Good for small molecules (Proteins / Peptides) (Steroids)
Ion Max Source Design - APCI Probe
Which is Best? • It depends on the exact application. • Increasing polarity and molecular weight and thermal instability favors electrospray. – Most drugs of abuse are highly polar and are easily analyzed using electrospray. – High molecular weight proteins also require electrospray • Lower polarity and molecular weight favors APCI or APPI. • Lower background, but compounds must be more thermally stable.
Mass Spectrometry – Block Diagram
Typical Mass Accuracy and Resolution Type of MS Mass accuracy Resolution Utility for 0.1 amu 6,000 Identify Quadrupole 0.1 amu 8,000 Identify Traps 0.0001 amu <20,000 TOF Empirical formula/ TOF 60,000 Q-TOF composition 0.0001 amu 10,000 Empirical formula/ Sector composition 0.0001 amu 1,000,000 Empirical formula/ Orbitrap composition
MASS ANALYSER QUADRUPLE
TSQ Quantiva MS—Powered by AIM Technology Active Ion Management (AIM) Systematic optimization of all electric fields, in concert, to produce breakthrough performance.
TSQ Triple Quadrupole (available on YouTube) http://www.youtube.com/watch?v=LFB14D8pkoc
Scan Modes in Quadrupole Scan Mode Q1 Q2 Q3 Purpose Scanning Pass All Pass All MW Info. Full Scan Fixed m/z Pass All Pass All Quantitation SIM Fixed m/z Pass All (+ CE) Scanning Structural Info. Product Fixed m/z Pass All (+ CE) Fixed m/z Targeted Quantitation SRM Scanning Pass All (+ CE) Scanning Analyte Screening Neutral Loss Scanning Pass All (+ CE) Fixed m/z Analyte Screening Precursor
Full Scan Mode RT: 0.00 - 20.02 1.24 NL: 100 7.35E7 95 TIC MS HS-helin- 90 1024-1 85 80 4.60 75 70 Chromatogram 65 Relative Abundance 60 55 50 45 40 35 2.62 30 3.79 25 3.10 20 5.38 15 7.78 10 7.58 5 8.23 15.95 16.93 18.34 18.94 8.90 9.81 12.73 14.05 15.09 0 0 2 4 6 8 10 12 14 16 18 20 Time (min) One Click 240 100 Spectrum H + Base peak O H N H tB u at m/z 240 (MH + ) H O % H O 241 0 60 80 100 120 140 160 180 200 220 240 260 280 300
Full Scan (Q1 or Q3) Full Scan Mode Purpose: Survey scan of a chromatographic peak Q1 Scanning RF Only Q3 RF Only Full scan Q1: Q1 RF Only RF Only Q3 Scanning Full Scan Q3:
Selected Ion Monitoring – SIM SIM Mode Purpose: Quantitation on a specific m/z range of ions Q1 Set RF Only + CE Q3 RF Only SIM Q1: Q1 RF Only RF Only + CE Q3 Set SIM Q3:
Selected Ion Monitoring – SIM SIM is in essence a full scan acquisition on a relatively narrow mass window (defined as center mass / scan width) Pass All Fixed m/z Pass All ¤ Advantages ¤ Disadvantages ⁄ ⁄ Targeted analyte monitoring Can suffer from interferences ⁄ ⁄ High duty cycle Not as sensitive or selective as SRM
Full Scan versus 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)
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