Cad introduction 2019 30 min

Charged aerosol detector

1. Jan Pettersson Nordic HPLC and Chromeleon Support Introduction to Charged Aerosol Detection (CAD)

2. Outline

3. Introduction to Charged Aerosol Detection Comparison of Charged Aerosol Detection to UV and MS Used to quantitate any non-volatile and many semi-volatile analytes with LC Provides consistent analyte response independent of chemical structure and molecule size Neither a chromophore, nor the ability to ionize, is required for detection Dynamic range up to four orders of magnitude from a single injection ( sub-ng to µg quantities on column ) Mass sensitive detection – CAD provides relative quantification without the need for reference standards Compatible with gradient conditions for HPLC, UHPLC, and micro LC

4. Same response for all components – independent of chemical structure Very desirable for many applications (i.e., when use of individual standards is not feasible) Drug libraries, synthetic mixtures Impurities and degradants Herbal medicines Natural products Polymers and surfactants Lipids Allows estimation of quantity Key Concepts – Uniform Response 7.2% RSD variation in CAD response among a wide diversity of non-volatile analytes

5. Key Concepts – Wide Dynamic Range with Charged Aerosol Detection

6. Evolution of Charged Aerosol Detectors Corona™CAD Introduction of the first commercial charged aerosol detector for HPLC with full control via front panel interface. Designed for near-universal detection on any HPLC system using isocratic or gradient separations 2005 2006 Corona™ Plus Expanded solvent compatibility with heated nebulization, software drivers for popular CDS systems and external gas conditioning module for improved precision. Corona™ ultra UHPLC compatible, stackable design, enhanced sensitivity, touch- screen user interface with real-time chromatogram display, incorporated precision internal gas regulation system 2009 ESA Biosciences, Inc. acquired by Dionex Corp. Corona™ ultra RS Unified with Dionex™ UltiMate™ 3000 UHPLC+ system, added on-board diagnostics / monitoring, automated flow diversion capability and selection of linearization parameters 2011 Thermo Fisher Scientific acquires Dionex Corp . 2013 Thermo Scientific™ Extended micro flow rate range; total Dionex™ redesign with concentric nebulization and optimized Corona™ Veo RS spray chamber for enhanced sensitivity, heated evaporation and electronic gas regulation 2015 Thermo Scientific™ Full integration with Thermo Scientific™ Vanquish™ Vanquish™ Charged UHPLC platform, slide-in module design, reduced Aerosol Detector flow path for optimum operation

7. Corona Veo RS Corona Veo Technology – Improved Flow Path

8. How the Charged Aerosol Detection Technology Works 9. Collector Charge on analyte particles is measured by a sensitive electrometer 2. Nebulizer Spray of fine droplets formed 3. Spray Chamber Aerosol conditioning/droplet selection 5. Drying Tube Droplets evaporate leaving dry particles 4. Drain Large droplets are eliminated Inlet: HPLC Column Effluent and N 2 (g) 7. Mixing Chamber Positive charge transferred to analyte particles 6. Corona Charger Positive charge added to gas stream 8. Ion Trap Analyte particles selected, high mobility species removed

9. How the Charged Aerosol Detection Technology Works Evaporation tube Nebulizer Corona Charger

10. The inlet liquid and nebulization gas streams coaxially form into a stable aerosol at the nebulizer tip Small droplets are transported upward into the heated evaporation sector Larger droplets fall and are expelled by a precision micro-pump Coaxial N 2 flow Capillary Inlet Aerosol FocusJet Concentric Nebulizer Tip Active drain pump Aerosol To Evaporation Sector Gas In (N 2 ) Inlet Concentric Nebulizer FocusJet ™ Concentric Nebulization System

11. Corona CAD Evaporation Nebulisation Evaporation The residual mobile phase is evaporated resulting in particles of uniform size being passed down the drying tube. Drying Tube Residual Mobile Phase High analyte mass concentration with residual mobile phase Low analyte mass concentration with residual mobile phase High analyte mass concentration results in larger particle and surface area Low analyte mass concentration results in smaller particle and surface area

12. Corona CAD Particle Charging Nebulisation Evaporation Particle Charging Concurrent with sample nebulisation, a second stream of the nitrogen is split and this gas become ionized by the high voltage of the Corona Charger. Next the analyte particles and ionized gas collide in a mixing chamber where the charge is transferred to the particle. Corona Needle Secondary gas stream positively charged by a high-voltage Platinum corona wire Mixing Chamber Positive charge is transferred to the analyte particles by charged opposing secondary gas stream

13. Mixing Chamber Each dried particle/ consists of multiple species Ion jet Charged particle Particle size proportional to mass of analyte + background residue Charge per particle proportional to particle size independent of composition Charged particles are measured, not gas phase ions as in MS Corona CAD Particle Charging

14. Corona CAD Particle Monitoring Nebulisation Evaporation Particle Charging Particle Monitoring After passing by an ion trap to remove excess ionized gas, the charged particles impact with the collector. The charge is then transferred and measured by a sensitive electrometer. Collector Analyte particles transfer their charge Ion Trap Negatively charged ion trap removes high mobility particles Electrometer Charge is drawn off and measured by a sensitive electrometer Signal Out Signal is directly proportional to quantity of analyte in sample

15. Corona CAD is different from APCI The particle is charged , not individual molecules ionized Charging of particles is not a function of relative proton affinity APCI ( Atmospheric Pressure Chemical Ionization)

16. Conventional Gradient Elution Inverse Gradient Compensation Independent of analyte properties Dependent on solvent composition (like ELSD) Inverse gradient compensation to normalize response Inverse Gradient Compensation for Uniform Response

17. Inverse Gradient Compensation for Uniform Response - 5.0 10.0 25.0 pA 1 - 1 - 2 - 3 - 4 - 5 - 6 - 7 - 8

18. Inverse Gradient Compensation for Uniform Response - 5.0 10.0 25.0 pA 1 - 1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 - 1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 - 5.0 10.0 20.0 35.0 pA min 2 - 1 - 2 - 3 - 4 - 5 - 6 - 7 - 8

19. Corona Veo / Corona Veo RS Detectors Concentric nebulization system improves sensitivity and precision Thermally controlled evaporation scheme widens the scope of applications Corona Veo RS model includes low flow capabilities for micro LC, as well as UHPLC Usability and serviceability have been enhanced CDS Drivers available for use with Non-Thermo Scientific HPLC/UHPLC systems

20. Vanquish CAD Model Flow Rate Range ( ml/min) Data Rate (Hz) Evap Temp (ºC) Positioning H 0.01 – 2.0 (microflow) 2 – 200 Settable from ambient +5–100 Ideal for R&D and methods development Labs Flex 0.2 – 2.0 2 – 100 Selectable 35, 50 or 70 Suitable for routine analysis in QC/QA Labs Vanquish Charged Aerosol Detectors H & F

21. CAD vs ELSD Measures the optical reflection of solute particles after the sample has been passed through a nebulizer Charged Aerosol Detection Measures charged particles by an electrometer generating a signal that is proportional to particle size (mass of analyte) after nebulization Evaporating chamber Siphon Heated Nebulizer Light source Detection chamber Particle size is proportional to mass of analyte + background residue Evaporative Light Scattering Detector Charged particles PMT Evaporation tube Nebulizer Electrometer Corona Charger Drain

22. For Rayleigh scattering: b = 2 For Mie scattering: b = 1 ⅓ For Refraction and reflection scattering: b = ⅔ ELSD exhibits a narrower linear calibration range than CAD Response Mass on Column Narrow linear segment ELSD Mass on Column Response Expanded linear segment CAD Nonvolatiles - Decreasing slope with increasing mass (b ~ ⅔) Detector Response Characteristics

23. Comparison Review Feature Charged Aerosol Evaporative Light Scattering Response Curvilinear Sigmoidal Dynamic Range >4 orders 2 –3 orders LoQ and LoD LoQ and LoD often lower (better) than estimated by SNR LoQ and LoD often higher (worse) than estimated by SNR Sensitivity ( LoD ) <1 ng >10 ng Semi-volatility Range Similar Similar Analyte Response Independent of structure Variable - dependent on compound Flow Rate Range (0.2 – 2 mL/min) One nebulizer Possibly several nebulizers Ease of Operation Simple Can be complex

24. Drug Composition Impurity Testing Formulation Counterions Surfactants / excipients Degradation / Stability Testing Characterization Glycan analysis Adjuvant analysis Lot-to-lot Variability Cleaning Validation Mass Balance Extractables / Leachables PEGylation and Drug-antibody Conjugates siRNA Lipid Delivery Vehicles QbD MIST Pharma and Biopharma Application Areas

25. Impurity Testing - API CAD 20 mg/mL ELSD 50 mg/mL ELSD 20 mg/mL 14 Apramycin and Impurities CAD 50 mg/mL Apramycin ELSD Response Disappears

26. Formulation – Counter Ions Drug Counter ions Instrumentation: Thermo Scientific ™ Dionex ™ UltiMate™ 3000 RSLC system Column: Acclaim Trinity P2, 3 μ m, 3 × 50 mm Col. Temp: 30 ºC Flow Rate: 0.6 mL/min Inj. Volume: 2 μ L Mobile Phase A: Water Mobile Phase B: 100 mM ammonium formate, pH 3.65 Gradient: Charged Aerosol: Thermo Scientific ™ Dionex ™ Corona Veo ™ RS; 55 ºC, 5 Hz, 2 s, PF 1.5 Sample: 20 to 100 ng/µL each in deionized water Peaks Phosphate 7. Nitrate Sodium 8. Citrate Potassium 9. Fumarate Chloride 10. Sulfate Malate 11. Magnesium Bromide 12 .Calcium Ref: Column Manual Time (min) -8.0 0.0 1.0 11 15 %A 90 90 90 %B 10 10 10 100 100 Anions, Cations, Organic and Inorganic Ions Simultaneously

27. Formulation – API and Counter Ions Instrumentation: Thermo Scientific™ UltiMate™ 3000 RSLC system Column: Thermo Scientific™ Acclaim™ Trinity P2, 3 μ m, 3 × 50 mm Col. Temp: 30 ºC Flow Rate: 0.6 mL/min Inj. Volume: 5 μ L Mobile Phases: A: Acetonitrile B: Water C: 100 mM ammonium formate, pH 3.65 Gradient: UV Detector: UV Diode Array; 254 nm, 5 Hz, 0.5 s Charged Aerosol: Thermo Scientific™ Dionex™ Corona™ Veo RS; 55 ºC, 5 Hz, 2 s, PF 1.5 Peaks: 1 aspartate 24 µg/mL 2 sodium 3 saccharin 24 4 amphetamine 122 5 sulfate 26 Ref: AN20870 Time (min) A B C -8.0 35 59 6 0.0 35 59 6 0.5 35 59 6 5.0 35 65 10 20 80 12 20 80 Complimentary Detection by CAD and UV/Vis CAD UV @ 254 nm Adderall and Counterions

28. Characterization – Vaccine Adjuvants UV @ 210 nm Charged Aerosol 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time [min] -100 100 200 A b s o r b a n c e [ m A U ] Saponins Cholesterol -10 20 40 60 80 90 C u r r e n t [ p A ] Saponins Lyso -PC Cholesterol Oxidation Products Cholesterol DPPC Column: Thermo Scientific ™ Accucore ™ PFP 2.6 μ m, 2.1 × 100 mm Mobile phase A: 0.1 % formic acid in deionized water Mobile phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol, Optima LCMS Gradient: 25-70-70% B 0-4-9 min Flow rate: 1.0 mL /min Inj. volume: 0.2 μ L Col. temp: 50°C Evap. Temp: 50°C Detector: Thermo Scientific™ Vanquish™ CAD, 2Hz, 5s, PF 1 CAD detects key analytes that UV misses

29. Simple Carbohydrates Lipids Profiling methods Targeted methods Artificial Sweeteners Food and Beverage Application Areas

30. Food and Beverage – Simple Carbohydrates Column: Amino, 3 μ m, 3 × 250 mm Mobile Phase: Acetonitrile:water (92:8) Flow Rate: 0.8 mL/min Inj. Volume: 2 μ L Col. Temp: 60 ºC Post-column Temp: 25 ºC Evap. Temp: 75 ºC Sample Preparation: Add 20 mL of 85% acetonitrile to 1 gram juice Analysis of Simple Sugars Simplified sample preparation “Dilute-and-shoot” method