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Column selectivity HPLC Vanquish
- 1. The world leaderin serving science Column Selectivity Naghmeh Berglund February 2019 Finland
- 2. 2 Overview • When selectinga column • Solid Core Enhanced Technology • Why C18
- 3. 3 What are weLooking for when Selecting a Column C18 C8 C4 C30 HilicIEX PFP SEC Mixed Mode • Need retention between analyte and column • Reverse phase, hydrophobic interactions • Polar compounds – HILIC, Ion exchange • Column needs to differentiate between similar molecules • Look to the differences in the analyte’s to help with the choice of separation column • Column needs to be stable in conditions being used • Overloading • pH effects Remember: Silica dissolves above pH 7 • Temperature effects
- 4. 4 What are weLooking for when Selecting a Column (ii) • Resolution • The degree of separation between two adjacent peaks • Higher R, better separation • Resolution ≥ 1.50 • Separation quality good enough to accurately measue peak area or peak height of each peak • Key-factors in resolution • Retention • Efficiency • Selectivity
- 5. 5 What are weLooking for when Selecting a Column (iii) • Retention or capacity factor(k) • Amount of time spend by an analyte interacting with the stationary phase • High k value indicates more retention • Selectivity or separation factor (α) • Ability to ‘chemically’ distinguish between sample components • Greatest impact on resolution • Efficiency (N) • The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travels through the HPLC system and column • The plate number is a measure of the peak dispersion on the HPLC column, which reflects the column performance
- 6. 6 Historical Overview inSilica Stationary Phases for Liquid Chromatography (LC) https://www.youtube.com/watch?v=efUrl_djzQ0 Large irregular particles 10µm porous particles 5µm porous particles 3µm porous particles Sub-2 µm porous particles Solid Core particles Sub-2 µm Solid Core particles 1960 1970 1980 1990 2000 2010 2020 Over the years, stationary phase particles have become: • Spherical • Smaller (sub-2 µm) UHPLC • Available with solid core and porous layer • Now combining Sub-2 µm and Solid Core To provide better separations Core-enhanced Technology 2.6 µm 80Å / 150A 1.5 µm 80 Å 4.0 µm 80Å
- 7. 7 Effect of particlesize distribution Fully Porous Particles D90/10 ~ 1.5 Core Enhanced Technology D90/10 ~ 1.1
- 8. 8 Speed, Sensitivity, PeakCapacity, Low Backpressure
- 9. 9 1.5µm Accucore VanquishColumn Technology • Increasing efficiency = Better separations 40% efficiency gain
- 10. 10 The most popularcolumn is an octadecyl carbon chain (C18)-bonded silica (USP classification L1) with 297 columns commercially available. This is followed by C8-bonded silica (L7 - 166 columns), pure silica (L3 - 88 columns), cyano-bonded silica (L10 - 73 columns) and phenyl-bonded silica (L11 - 72 columns) https://en.wikipedia.org/wiki/Reversed-phase_chromatography Silica C18 Columns – The Answer for all Separation Questions https://www.youtube.com/watch?v=efUrl_djzQ0
- 11. 11 • Reversed Phaseis the most popular mode of liquid chromatography • C18 covers 80-90% of Reversed Phase • Your trustworthy C18 columns is typically the first choice to target new analytes • Retention is primarily based on the analyte’s hydrophobicity So, is C18 Really the only Answer Phenyl
- 12. 12 Phase Charactarisation Hydrophobic retention(HR) Hydrophobic selectivity (HS) Steric Selectivity (SS) Hydrogen bonding capacity (HBC) - Good measure of degree of endcapping/ indicative of available surface area Activity towards bases - Indicator of free silanols Activity towards chelators - Indicator of silica metal content Ion Exchange Capacity (pH 7.6) - Indicator of total silanol activity Activity towards acids - Indicator of interactions with acidic compounds Ion Exchange Capacity (pH 2.7) - Indicator of acidic silanol activity Hydrophobic Interactions Secondary Interactions Acidic Interactions Hydrophobic retention Base activity Acid interaction HR BA AI Hydrophobic selectivity Chelation Ion exchange capacity pH 2.6 HS C IEX (2.6) Steric selectivity Ion exchange capacity (pH 7.6) SS IEX (7.6) Hydrogen bonding capacity HBC
- 13. 13 Phase Characterization RP-MS Optimized forMS detection, excellent combination of speed and quality of separation Phenyl-Hexyl Unique selectivity for aromatic and moderately polar analytes C18 Optimum retention for non-polar analytes PFP Alternative selectivity to C18, particularly for halogenated analytes aQ Compatible with 100% aqueous mobile phases, special selectivity for polar analytes HILIC Enhanced Retention of polar and hydrophilic analytes
- 14. 14 Bonded Phase: • Encapped •Embedded Silica Support: • Type I or Type II silica • Silica metal ion content • Fully porous, polymeric or superficially porous support • Pure Silica or organic/inorganic hybrid • Particle size and particle size distribution • Pore size, surface area Column selectivity
- 15. 15 Wide range ofAccucore separation functionalities • Accucore RP-MS • Accucore Polar Premium • Accucore C18 • Accucore Phenyl-X • Accucore C8 • Accucore C30 • Accucore aQ • Accucore HILIC • Accucore Urea-HILIC • Accucore Phenyl-Hexyl • Accucore PFP 2.6µm Accucore 150-C18 Accucore 150-C4 Accucore 150-Amide-HILIC 4µm Accucore XL C18 Accucore XL C8 1.5µm Accucore Vanquish C18 New Accucore Biphenyl
- 16. 16 Structural isomers onC18 and Biphenyl column Molar mass 346.467g/mol
- 17. 17 New Accucore BiphenylColumn Robustness
- 18. 18 Accucore Biphenyl Lot-to-LotConsistency
- 19. 19 Different Selectivity canProvide a More Complete Picture
- 20. 20 Accucore C18 vsAccucore Phenyl-X Columns: Accucore Phenyl-X Accucore C18 Dimensions: 2.6 µm, 100 x 2.1 mm Mobile Phases: A: Acetonitrile B: Methanol C: Water Isocratic: 15% A, 40% B, 45% C Flow: 400 µL/min Temperature: 40 °C Injection: 1 µL Detector: UV 220 nm mAU 0 20 40 60 80 1.47 Minutes 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 mAU 0 20 40 60 80 100 1 1 2 3 2 4 34 Rs 1.25 Rs 2.04 1. Estriol (E3) 2. Estradiol ( E2) 3. Estrone (E1) 4. Ethynylestradiol Accucore Phenyl-X Accucore C18 Phenyl-X shows different selectivity and better resolution
- 21. 21 Accucore C18 vsAccucore C30 Columns: Accucore C30 Accucore C18 Kinetex C18 Dimensions: 2.6µm , 100 x 3.0 mm Mobile Phases: Methanol:buffer, 98:2 Buffer = 2 mM ammonium acetate Flow: 650 µL/min Temperature: 20 ºC Injection: 5 µL Detector: UV 250 nm Peaks: 1. Vitamin K2, 50 µg/mL 2. Vitamin K1, 50 µg/mL Other peaks formed by UV irradiation Vitamin K2 Vitamin K1 1 2+2’ 2 1 2’ Different selectivity for K2 isomers C30 shows better separation for K1 isomers Kinetex C18 Accucore C30 0.00 1.25 2.50 3.75 5.00 6.25 8.00 -25 50 100 150 200 250 300 350 mAU min Accucore C18 2+2’ 0 Kinetex® is a registered trademark of Phenomenex Inc., USA
- 22. 22 Summary C18 is typicallyan excellent general purpose column https://www.youtube.com/watch?v=efUrl_djzQ0 • Look beyond the C18 • Ask for required application or analytes • Also as a complementary column for confimation of results • And do not forget alternative column vendor selection • Especially for C18
- 23. 23 Thank You ForYour Attention