Optimizing solid core_30955
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The document discusses the evolution and optimization of solid core materials in chromatography, focusing on their advantages over traditional porous materials. It highlights benefits such as reduced pressure requirements, improved separation efficiency, and various particle morphologies available for enhancing performance. The future of solid core technology is also examined, emphasizing developments in particle size and column configurations to meet the needs of modern analytical challenges.
Optimizing solid core_30955
- 1. Optimizing the Performance of Solid Core for Today and Tomorrow Mike Oliver Product Manager, Sample Preparation and Accucore LC Products Tony Edge R&D Principal June 2014
- 2. Introduction • The History of Solid Core • The concept Th i i l lid ti l d th h ll th f d• The original solid core particles and the challenges they faced • Solid Core Chromatographyg p y • Understanding the benefits of the technology • Bar for bar greater efficiency • How to optimize the separation• How to optimize the separation • Understanding chemistry • Optimization of the morphology • The Future • Where will solid core take us?• Where will solid core take us? • New particle morphologies – their synthesis explained 2
- 3. Solid Core Material – The Concept • Features • Less bed consolidation• Less bed consolidation • Better packing of particles • Less void volume in column • Reduced pore depth • BenefitsBenefits • More efficient chromatography • Allows the use of low pressure systems • Competitive Edge • Bar for bar gives better separations than porous materialsg p p 3
- 4. Liquid Chromatography Particle Design 2.6 µm 80 Å Solid Core Particles 80 Å 2.6 µm 150 Å Conventional Fully Porous Non-Porous Solid Core 4 µm 80 Å 1.x µm 80 Å Reduce Size to improve kinetics at expense of operating pressure Low sample capacity Very high pressure Small particle kinetics Reasonable pressure Very High Sample Capacity Lower Efficiency Low Sample Capacity Very High Efficiency High Sample Capacity High Efficiency 4 operating pressurey y g y g y
- 5. History of Solid Core Technology • 1960s • Horvath and Lipsky introduce the concept of pellicular/shell particles • 1970s • Core-shell particles developed:- • Zipax (DuPont later Rockford laboratories and finally acquired by HP/Zipax (DuPont later Rockford laboratories and finally acquired by HP/ Agilent), Corasil (Waters), Perisorb (Merck) • Improvement in the manufacturing of high-quality fully porous spherical particles • inhibits success of the shell particles• inhibits success of the shell particles • 10 μm fully porous spherical particles • 1980s • 5 μm porous particles • 1990s 3 μm porous particles• 3 μm porous particles • 2000−Present • sub-2 μm porous particles and core shell 5
- 6. The Theory … the Knox Equation A term B term H C term H=Au1/3+B/u+CuCu B AuH 3 1 H H=Au1/3+B/u+CuCu u AuH Linear velocity / mm/s 6 Linear velocity / mm/s
- 7. A–Term Eddy Diffusion or Multiple Paths Packing Efficiency D90/10~1.5 Porous Silica Packing Efficiency D90/10~1.1Accucore 7
- 8. A–Term Eddy Diffusion or Multiple Paths Enhanced roughness of their surface compared to porous particles results in less bed consolidation as particles willparticles, results in less bed consolidation as particles will tend to stick together 8
- 9. B–Term Longitudinal Diffusion • The B−term depends on; • Void volume of the column 9 • Void volume of the column
- 10. C–Term Resistance to Mass Transfer • The C term depends on; Diff i diff i th i th ili• Differences in diffusion path in the silica pores • Differences in the radial diffusion path in the liquid • Size of molecule 10 • Significant for large molecules but not for small molecules
- 11. Experimental Van Deemter Plots 20.0tical 10 0 15.0 Theoret 5.0 10.0 uivalent Plates 0.0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 eightEq 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 He Linear velocity of mobile phase (mm/s) Accucore RP-MS 2.6µm 5µm 3µm <2µm Columns: 100 x 2.1 mm Mobile phase: H2O / ACN (1:1) Temperature: 30 °C Detection: UV at 254 nm Flow rate range: 0.1 to 1.0 mL/min Highest efficiency and lowest rate of efficiency loss with flow rate for solid core 11 Flow rate range: 0.1 to 1.0 mL/min Analyte: o-xylene rate for solid core
- 12. Pressure Comparison 900 1000 BAP 2 00 2 11 600 700 800 900 600 bar limit pp ddL 3 0 23 0 400 500 600 ressure(bar) HPLC pressure limit 100 200 300 Pr u = 8 7mm/s 0 0 200 400 600 800 1000 Flow rate (µL/min) Accucore RP MS 2 6µm <2µm 3µm 5µm u = 8.7mm/s Accucore RP-MS 2.6µm <2µm 3µm 5µm Columns: 100 x 2.1 mm Mobile phase: H2O / ACN (1:1) Temperature: 30 °C Wide flow rate range with P < 600 bar 12 Temperature: 30 °C
- 13. Van Deemter – Limitations • Classical interpretation of how a column is performing • 3 parameters • A – Eddy diffusiony • B – Longitudinal diffusion • C – Resistance to mass transfer • Optimization of these parameter will give the best peak shape/efficiency • However it does not take into account; • Analysis time P t i ti t• Pressure restrictions on a system 13
- 14. Kinetic Plots • Allows for fairer comparisons of analytical systems • Van Deemter just compares pure separation ability• Van Deemter just compares pure separation ability • Incorporates time of analysisp y • Analysts want FASTER chromatography • Van Deemter plots do not specify the time of analysis • Incorporates pressure limitations of systems • Van Deemter does not account for a pressure limitationp on system • Based on three very simple classical equations• Based on three very simple classical equations 14
- 15. Kinetic Plots – Retention Time 1000010000 1000 (s)ofpeak( 100 iontime Accucore allows optimisation of retention times Retenti Solid core produces sharper peaks in less time 10 1,000.00 10,000.00 100,000.00 Efficiency p 15 Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency
- 16. Impedance • Devised by Knox and Bristow in 1977 Defines the resistance a compound has to moving• Defines the resistance a compound has to moving down a column relative to the performance of that column • Allows for pressure to be incorporated Often plotted with a reverse axis• Often plotted with a reverse axis • Mimics van Deemter plot • Minimum value optimum conditions 2 Pt E p • Often plotted as a dimensional form 2 N E • t/N2 • t0 or tr both used 16
- 17. 100,000 Kinetic Plots – Impedance 100,000 2 0 N Pt E ce 2 N 10,000 mpedanIm S lid i lSolid core requires less pressure to obtain sub 2 µm efficiencies 1,000 100.001,000.0010,000.00100,000.001,000,000.00 Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency 17 µ µ µ µ Efficiency
- 18. The Impact of Selectivity on Resolution Efficiency SelectivityRetentionEfficiency SelectivityRetention 2 5 3.0 2 5 3.0 N R= k’ k’+1 -1 4 N R= k’ k’+1 k’ k’+1 -1 -1 4 2.0 2.5 (R) 2.0 2.5 (R) k +1 4 k2 k +1k +1 4 k’22 1.5 N solution( 1.5 N solution( = k2 k’1 = k2 1 = 2 1 0.5 1.0 k Res 0.5 1.0 k ResSelectivity () has the greatest impact on 1.00 1.05 1.10 1.15 1.20 1.25 0.0 N 1.00 1.05 1.10 1.15 1.20 1.25 0.0 N improving resolution. 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 N k 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 N k S 18 Stationary phase, mobile phase, temperature
- 19. The Growth of Solid Core Technology • Particle sizes available • 1.3, 1.6, 1.7,2.6, 2.7, 4, 5 P i il bl• Pore sizes available • 80, 120, 150, 300 • Stationary Phases Availabley • Reversed phases • C4, EC-C8, C8, EC-C18, SB-C18, XB-C18, C18, C18+, C30, RP-MS, AdvanceBio C18 HT C28 Peptide ES C18AdvanceBio, C18-HT, C28, Peptide ES-C18 • Polar encapped / embedded phases • Polar aQ, Polar Premium, SB-Aq, Bonus-RP, RP-Aqua • HILIC / Normal phases • Silica, Amide, Urea-HILIC, EC-CN, Penta-HILIC • π ‐π phasesp ases • Phenyl, Biphenyl, Phenyl-Hexyl, PFP, Phenyl-X, Diphenyl • Currently >50 different stationary phases available 19 • >7 manufacturers
- 20. Stationary Phase Characterization • Hydrophobic retention (HR) Hydrophobic Interactions y p ( ) • k’ of neutral compound • Hydrophobic selectivity (HS) • α two neutral compounds that have different log P • Steric Selectivity (SS) • α sterically different moleculesα sterically different molecules • Hydrogen bonding capacity (HBC)y g g y ( ) • α molecule that hydrogen bonds and a reference • Good measure of degree of endcapping 20 • Gives indication of available surface area
- 21. Stationary Phase Characterization • Activity towards bases (BA) Interactions with Bases and Chelators • Activity towards bases (BA) • k’, tailing factor (tf) of strong base • Indicator of free silanols • Activity towards chelators (C) • k’, tailing factor (tf) of chelator • Indicator of silica metal content 21
- 22. Stationary Phase Characterization Interactions with Acids and Ion Exchanges • Activity towards acids (AI) • k’, tf acid • Indicator of interactions with acidic compounds• Indicator of interactions with acidic compounds • Ion Exchange Capacity (IEX pH 7.6)g p y ( p ) • α base / reference compound • Indicator of total silanol activity • All silanols above pKa I E h C it (IEX H 2 7)• Ion Exchange Capacity (IEX pH 2.7) • α base / reference compound • Indicator of acidic silanol (SiO-) activity 22 • Indicator of acidic silanol (SiO ) activity
- 23. Column Characterization (Visualization) HR /10 HSAI Accucore C18 HR /10 HSAI Accucore RP-MS SSIEX (2.7) SSIEX (2.7) HBC IEX (7.6)BA C HBC IEX (7.6)BA C HR /10 HSAI Accucore PFP HR /10 HSAI Accucore Phenyl-Hexyl HS SSIEX (2.7) AI HS SSIEX (2.7) AI HBC IEX (7.6)BA C HBC IEX (7.6)BA C 23
- 24. Widest Range of Solid Core Selectivity Options 500 mAU 1,2,3 curcuminoids 2 00 2.50 HR /10 HSAI Accucore RP-MS Solid Core C18 0.50 1.00 1.50 2.00 HS SSIEX (2.7) AI Accucore C18 Accucore 150-C18 Accucore C8 Accucore 150-C4 Accucore Polar Premium 1 0.00 HBCC Accucore aQ Accucore Polar Premium Accucore Phenyl-Hexyl Accucore PFP 2 3 Polar Premium shows different selectivity and separates the peaks IEX (7.6)BA Accucore Phenyl-X Accucore C30 0.0 1.0 2.0 3.0 0 Minutes 24
- 25. Database for Column Characterization http://www.usp.org/app/USPNF/columnsDB.html • Not all modes of interactions are covered, specifically; i i HILIC N l h 25 • π‐π interactions, HILIC, Normal phase
- 26. System Considerations • Column: Accucore RP-MS 2.6 μm, 100 x 2.1 mm • Gradient: 65–95 % B in 2.1 min Dwell volume: 100 µL 95 % B for 0.4 min • Flow rate: 400 µL/min Accela 1250 Dwell volume: 800 L Surveyor Accela Surveyor Agilent 800 µL Minutes 0.00 1.00 2.00 3.00 4.00 Accela 1250 Surveyor Agilent 1100 Run time (min) 2.5 3.0 3.5 Dwell volume: 1000 µL min0 0 5 1 1 5 2 2 5 3 3 5 Agilent 1100 Average PW (1/2 Height) 0.02 0.02 0.04 min0 0.5 1 1.5 2 2.5 3 3.5 Solid core can deliver performance on a b f diff t t 26 number of different systems
- 27. System Considerations • Minimize volume dispersion Always Optimize System Configuration • Tubing–short L, narrow ID • Low injection volume • Low volume flow cell• Low volume flow cell • Optimize detector sampling rate Need enough points to define peak (minimum of 10, >20 for quantitation) 5 pts Fast scanning MS • Low dwell volume pump for fast 45 pts 9 pts Low dwell volume pump for fast gradients 27
- 28. Reducing the Particle Size • Industry is fixated with pressure • This is an unwanted artefact of running with smaller particles R d l lif ti d i t t lif ti i f i ti l h ti• Reduces column lifetime, reduces instrument lifetime, causing frictional heating • System dispersion is critical with smaller particlesy p p • Injection volumes, connectors, detector volumes become more important C t l f t t• Control of temperature • Increase in pressure results in greater column temperature gradients • Need to ensure that column temperature is controlled effectivelyp y • Isothermal / adiabatic control Is there a finite limit to particle size reduction? 28 reduction?
- 29. Analyzing Biomolecules • Move to produce bigger molecules • Difficult to copy• Difficult to copy • Greater success rates • Chromatography requirements • Need less retentive phase • Need wide pores to cope with larger molecules• Need wide pores to cope with larger molecules 29
- 30. Peptides – Resolution & Peak Shape RT: 0 11 15 03RT: 0.11 - 15.03 1.12 8.74 3.76 7.09 13.468.91 11 20 Accucore 150-C18 11.20 13.63 1.01 C ti l S lid C C18 1.01 8.52 6.983.60 13.13 15.0310.84 Conventional Solid Core C18 100000 10.20< 2 µm Wide Pore Fully Porous C18 0 50000 uAU 8.491.59 14.4912.035.25 14.31 y 2 4 6 8 10 12 14 Ti ( i ) -50000 30 Time (min)
- 31. Proteins – Excellent Resolution vs < 2 µm Wide Pore • Sharper and higher peaks 80000 100000 Accucore 150-C4 Backpressure: 185 bar higher peaks than < 2 µm Wide Pore Fully Porous C4 40000 60000 uAU Porous C4 • Better resolution and sensitivity 100000 0 20000 • Significantly lower backpressure60000 80000 100000 < 2 µm Wide Pore Fully Porous C4 Backpressure: 320 bar p 40000 60000 0 1 2 3 4 5 6 7 8 9 10 Ti ( i ) 0 20000 31 Time (min)
- 32. Effect of Pore Depth for Small and Large Molecules 12.00 10.00 equation small molecule, large ρ small molecule, small ρ large molecule, large ρ 6 00 8.00 n Deemter e large molecule, small ρ 4.00 6.00 pnents of va 2.00 B+C comp 0.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 Reduced Velocity, ν 32
- 33. What is the Solution? • Fractals • Mandelbrot / Julia sets C l t h t bl l ti i• Colors represent how stable solution is 2 czz 1 i biac 33 1i
- 34. Increasing Surface Area • Koch curve • A straight line has a dimension of 1• A straight line has a dimension of 1 • This line is infinitely long • Curve has a dimension of 1.26 • Fractal shape • Same surface no matter what the scale• Same surface no matter what the scale being observed • Reduced mass transfer effects? Eff ti l if f ?• Effectively uniform surface? • Reduced issues with mass transfer into and out of pores? 34
- 35. Approaches to Growing Solid Particles • Sol-Gel process under acidic conditions 35
- 36. Growing Solid Silica • Base activated (Stöber) Process • Porous particles a polymeric surfactant (porogen) is typically added • e.g. Hexadecyltrimethylammonium bromide, Pluronic 123, • This allows formation of micelles which propagate pore structure on• This allows formation of micelles which propagate pore structure on growth of particle • Surfactant washed out of end material to leave final structure which is then calcined 36 then calcined
- 37. Growing a Particle Solid particle grows by stages Nanotechnology 22 (2011) 275718 37 gy ( )
- 38. The Ingredients for a New Particle • MPTMS - (3-Mercaptopropyl)trimethoxysilane • MPTES - (3-mercaptopropyl)triethoxysilane • MPMDS - (3-mercaptopropyl)methyldimethoxysilane • TEOS - tetraethyl orthosilicate• TEOS - tetraethyl orthosilicate • TMOS - tetramethyl orthosilicate • CTAB - (3-glycidyloxy propyl) trimethoxy silane, cetyltrimethylammonium bromidecetyltrimethylammonium bromide • PVA - poly(vinyl alcohol) (PVA, MW 10 K) 38 p y( y ) ( , W )
- 39. The Recipe - Typically • 0.25g PVP + 0.1g CTAB was dissolved in 5 ml DI water 8 ml methanol was added with stirring and mixture left to cool to room• 8 ml methanol was added with stirring and mixture left to cool to room temperature • 2 ml ammonium hydroxide (5.6%) was added to the reaction and stirred vigorously for 15 minutes • 0.5 ml MPTMS was added drop wise over 30 seconds and the reaction stirred for 24 hours at room temperaturestirred for 24 hours at room temperature • Resultant particles were collected and washed by centrifugation with DI water then ethanol (both 3×10 ml, 4500 rpm, 3 minutes) A. Ahmed, W. Abdelmagid, H. Ritchie, P. Myers, H. Zhang J. Chrom. A, Volume 1270 (2012) 194–203 39
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- 41. 41
- 42. Recipe Variables Already Investigated • Increasing Stirring speed • Bigger nanospheres 200rpm Bigger nanospheres • Reduces particle size distribution 500rpm 1500rpm • Temperature • Raising the temperature reduces roughness of surface but provides more uniform particle sizeuniform particle size • Microwave and conventional heating provide similar particles 42 • microwave is much quicker
- 43. Varying the Recipe Standard Method 0.25 g PVA (Mw 10k) 0 10 g CTAB0.10 g CTAB 5 ml DI Water 8 ml Methanol 2 ml NH4OH (5.6%)2 ml NH4OH (5.6%) 0.5 ml MPTMS Increasing alkalinity Smaller, less densely packed nanospheres on surface 43
- 44. Varying the Recipe Altering pH Larger more densely packed nanospheresLarger, more densely packed nanospheres on surface Altering polymer concentration Smooth spheres, no nanospheres, poor dispersity 44
- 45. Varying the Recipe Altering polymer concentration Smooth spheres no nanospheres poorSmooth spheres, no nanospheres, poor dispersity Altering additive concentrationg Small, monodisperse, fuzzy particles 45
- 46. Varying the Recipe Altering polymer type and concentration Fused smooth spheresFused smooth spheres Altering polymer type and concentrationAltering polymer type and concentration Very small nanospheres on surface 46
- 47. Varying the Recipe Addition of extra polymer Very small nanospheres on surfaceVery small nanospheres on surface Changing pH Fused smooth spheres 47
- 50. Separation on an SoS Particle 50
- 51. Separation on an SoS Particle Monodisperse SOS, C4 bonded, endcapped 1. GY 40 °C, 300 μL/min 2. VYV Gradient: 10−40% B in 4 minutes 3. met-Enk A. 0.02M KH2PO4 buffer, pH 2.7 + 0.1% TFA 4. leu-Enk B. Acetonitrile + 0.1% TFA 5. Angiotensin II 51
- 52. Conclusions • The History of Solid Core • The concept Th i i l lid ti l d th h ll th f d• The original solid core particles and the challenges they faced • The introduction on a new generation of solid core materials • Solid Core Chromatography • Understanding the benefits of the technology • Bar for bar greater efficiency• Bar for bar greater efficiency • How to optimize the separation • Understanding chemistry • Optimization of the morphology • The Future• The Future • Where will solid core take us? • New particle morphologies – their synthesis explained 52
- 53. Acknowledgements Haifei Zhang Adham Ahmed Kevin SkinleyHaifei Zhang Adham Ahmed Kevin Skinley, (Laura), Peter Myers Richard Hayes University of LiverpoolUniversity of Liverpool Harry Ritchie Trajan Scientific 53 Trajan Scientific