HPLC & LC-MS. Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment

Transcription

1 HPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich

2 Overview of Presentation Chromatographer needs triangle What in your work has driven the need for us to innovate? Levers What particle parameters do we have to pull to fulfill needs? Why do these parameters work? Results Two specific HPLC innovations 2

3 Chromatographer Needs Triangle Sensitivity Speed 3 Resolution

4 Levers: Particle Parameters Physical Attributes: 1. Particle Size & Size Distribution 2. Architecture Unique structures or morphology }Kinetic Chemical Attributes: 3. Composition What the particle is made out of 4. Particle surface and bonded phases }Thermodynamic 4

5 Levers: Particle Parameters Needs Fundamentals Innovation Particle Parameters Speed Sensitivity Selectivity Ruggedness Composition???? Size & Size Distribution???? Architecture???? Surface Chemistry & Bonded Phases???? 5

6 Resolution: Competing Processes Result of two competing processes in the column Differential migration ( t R ) t R Band dispersion (w) Inject A and B A B A B ta t B 6 tr L while w L 1/2 w A w B

7 Resolution: Simple Equation Maximize: Differential migration ( t R ) Minimize: Band dispersion (w) R s = t R 0.5 (w A + w B ) Thermodynamic (chemistry) Kinetic 7

8 Resolution and Column Efficiency But how to get at a physical means to improve resolution? R s = N k k N = L/H H = A + B/u + Cu N 1/H small H values are good. 8

9 Flow Flow van Deemter Equation Terms A = Eddy (axial) diffusion Particle shape, size, distribution Bed uniformity B = Longitudinal diffusion Flow rate and mobile phase H = A + B/u + Cu Outside the particle Inside the particle C = Mass transfer Particle size Particle porosity Stationary phase thickness Small H values are good, so we need to minimize the A, B & C terms. 9

10 Visual Depictions of HPLC Packed Bed Typical HPLC packed bed Well-ordered packed bed Flow 10

11 Depiction of Extra-Particle Volume Uniform particles Minimize extra-particle volume Smaller particles are better Non-uniform particles High and variable extra-particle volume 11

12 Visualizing the van Deemter Relationship H = A + B/u + Cu H Flat (C) Low (A) 12 u (linear velocity)

13 Levers: Particle Parameters Physical Attributes: 1. Particle Size & Size Distribution 2. Architecture Unique structures or morphology }Kinetic Chemical Attributes: 3. Composition What the particle is made out of 4. Particle surface and bonded phases }Thermodynamic 13

14 Resolution Equation & Selectivity 3.0 R s = N k k+1 4 k = (t R t 0 )/t 0 = k 2 /k 1-1 Resolution (R) N k K 10K 15K 20K 25K Yun Mao, PhD Dissertation, University of Minnesota, N k

15 Influencing Selectivity How do we influence selectivity ( )? Bonded phase (surface interactions) Mobile phase C18 2, 3 Change bonded phase, change Silica

16 Influencing Selectivity How do we influence selectivity ( )? Bonded phase Mobile phase Influence ionization state of analyte Influence solvent polarity (solvation) >ph 8 silica unstable reversed phase retention (hydrophobicity) AH BH + BH + + OH - < > B + H 2 O acidic conditions B A- A - + H 3 O + < > AH + H 2 O basic conditions 16 ph

17 Resolution Levers: Particle Parameters Physical Attributes: 1. Particle Size & Size Distribution 2. Architecture Unique structures or morphology }Minimize band dispersion Chemical Attributes: 3. Composition What the particle is made out of 4. Particle surface and bonded phases }Increase selectivity R s = t R 0.5 (w A + w B ) 17

18 Summary What Particle Levers We Can Pull van Deemter Small particles Low particle size distribution Well-packed bed Minimal resistance to mass transfer H = A + B/u + Cu R s Choices in bonded phases Ability to operate at ph extremes R s = N k k Fused-Core technology (Ascentis Express, established 2007) Hybrid HPLC particles (Kromasil Eternity) Both use existing HPLC instrumentation (no capital investment) 18

19 Particle Innovation Chronology Particle Size and Architecture speed, efficiency 5 μm 3 μm <2 μm Fused-Core Ascentis Express monoliths, CEC Permeability wall Particle Composition expand ph range silica polymers oxides Hybrids Kromasil Eternity 19

20 Fused-Core Ascentis Express Particles H = A + B/u + Cu Comparison of architecture of Fused-Core particles vs. porous 3 μm particles 20

21 Architecture: Speed Implications H = A + B/u + Cu Van Deemter curve at 35 C column USHW μm Fused-Core H (μm) Virtually no performance loss at higher flow rates u (mm/sec.) 21 Data provided by Prof. Luigi Mondello, U. Messina, Messina, Italy

22 Architecture: Speed Implications P 1/d 2 p Pressure rises faster than efficiency and resolution as particle size is reduced P 1/d p 2 N 1/d p 16,000 14, μm Fused-Core Ascentis Express particles permit high speed operation at conventional pressures. Pressure (psi) 12,000 10,000 8,000 6,000 ~ 1 ml/min. 2.7 μm Fused-Core 4,000 3 μm 2, Mobile Phase Velocity (mm/sec)

23 Size & Architecture: Speed Implications Scaling methods to fast LC on traditional HPLC systems 5 μm C18, 15 cm 1.5 ml total run volume N~11,000; R s ~ 1.8; 885 psi Time (min) At least 4-fold increase in throughput with 3-fold solvent-saving due to short column length. Fused-Core Ascentis Express, 5 cm 0.5 ml total run volume N~11,000; R s ~1.8; 1750 psi Time (min)

24 Size & Architecture: Speed Implications (cont d) Virtually no loss of resolution at 4X normal flow rate: benefit of flat van Deemter Fused-Core Ascentis Express, 5 cm 30 sec.; 3700 psi (1.2 ml/min.) 9-fold increase in throughput compared to the original method on 5μm particle Time (sec) Fused-Core Ascentis Express, 5 cm 18 sec.; 6400 psi (2 ml/min.) 15-fold increase in throughput compared to the original method (not possible with 5 or 3 μm due to loss of N) Time (sec)

25 Number Tight Particle Size Distribution of Fused- Core (A-term Implications) H = A + B/u + Cu Ascentis Express: 2.7 μm +/- 6% Bed uniformity (A-term) Frits (lifetime) 500 Typical porous silica particle: Average +/- 19% Particle Diameter, μm

26 SEM of Fused-Core Particles X-section 26

27 Ruggedness: Ascentis Express Chosen for High Speed LC-MS DMPK Studies* Express columns chosen by GSK group for ruggedness and performance The authors conclude: The partially porous stationary phase material has demonstrated equivalent resolving power to sub-2μm materials under the ballistic gradient chromatography conditions employed, and shown to exhibit excellent resilience and performance over the analysis of thousands of protein precipitated plasma extracts, suggesting that this type of column is a valuable tool for pharmaceutical bioanalysts. 27 * D.N. Mallett (GSK), C. Ramírez-Molina, J. Pharm. Biomed. Anal., 2009, 49(1),

28 Sensitivity Demonstration Ascentis Express Peptide ES-C18 vs. 5 μm C18 (expanded scale) Ascentis Express Peptide ES-C18 ~20 peaks N 1/w 2 Area = ½ h w Time (min) Time (min) C18, 5μm ~10 peaks Time (min) Time (min)

29 Selectivity Choices in Fused-Core Particles R s = N k k C18 C8 Phenyl-hexyl RP-Amide HILIC Peptide C18 Ascentis Express C18 Ascentis Express Phenyl-Hexyl Ascentis Express RP-Amide Time (min)

30 Resolution Advantages of Fused-Core Conventional C18, 3 μm Ascentis Express C18 (2.7 μm) Peak ID 1. Uracil 2. Nordoxepin (50 mg/l) 3. Desipramine (50 mg/l) 4. Nortiptyline (50 mg/l) 5. Doxepin (50 mg/l) 6. Norclomipramine (50 mg/l) 7. Imipramine (50 mg/l) 8. Amitriptyline (50 mg/l) 9. Clomipriamine (50 mg/l)

31 UHPLC Users: Want more resolving power At the system pressure limit, Ascentis Express provides more resolving power because you can use longer columns. mau 250 nm mau 250 nm Time (min) Time (min) 3 4 Ascentis Express C18 75 cm (five 15 cm cols.) 0.6 ml/min. 903 bar (13,500 psi) k (toluene) = 5.3 G sub-2μ C18 30 cm (two 15 cm cols.) 0.3 ml/min. 780 bar (11,300 psi) k (toluene) = mm I.D. columns, water:acetonitrile 56:44, 60 C, peaks: uracil, acetophenone, benzene, toluene (deuterated analogs)

32 Summary Fused-Core Particles: Ascentis Express Particle Parameters Speed Sensitivity Selectivity Ruggedness Size & Size Distribution 2.7 μm = low pressure Low A-term μm, uses 2 μm frits Architecture Fused (solid) core = Low C-term (flat van Deemter) Fused (solid) core = Low C-term - - Surface Chemistry & Bonded Phases - - Choices in bonded phases - 32

33 Fused-Core Features & Benefits Summary Features: 1. Unique particle architecture 2. ~ 3 μm particle size 3. Tight particle size distribution Benefits: 1. Efficiency equal to sub-2 μm particles 2. Even higher efficiency possible than sub-2 μm particles by using longer columns 3. Maintains efficiency at high flow rates 4. One-half the pressure of sub-2 μm particles 5. Uses conventional HPLC instruments (e.g. what you currently have) 33

34 Ascentis Express Application Areas Process Chemistry Pharmaceutical/Med. Chem. Food & Beverage Natural Products/Agriculture Forensic Bioanalytical (DMPK, clinical) Biochromatography Environmental Others Using UHPLC: want more resolving power want more speed want more rugged columns want to transfer methods onto conventional systems (e.g. QA/QC) Using conventional columns: want more speed want more resolving power basically want UHPLC performance on conventional systems 34

35 35 Hybrid Kromasil Eternity Particles

36 Selectivity and High ph R s = N k k

37 Particle Stability at High ph Comparison with conventional technology Mobile phase: acetonitrile in 10 mm ammonium acetate, ph 10.5, 45 C 37

38 Summary Hybrid HPLC Particles: Kromasil Eternity Particle Parameters Speed Sensitivity Selectivity Ruggedness Composition - - High ph operation High ph operation 38

39 Summary: Etiology of Two Particle Innovations Customer needs drive innovations Leverage the fundamental equations: van Deemter: Resolution: H = A + B/u + Cu R s = N k k Fused-Core Ascentis Express High speed High efficiency Conventional and UHPLC systems 39 Kromasil Eternity Hybrid HPLC technology Durable particles for high ph separations

40 Other Supelco/Fluka HPLC and MS Innovations Chromatography: Chiral Unique CSPs for HPLC, GC, LC-MS, SFC, Prep (Astec line) Ionic liquids for GC extremely polar selectivity Fast GC phases and techniques Carbonaceous adsorbents (Carboxen ) LC-MS and MALDI-MS: Phospholipid removal (HybridSPE-PPT ) Chiral LC-MS (Astec CHIROBIOTIC ) High-purity MS solvents and additives (CHROMASOLV ) Ionic liquids for MALDI and ESI Packed pipette tips for proteomics High performance fittings and accessories 40

41 Acknowledgements/Collaborators Dr. Jack Kirkland, AMT, Inc. Prof. Luigi Mondello, U. Messina, Messina, Italy Dr. Richard Henry, Consultant Jared Benedict, Ph.D., Eka Nobel Supelco and Fluka R&D Teams Fused-Core is a trademark of Advanced Materials Technologies, Inc. (AMT) 41

42 When to Choose Fused-Core Ascentis Express Process Chemistry Pharmaceutical/Med. Chem. Food & Beverage Natural Products/Agriculture Forensic Bioanalytical (DMPK, clinical) Biochromatography Environmental Others Using UHPLC: want more resolving power want more speed want more rugged columns want to transfer methods onto conventional systems (e.g. QA/QC, prep) Using conventional columns: want more resolving power want more speed want more sensitivity want UHPLC performance on conventional systems Using either UHPLC or HPLC: want different selectivity want solvent savings 42

43 UHPLC Users: Want more resolving power Ascentis Express provides twice the resolving power as sub- 2 μm particles at equal pressures Time (min) Time (min) Ascentis Express C18, 10 cm 1400 psi Sub-2 μm C18, 5 cm 1650 psi G G mm I.D. columns water:acetonitrile 55:45 (top) or 54:46 (lower) 0.2 ml/min. ambient temperature UV at 200 nm 1 μl inj. peaks: estradiol, ß-estradiol, impurity, estrone, estrone degradant

44 UHPLC Users: Want more speed Ascentis Express provides twice the speed as sub-2 μm particles at equal pressures. G Ascentis Express C18, 10 cm 0.4 ml/min psi Sub-2 μm C18, 10 cm 0.2 ml/min psi 44 G mm I.D. columns water:acetonitrile 49:51 (top) or 55:45 (lower) ambient temperature UV at 200 nm 1 μl inj. peaks: estradiol, ß-estradiol, impurity, estrone, estrone degradant

45 UHPLC Users: Want more rugged columns Ascentis Express has the efficiency and resolution of sub-2 μm particles, but with much lower pressure drop and is much more resistant to plugging. With the repeated injections of plasma extracts shown here, the authors observed increasing and excessive pressure and retention variation on the sub-2 μm column. Sub-2 μm C18 ~700 bar (>10,000 psi) 65 C Ascentis Express C18 ~447 bar (6500 psi) 40 C 5 cm x 2.1 mm I.D. columns; 0.1% formic acid in water:acetonitrile, gradient of 5 95% acetonitrile in 1 min.; MS/MS detection; 1.1 ml/min.; 40 C or 65 C; peaks: bromo-guanosine, labetalol, reserpine, SB243213A (GSK) 45 D.N. Mallett (GSK), C. Ramírez-Molina, J. Pharm. Biomed. Anal., 2009, 49(1),

46 UHPLC Users: Want to transfer methods onto conventional systems (e.g. QA/QC, prep) Methods on Ascentis Express are easily scalable to conventional HPLC systems, and vice versa. Ascentis Express C18, 10 cm Pressure: 167 bar (2450 psi) N (peak 5): 22,700 G μm C18, 25 cm Pressure: 128 bar (1880 psi) N (peak 5): 22,150 G mm I.D. columns; water:acetonitrile 65:35; 1 ml/min.; ambient temperature; UV at 254 nm; peaks: oxazepam, alprazolam, cloazepam, N-desmethyldiazepam, diazepam

47 Conventional HPLC Users: Want more resolving power Higher efficiency means higher peak capacity and more information from Ascentis Express methods than from conventional HPLC columns Ascentis Express Peptide ES-C18 ~20 peaks C18, 5μm ~10 peaks Time (min) Time (min) Time (min) Time (min) 5 cm x 2.1 mm I.D. columns; 0.1% formate in water:acetonitrile gradient; 1 ml/min.; 35 C; ESI(+)-TOF; sample: HAS tryptic digest

48 Conventional HPLC Users: Want more speed At least eighttimes the speed on conventional HPLC systems. Ascentis Express C18, 5 cm 1.2 ml/min. 24 second run G μm C18, 15 cm 0.4 ml/min. 3.8 minute run 48 G mm I.D. columns water:acetonitrile 31:69 35 C peaks: uracil, acetophenone, benzene, toluene, naphthalene

49 Conventional HPLC Users: Want UHPLC performance (speed) on conventional systems At least 4-fold increase in throughput with 3-fold solventsaving due to short column length Time (min) 5 μm C18, 15 cm 0.4 ml/min. 4 min. run time 1.5 ml total run volume N~11,000; R s ~ 1.8; 885 psi Ascentis Express, 5 cm 0.6 ml/min. 1 min. run time 0.5 ml total run volume N~11,000; R s ~1.8; 1750 psi Time (min) 49 5 cm x 3 mm I.D. columns; water:acetonitrile 20:80 (top) or 31:69 (bottom); 0.4 ml/min. (top) or 0.6 ml/min. (bottom); 35 C; uracil, acetophenone, benzene, toluene, naphthalene

50 Conventional HPLC Users: Want UHPLC performance (speed) on conventional systems Virtually no loss of resolution at 4X normal flow rate: benefit of flat van Deemter. 9-fold increase in throughput compared to the original method on 5 μm particle Time (min) 5 μm C18, 15 cm 0.4 ml/min. 4 min. run time 885 psi Ascentis Express, 5 cm 1.2 ml/min. 30 sec. run time 3700 psi Time (sec)

51 Conventional HPLC Users: Want better sensitivity Higher efficiency provides better sensitivity on Ascentis Express vs. 3 μm or 5 μm totally porous particles. G cm x 4.6 mm I.D. columns; water:acetonitrile 65:35; 1.8 ml/min.; 35 C; UV at 254 nm; 5 μl inj.; peaks: uracil, acetophenone, benzene, toluene

52 UHPLC or HPLC Users: Want different selectivity Variety of phase chemistries of Ascentis Express: Ascentis Express C C18 C8 Phenyl-hexyl Amide HILIC Ascentis Express Phenyl-Hexyl Peptide C Ascentis Express RP-Amide Time (min)