High-Throughput Separations for QA/QC, LC/MS, LC/MS/MS, Combinatorial Chemistry and Rapid Method Development

Transcription

1 Robert Ricker Bio-Applications Scientist CABU April 5, 2 Rapid Resolution HPLC High-Throughput Separations for QA/QC, LC/MS, LC/MS/MS, Combinatorial Chemistry and Rapid Method Development Slide 4

2 Ultra-Fast Chromatography Powerful Tool Less labor cost Less cost per column Less solvent usage / disposal 157P1.PPT Slide 5

3 Ultra-Fast Chromatography Acceptance Rugged 3.5 µm Particles Better HPLC s Perceived QA/QC, LC/MS, CombiChem 157P1.PPT Slide 6

4 Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 7

5 Why Rapid-Resolution Chromatography? Increases Productivity Gaining Acceptance Easily Achieved with Few Tradeoffs 4.6 x 25mm 5 µm 4.6 x 15mm 3.5 µm Equal Resolution Slide 8 157P1.PPT

6 Why Rapid-Resolution Chromatography? Increases Productivity Gaining Acceptance Easily Achieved with Few Tradeoffs 4.6 x 15mm 3.5 µm Config Flow rate Selectivity Temperature Run Time Slide 9 157P1.PPT

7 Strategy for Ultra-Fast Separations Drive Resolution Up Then Reduce Run Time Rs factors that DON T increase run time! Rs Rs Rs factors that DECREASE run time! Slide 1

8 How Resolution Varies with k, N, and α t Initial Increase k Increase N Increase α time Slide P1.PPT

9 Strategy for Ultra-Fast Separations Analyzing the Fundamental Resolution Equation Plates Selectivity Retention R s = N 4 (α-1) k k +1 Particle Size Bonded Phase Temperature Mobile Phase Mobile-Phase Concentration Column Length Temperature ph Slide 12 99P1.PPT

10 Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 13

11 Change Bonded Phase to Improve Selectivity StableBond Family of Selectivities 3Å / 8Å 3Å / 8Å Bulky diisobutyl (with C18) or diisopropyl (with C8, C3, CN, phenyl) side-chain groups are used to stabilize both long and short-chain monofunctional ligands. 8Å 3Å / 8Å 3Å / 8Å Slide 14

12 ZORBAX StableBond Optimizes Resolution with Five Bonded-Phases StableBond SB-C18 StableBond SB-C8 StableBond SB-Phenyl StableBond SB-C3 StableBond SB-CN Columns: 4.6 x 25 mm, 5 µm Mobile Phase: - 1% B in 18.8 min A: 5 mm NaH 2 PO 4, ph 2.5 in 95% H 2 O / 5% ACN B: 5 mm NaH 2 PO 4, ph 2.5 in 47% H 2 O / 53% ACN Flow Rate: 1.5 ml/min Temperature: 26 C UV Detection: Sample: 254 nm 1. Procaine 2. Lidocaine 3. d-cinchonine 4. Butacaine 5. Tetracaine Time (min) Each different bonded-phase changes selectivity and resolution of these compounds. Slide 15

13 ZORBAX Eclipse XDB Offers Alternative Selectivity to ZORBAX StableBond Columns: 4.6 x 75 mm, 3.5 µm Mobile Phase: 8% 25 mm NaH 2 PO 4, ph 3. : 2% MeOH Flow Rate: 1. ml/min Temperature: 35 C Detection: UV 254 nm Sample: 1. Barbital 2. Sulfamethoxazole 3. Caffeine 2 Eclipse XDB-C8 3 2,3 SB-C Time (min) Time (min) The selectivity differences between these two bonding technologies provide increased opportunities for maximizing sample resolution. Slide 16

14 Comparison of Angiotensins Separation with TFA and NH4OH 5.E6 TIC (2-15 m/z) 4.E6 Acidic Conditions A-.1% TFA in water 3.E6 B-.85% TFA in 8%AcN Basic Conditions A- 1 mm NH 3.E7 4 OH in water 2.E6 1.E E7 4.E7 B- 1 mm NH 4 OH in 8%AcN 2.E7 1.E AII AI AII + AIII AIII AI min Zorbax Extend C18 (2.1 x 15 mm) HP 11 MSD: Pos. Ion ESI Vf 7V, Vcap 4.5 Kv N 2 =35psi, 12L/min. 325 C Gradient: 15-5%B / 15 min..2 ml/min Temp: 35 C Sample: 2.5 µl (5 pmol each) min Slide 17

15 Break Number 1 For Questions and Answers Press *1 on Your Phone to Ask a Question Slide 18

16 Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 19

17 New Selectivities Using Stable, Short-Chain Bonded Phases at High Temperature, Low ph 35 C Resolution 1 8 Improvement 8 1 6º C 3 SB-CN SB-C Myoglobin 9. Calmodulin 1. Carbonic Anhydrase Slide 2 166P1.PPT

18 Comparison of Gradient Performance at Different Temperatures mau C mau 1 5 mau C 1 9 C B6 2. B3 3. Pantothenic Acid 4. Folic Acid 5. B2 6. B12 Zorbax SB-C8 (4.6x5mm) A=9% H 2 O : 1% MeOH containing 25 MM sodium phosphate buffer B=1% H 2 O : 9% MeOH containing 25 MM sodium phosphate buffer [-35%B in 5 min] Detect.: 21 nm min Slide S1.PPT

19 Ion-Pairing Chromatography of B-Vitamins at Elevated Temperature for High-Throughput Analyses Isocratic Ion-Pairing Zorbax SB-C18 (4.6 x 75 mm) mixture containing 7.4 mm hexane sulfonate and.7% phosphoric acid min. Slide S1.PPT

20 Ultra-Fast Analysis of Parabens 1 Ambient Conditions: LC: Hewlett-Packard HP11 Columns: Zorbax SB-C18, 3.5 µm, 4.6 mm x 3 Mobile Phase:.1% H 3 PO 4 : ACN (5:5) UV: 254 nm; Flow: 2. ml / min. Inj Vol: 1 µl Absorbance C Sample: 1. Methylparaben 2. Ethylparaben 3. Propylparaben 4. Butylparaben.5 1 min Parabens: Preservatives used in food and personal care products against microbes Slide P1.PPT

21 Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 24

22 Increasing Analysis Speed in HPLC while Maintaining Resolution 5µm 3.5µm 5µm 3.5µm Dimension 25 x 4.6 mm 15 x 4.6 mm 15 x 4.6 mm 75 x 4.6 mm Analysis Time (min) 3 min. 4% reduction 18 min. 18 min. 5% reduction 9 min. Solvent Waste, ml 3 ml 4% reduction 18 ml 18 ml 5% reduction 9 ml N 2, { Resolution α N 1/2 Unchanged 12, 1, {2, 9% Difference Slide S1.PPT

23 Rapid Resolution of Proteins / Peptides ZOR B AX 3S B -C8 4.6 x 25 mm, 5 µm R apid R esolution 4.6 x 15 mm, 3.5 µm 4 min. 24 min. 1. Met-enkephalin 2. Leu-enkephalin 3. Angiotensin II 4. Neurotensin 5. RNase 6. Insulin (Bov) 7. Lysozyme 8. Calmodulin 9. Myoglobin 1. Carbonic Anhydrase R apid R esolution 4.6 x 5 mm, 3.5 µm 9 min Slide P1.PPT

24 Reduce Column Length to Reduce Run Time Rapid Separation of Analgesics SB-C x 15 mm all 7 components resolved in less than 1 min 4.6 x 3 mm Conditions: LC: Hewlett-Packard HP11 Columns: Zorbax SB-C18, 3.5 µm Mobile Phase: 1 mm octane sulfonic acid, Na salt, ph 2.5 : ACN (8:2) UV: 275 nm; Flow: 2. ml / min.; 7 C Inj Vol: 1 µl 4.6 x 5 mm Absorbance Sample: 1. Acetaminophen (4-acetamidophenol) 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Aspirin (acetosalicyclic acid) 6. Salicylic acid 7. Phenacetin (acetophenetidin) 4.6 x 75 mm 4.6 x 15 mm min Slide P2.PPT

25 Improved Column Lifetime Using Ultra-Pure Silica Sol (Rx-SIL) Rx-SIL ( Silica Sol ) Xerogel STRUCTURE: UNIFORM SUB PARTICLES SPONGE-LIKE, POLYMERIC NETWORK POROSITY (%): 5 7 SURFACE AREA (M 2 /G) FOR 1Å PORE SIZE: STRENGTH: HIGH ph RESISTANCE: PURITY: PORE SIZE DISTRIBUTION: 15 HIGH GOOD HIGH NARROW 3 WEAK POOR LOW - HIGH BROAD Silica-based packings made using agglutination of sol gel particles exhibit increased column lifetime Slide P1.PPT

26 Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 29

27 Columns Packed with Smaller Particles Retain Efficiency at High Flow Rate 3 micron N 5 micron FLOW 1 micron Slide 3

28 Gaining Full Potential in HPLC by Increasing Flow Rate Pressure 9 bar mau Rs 2,1 = 2.2 Rs 7,6 = 2.2 Flow Rate 2 ml/min 133 bar mau ml/min 181 bar mau Rs 2,1 = 2.1 Rs 7,6 = sec min 4 ml/min ZORBAX SB-C18, 3.5 µm, 4.6 x 3 mm Slide 31

29 Improving Resolution Using k* Resolution Relationship for Gradient E lution R N 4 α*k* t G F k* = ~ S Φ V m V G V m b = 1 / k* = gradient steepness Φ = change in volume fraction of organic S = constant F = flow rate t G = gradient time (min.) V m = column void volume Thus, all of these increase k*: 1. Longer gradient time t G 2. Shorter column V m 3. Higher flow rate F 4. Shorter organic range Φ Slide P1.PPT

30 Adjusting Gradient Parameters for High Throughput mau 1 5 mau 1 5 mau % B in 5 min, 1mL / min 4-35% B in 2 min, 1mL / min -35% B in 5 min, 2mL / min B6 2. B3 3. Pantothenic Acid 4. Folic Acid 5. B2 6. B12 Zorbax SB-C8 (4.6 x 5mm) A=9% H 2 O : 1% MeOH containing 25 mm Na phosphate buffer B=1% H 2 O : 9% MeOH containing 25 mm Na phosphate buffer [-35%B in 5 min] Temp.: 9 C Detect.: 21 nm min Slide S1.PPT

31 Break Number 2 For Questions and Answers Press *1 on Your Phone to Ask a Question Slide 34

32 Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 35

33 LC for Combinatorial Chemistry 4-1, compounds/month Desire consensus method capable of separating a group of molecules of varying properties - nonideal method development May require follow-up methods capable of high resolution separation Goal is rapid resolution in 1-4 minutes Preparative separations usually 15-5 mg Slide 36

34 Optimized Column Configuration and Operating Parameters for High-Throughput Analyses* Vendor A-C x 3 mm Flow: 4 ml / min. Gradient: -1% B / 2 min. Detect.: 22 nm A: 1/9, MeOH / H 2 O +.2%H 3 PO 4 B: 9/1, MeOH / H 2 O +.2% H 3 PO 4 2 mm each compound, 5µL inj. Relative Absorbance 1.2 min <1 min 3 min Eclipse XDB-C x 3 mm Eclipse XDB-C x 3 mm LC: 11 Flow: 4 ml / min. Gradient: -1% B / 2 min. Detect.: 22 nm LC: 11 Flow: 4 ml / min. Gradient: -1% B / 1 min. Detect.: 268 nm * Note improved baseline for HP 11 DAD min Slide S1.PPT

35 Heterogeneity of Anthocyanins R OH HO A + O B R O - Glyc. OH Anthocyanidin R R Cyanidin OH H Delphinidin OH OH Malvidin OCH 3 OCH 3 Pelargonidin H H Peonidin OCH 3 H Petunidin H OCH 3 Slide P1.PPT

36 Computer-Predicted vs. Observed Separations of a Complex Anthocyanin Sample Predicted Zorbax SB-C x 25mm 26 C, F=1.5 Absorbance 525 nm Observed A= 5% HCOOH B= 1% MeOH Time % B 14% 17% 23% 47% 47% 14% min 6 Final predicted vs. observed anthocyanin separation with <6 min. run time and separation of overlapped peaks. Slide P2.PPT

37 Comparison of Column Configuration in Rapid LC/MS Analysis of Anthocyanins Relative Abundance Base-Peak Chromatograms ZORBAX SB-C x 15mm min 4.6 x 5mm min Multisegment Gradients of A: 5% formic acid, B: 1% MeOH Slide 4

38 EICs (extracted ion chromatograms) Based on Anthocyanin Fragments ZORBAX SB-C18 Myrtillus extract 4.6 x 5mm Malvidin m/z 331 Peonidin m/z 31 Petunidin m/z 317 Cyanidin m/z 287 Delphinidin m/z 33 BPC PLQ T he MS detector allows component analys is without the s ame high res olution neces s ary for U V detection Slide 41

39 Anthocyanins: Improved Separation Using New Mobile Phase Abs (525 nm) 95 min min 75 min A= 5% HCOOH B= 1% MeOH Time A= 3% H 3 PO 4 B= 1% MeOH Time % B 14% 17% 23% 47% 47% 14% % B 23% 26% 6% min Zorbax SB-C18, 4.6 x 25 mm, 26 C, F=1 Slide 42

40 Anthocyanins: Improved Separation With Shorter Columns A = 3% H 3 PO 4 B = 1% MeOH 75 min 4.6 x 25mm, 5µm Time Percent B 23% 26% 6% Excellent resolution obtained by change of mobile phase, allows use of shorter columns with smaller particle-size, for higher throughput. 45 min 4.6 x 15mm, 3.5µm Time Percent B 23% 26% 6% 22.5 min 4.6 x 75mm, 3.5µm Time Percent B 23% 26% 6% Slide 43

41 Anthocyanins: Ultra-Fast Separation at High Flow Rates Zorbax SB-C18, 3.5 µm Cartridge Columns Abs (525 nm) mau x 3 mm F= 1 ml/min 12 min mau 4 ml/min 7.5 min mau 1 mau min 6.5 min 4.6 x 15 mm F= 1mL/min 4 ml/min %B in 15 min (3mm) - 3 %B in 7.5 min (15mm) A= 1% TFA, B= ACN, 1%TFA A= 1% TFA, B= ACN, 1%TFA min Slide 44

42 Ultra-Fast LC-MS Analysis of Analgesics (2 sec) Zorbax XDB-C8 (2.1 x 15 mm) Response Time <.1 min Column 2.1 x 15 mm 3.5 µm Flow.834 ml/min Temp 7 C Mobile Phase 18% ACN +1% TFA mau DAD 275 nm MS - BPC ES Positive Mode Cycle Rate 4.76 cycle/sec Fragmentor 3V Capillary 35V Gas Flow 13 L/min Nebulizer Temp 35 C min* Slide S1.PPT

43 Ultra-Fast LC-MS Analysis of Analgesics (2 sec) Zorbax XDB-C8 (2.1 x 15 mm) mau 5 DAD 275 nm Response Time <.1 min Column 2.1 x 15 mm 3.5 µm Flow.834 ml/min Temp 7 C Mobile Phase 18% ACN +1% TFA Acetominophen 2-Acetaminophenol Caffeine Acetanilide MS - BPC 152 EIC 195 EIC 136 EIC Cycle Rate 4.76 cycle/sec Fragmentor 3V Capillary 35V Gas Flow 13 L/min Nebulizer Temp 35 C 5 Phenacitin 18 EIC min Slide S1.PPT

44 Summary S ave time and money by choos ing the s hortes t pos s ible column with s malles t particle s ize to achieve the des ired s eparation 15- vs. 15-mm length 3.5µm vs. 5µm particles Adjus t chromatographic parameters to obtain optimal s eparation bonded phas e functionality mobile-phas e ph temperature flow rate highly selective detection (e.g., RI, F luorescence, MS ) Slide P1.PPT

45 Break Number 3 For Questions and Answers Press *1 on Your Phone to Ask a Question Slide 48

46 Wrap-up E-Seminar Questions Slide 49