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
Ultra-Fast Chromatography Powerful Tool Less labor cost Less cost per column Less solvent usage / disposal 157P1.PPT Slide 5
Ultra-Fast Chromatography Acceptance Rugged 3.5 µm Particles Better HPLC s Perceived QA/QC, LC/MS, CombiChem 157P1.PPT Slide 6
Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 7
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
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
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
How Resolution Varies with k, N, and α t Initial Increase k Increase N Increase α time Slide 11 955P1.PPT
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
Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 13
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
ZORBAX StableBond Optimizes Resolution with Five Bonded-Phases StableBond SB-C18 StableBond SB-C8 StableBond SB-Phenyl StableBond SB-C3 StableBond SB-CN 1 1 1 1 3 1 3 2 2 2 2 3 2 3 3 5 4 4 5 4 5 5 5 4 4 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 5 1 15 Time (min) Each different bonded-phase changes selectivity and resolution of these compounds. Slide 15
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-C8 1 1 5 1 Time (min) 5 1 15 Time (min) The selectivity differences between these two bonding technologies provide increased opportunities for maximizing sample resolution. Slide 16
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.E6 2.5 5 7.5 1 12.5 5.E7 4.E7 B- 1 mm NH 4 OH in 8%AcN 2.E7 1.E7 5.499 AII 8.477 9.621 AI AII + AIII AIII 12.5 13.261 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) 2.5 5 7.5 1 12.5 min Slide 17
Break Number 1 For Questions and Answers Press *1 on Your Phone to Ask a Question Slide 18
Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 19
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 9 9 3 SB-C3 8 9 1 8. Myoglobin 9. Calmodulin 1. Carbonic Anhydrase 8 9 1 Slide 2 166P1.PPT
Comparison of Gradient Performance at Different Temperatures mau 1 5 4 C 1 2 3 4 5 6 mau 1 5 mau 1 5 65 C 1 9 C 1 2 2 3 3 4 1 2 3 4 5 4 5 6 5 6 1. 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 21 168S1.PPT
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 22 1599S1.PPT
Ultra-Fast Analysis of Parabens 1 Ambient 2 3 4 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 1 2 3 4 6 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 23 1651P1.PPT
Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 24
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 25 788S1.PPT
Rapid Resolution of Proteins / Peptides ZOR B AX 3S B -C8 4.6 x 25 mm, 5 µm 1 2 3 4 5 6 7 9 1 8 5 1 15 2 25 3 35 4 45 5 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 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 R apid R esolution 4.6 x 5 mm, 3.5 µm 9 min. 1 2 3 4 5 6 7 8 9 1 Slide 26 976P1.PPT
Reduce Column Length to Reduce Run Time Rapid Separation of Analgesics SB-C18 4.6 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 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 min Slide 27 1637P2.PPT
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 28 167P1.PPT
Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 29
Columns Packed with Smaller Particles Retain Efficiency at High Flow Rate 3 micron N 5 micron FLOW 1 micron Slide 3
Gaining Full Potential in HPLC by Increasing Flow Rate Pressure 9 bar mau 3 2 1 Rs 2,1 = 2.2 Rs 7,6 = 2.2 Flow Rate 2 ml/min 133 bar mau 3 2 1 3 ml/min 181 bar mau 3 2 1 Rs 2,1 = 2.1 Rs 7,6 = 2.2 3 sec.1.2.3.4.5.6.7.8.9 min 4 ml/min ZORBAX SB-C18, 3.5 µm, 4.6 x 3 mm Slide 31
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 32 994P1.PPT
Adjusting Gradient Parameters for High Throughput mau 1 5 mau 1 5 mau 1 5 1 1 1 2 3 4 2 2 3 3 5-35% B in 5 min, 1mL / min 4-35% B in 2 min, 1mL / min -35% B in 5 min, 2mL / min 6 4 1 2 3 4 5 5 6 5 6 1. 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 33 169S1.PPT
Break Number 2 For Questions and Answers Press *1 on Your Phone to Ask a Question Slide 34
Outline Theoretical Selectivity (Bonded Phase and Mobile Phase) Temperature Column Configuration (Length and Particle Size) Flow Rate Practical Examples 157P1.PPT Slide 35
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
Optimized Column Configuration and Operating Parameters for High-Throughput Analyses* Vendor A-C18 4.6 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-C18 4.6 x 3 mm Eclipse XDB-C18 4.6 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 1 2 3 4 * Note improved baseline for HP 11 DAD min Slide 37 1577S1.PPT
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 38 1411P1.PPT
Computer-Predicted vs. Observed Separations of a Complex Anthocyanin Sample Predicted Zorbax SB-C18 4.6 x 25mm 26 C, F=1.5 Absorbance 525 nm Observed A= 5% HCOOH B= 1% MeOH Time 1.2 35.3 64.6 64.9 65. % B 14% 17% 23% 47% 47% 14% 1 2 3 4 5 min 6 Final predicted vs. observed anthocyanin separation with <6 min. run time and separation of overlapped peaks. Slide 39 142P2.PPT
Comparison of Column Configuration in Rapid LC/MS Analysis of Anthocyanins Relative Abundance 2 175 15 125 1 75 5 25 25 2 15 1 5 Base-Peak Chromatograms 5 1 15 2 25 3 35 4 45 ZORBAX SB-C18 4.6 x 15mm min 4.6 x 5mm 5 1 15 min Multisegment Gradients of A: 5% formic acid, B: 1% MeOH Slide 4
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
Anthocyanins: Improved Separation Using New Mobile Phase Abs (525 nm) 95 min 1 2 3 4 5 6 7 8 9 min 75 min A= 5% HCOOH B= 1% MeOH Time 15.4 52.9 96.9 97.4 97.5 A= 3% H 3 PO 4 B= 1% MeOH Time 35 97 % B 14% 17% 23% 47% 47% 14% % B 23% 26% 6% 1 2 3 4 5 6 7 min Zorbax SB-C18, 4.6 x 25 mm, 26 C, F=1 Slide 42
Anthocyanins: Improved Separation With Shorter Columns A = 3% H 3 PO 4 B = 1% MeOH 75 min 4.6 x 25mm, 5µm Time 35 97 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 21 58.2 Percent B 23% 26% 6% 22.5 min 4.6 x 75mm, 3.5µm Time 1.5 29.1 Percent B 23% 26% 6% Slide 43
Anthocyanins: Ultra-Fast Separation at High Flow Rates Zorbax SB-C18, 3.5 µm Cartridge Columns Abs (525 nm) mau 5 4.6 x 3 mm F= 1 ml/min 12 min mau 4 ml/min 7.5 min mau 1 mau 2 4.5 min 6.5 min 4.6 x 15 mm F= 1mL/min 4 ml/min 2 4 6 8 1-3 %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
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 12 1 8 6 4 2 8 6 4 2 1 1 2 3 2 3 4 6 5 4 5 6 7 7 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.1.2.3.4.5 min* Slide 45 1888S1.PPT
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 5 4 4 5 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.1.2.3.4.5min Slide 46 1889S1.PPT
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 47 1295P1.PPT
Break Number 3 For Questions and Answers Press *1 on Your Phone to Ask a Question Slide 48
Wrap-up E-Seminar Questions Slide 49