Silica Monolith Columns for Silica Monolith Columns for Ultra High - PDF document

Silica Monolith Columns for Silica Monolith Columns for Ultra High Speed Separations Ultra High Speed Separations Outline Outline • Do more in less time • Onyx™technology & advantages • Product offerings • Applications • Summary

Do more with less time Do more with less time • Workload request from management – Before: purify/ analyze 10 – 20 samples per day – Now: purify/ analyze 100 – 200 samples per day • Reasons why: – Clinical trial moving from phase 2 to 3 – Automation of chemical synthesis – Increased regulatory monitoring Do more with less time Do more with less time • Solution # 1: – Buy 10 more HPLC systems • $500,000 (USD) cost • Additional analysts needed • Instrument maintenance • More lab space required

Do more with less time Do more with less time • Solution # 2 – Reduce HPLC analysis time • 30 minute method = 40 samples a day • 10 minute method = 130 samples a day • 5 minute method = 280 samples a day – Get more results with same resources Do more with less time Do more with less time 150 x 4.6mm 120 bar pressure 5 min equilibrate 15 min run 1 mL/min 50 x 4.6mm 250 bar pressure 2 min equilibrate 7 min run 2.5 mL/min Pressure 350 bar pressure 30 x 4.6mm too high!! 1.5 min equilibrate 5min run 3.5 mL/min

Do more with less time Do more with less time • Problems with solution # 2 – Loss of analyte resolution – Short column lifetimes – Insufficient re-equilibration – High backpressures at system limit Do more with less time Do more with less time • What if backpressure wasn’t a problem? – You could: • Use very fast flow rates to shorten run time • Re-equilibrate & clean column with fast flow rates • Change flow rates to optimize analyte separation

Onyx™ ™ technology and advantages technology and advantages Onyx Onyx™ ™ technology & advantages technology & advantages Onyx • What is Onyx? – A new solution for high-speed HPLC methods – A new solution for quickly analyzing dirty & complex samples – A silica-based monolithic LC column

Onyx™ ™ technology & advantages technology & advantages Onyx • Structure: – Monolithic silica rod with bimodal pore structure Onyx™ ™ technology & advantages technology & advantages Onyx • Structure: – Bimodal pore structure contains macropores and mesopore Macropores Mesopores

Onyx™ ™ technology & advantages technology & advantages Onyx • Macropore structure: Allows rapid flow (up to 9 mL/min) at low pressures Macropores are 2µm in diameter Dense network of pores through which mobile phase can Contaminants rapidly flow at easily pass low pressure and don’t clog column Onyx™ ™ technology & advantages technology & advantages Onyx • Mesopore structure: Creates large surface area for adsorption Mesopores are 130Å (13nm) in diameter Form the very porous structure of the column interior

Onyx™ ™ technology & advantages technology & advantages Onyx • Column housing – Rod is cladded in PEEK tubing with a special proprietary process and ready to use Onyx™ ™ technology & advantages technology & advantages Onyx • Specifications – 2.0 – 7.5 pH stability – 45 � C max temperature – 3000 psi (200 bar) max pressure – 1 – 9 mL/min flow rate range

Onyx™ ™ technology & advantages technology & advantages Onyx • Monolith vs. particle-based technology • Monolithic • Individual porous rod silica particles • High flow • High flow rates VS. resistance • Low • High backpressure backpressure • No inlet bed • Bed splitting splitting possible Onyx™ ™ technology & advantages technology & advantages Onyx • Versus traditional particle-based columns VS.

Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 1: High sample throughput Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 2: Short run times 2 mAU Onyx™ C18 20 1 3 4 • 5 minutes @ 4 mL/min 10 5 0 0 1 2 3 4 min Particle-based C18 mAU 30 • 35 minutes @ 2 mL/min 20 2 1 3 10 4 5 0 5 10 15 20 25 30

Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 3: Short equilibration time – Total run time = separation time + equilibration time Onyx™ Equilibration time 4 runs Separation time Traditional silica particle-based column 4 runs Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 4: Very low backpressure – Equivalent to 10µ particle-based material

Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 4 cont.: Very low backpressure – On average 60% less than particle-based columns Particle-based 5µ C8 Backpressure: 130 bar 200 mAU 2 3 150 Dimensions : 100 x 4.6mm Mobile phase: 100 1 4 A: 0.1% phosphoric acid 50 B: Acetonitrile Gradient: from (70:30) to (20:80) in 4min. 0 2.0 ml/min. Flow rate: 1 2 3 4 min Temperature : ambient 254 nm Detection: 1. phenol Injection: Onyx™ C8 2. 2,4-dinitrophenol Backpressure: 75 bar 3. 3,4-dinitrophenol mAU 4. pentachlorophenol 200 3 2 150 100 1 4 50 0 1 2 3 4 min Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 5: High efficiency – On par with 3µ particle-based materials

Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 5: High plate counts with coupling – Able to separate closely-eluting, complex samples 40,000 Plate count (N) Anthracene 30,000 20,000 10,000 Onyx™ ™ technology & advantages technology & advantages Onyx • Coupling with the Onyx column coupler – Couple several columns together – Enhance separation efficiency – Produce significantly higher plate counts – Backpressures maintained below HPLC system limit

Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 6: Minimal sample carryover – Pore structure reduces contamination issues mAU 3.5 3 Onyx™ C18 100 x 4.6 2.5 2 10µg on column 1.5 1 0.5 0 -0.5 0 0.5 1 1.5 2 2.5 min mAU 3.5 Almost No Carryover 3 Mobile phase: 40:60 0.1 % 2.5 TFA:Acetonitrile 2 Flow rate: 3.0 ml/mi 1.5 Temperature: 30oC 1 0.5 Detection: 254nm 0 Sample: Naproxen -0.5 0 0.5 1 1.5 2 2.5 min Onyx™ ™ technology & advantages technology & advantages Onyx • Advantage # 7: Optimal flow dynamics – No column frit needed – Resistant to matrix contaminant clogging – Injection of dirty samples permitted • Plasma, urine, foodstuffs extracts, environmental samples

Current product offerings Current product offerings Current product offerings Current product offerings • Reversed phase (RP) – C18 & C8 • 25, 50, & 100 x 4.6mm • For non-polar, basic, acidic, and metal chelating compounds • Selectivity comparable to particle-based RP-columns • Use standard conditions when developing a method

Current product offerings Current product offerings • Reversed phase (RP) capillary format – C18, 150 x 0.1mm (100µm) – For peptide digests & peptides – Directly couple to MS Current product offerings Current product offerings • 150 x 0.1mm special features 1. High flow rate sample loading (2-10µL/min) possible due to low backpressures • Eliminate use of trap loading columns • Accelerate re-equilibration at high flow rate 2. High efficiency and sensitivity across a wide range of flow rates (200nL/min – 10µL/min) • More peak identifications

Current product offerings Current product offerings • 150 x 0.1mm special features 3. Resistant to chromatographic perturbations caused by salts, lipids, sugars, etc. • Useful for 2-dimensional (2D) applications • Long lifetimes Current product offerings Current product offerings • Normal phase – Si • 100 x 4.6mm • For polar, non-ionic interactions • Can be used for in-situ surface modifications

Applications Applications Applications Applications • Steroids – Onyx™ C18, 100 x 4.6mm

Applications Applications • Sulfa drugs – Onyx™ C18, 100 x 4.6mm Applications Applications • Vitamins – Onyx™ C8, 100 x 4.6mm

Applications Applications • Anisoles – Onyx™ Si, 100 x 4.6mm Applications Applications • Flow rate flexibility – Improve separation and run time

Applications - - DMPK DMPK Applications • Hypertensives in plasma by LC/MS – Onyx™ C18, 100 x 4.6mm – Post protein precipitation Intens. x 10 5 3.0 Mobile phase: A: 0.1% Formic Acid B: Acetonitrile From 10-65% B in 4.0min Gradient: 4.0 ml/min Flow rate: Temperature: Ambient 2.0 LC/MS ESI+ Detection: Sample: 1. Pseudoephedrine (M/Z=166) 2. Propranolol (M/Z= 260) 3. Diltiazem (M/Z= 415) 1.0 4. Verapamil (M/Z=455) 0. 1 2 3 Time [min] Applications - - DMPK DMPK Applications • Diazepam & metabolite in urine by LC/MS – Onyx™ C18, 100 x 4.6mm – Direct inject/ “dilute and shoot” Human urine sample diluted 1;1 with water. 50 µL Injected. (dilute and shoot) Mobile phase A: 0.1% Formic H2O B: Acetonitrile From 5-95% B in 4.0min Gradient: 4.0 ml/min Flow rate: Temperature: Ambient LC/MS ESI+ Detection: Sample: 1. Nordiazepam ( Diazepam metabolite ) (M/Z=272) 2. Diazepam (M/Z=286)-