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

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• 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

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• Solution # 1:

– Buy 10 more HPLC systems

• $500,000 (USD) cost
• Additional analysts needed
• Instrument maintenance
• More lab space required

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• 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

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150 x 4.6mm 50 x 4.6mm 30 x 4.6mm

15 min run 7 min run 5min run 5 min equilibrate 2 min equilibrate 1.5 min equilibrate 1 mL/min 2.5 mL/min 3.5 mL/min 120 bar pressure 250 bar pressure 350 bar pressure

Pressure too high!!

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• Problems with solution # 2

– Loss of analyte resolution – Short column lifetimes – Insufficient re-equilibration – High backpressures at system limit

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• 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 Onyx™ ™ technology and advantages technology and advantages Onyx Onyx™ ™ technology & advantages technology & advantages

• 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 Onyx™ ™ technology & advantages technology & advantages

• Structure:

– Monolithic silica rod with bimodal pore structure

Onyx Onyx™ ™ technology & advantages technology & advantages

• Structure:

– Bimodal pore structure contains macropores and mesopore

Macropores Mesopores

Onyx Onyx™ ™ technology & advantages technology & advantages

• Macropore structure:

Allows rapid flow (up to 9 mL/min) at low pressures Macropores are 2µm in diameter Dense network

• f pores through

which mobile phase can rapidly flow at low pressure Contaminants easily pass and don’t clog column

Onyx Onyx™ ™ technology & advantages technology & advantages

• Mesopore structure:

Creates large surface area for adsorption Mesopores are 130Å (13nm) in diameter Form the very porous structure of the column interior

Onyx Onyx™ ™ technology & advantages technology & advantages

• Column housing

– Rod is cladded in PEEK tubing with a special proprietary process and ready to use

Onyx Onyx™ ™ technology & advantages technology & advantages

• Specifications

– 2.0 – 7.5 pH stability – 45C max temperature – 3000 psi (200 bar) max pressure – 1 – 9 mL/min flow rate range

Onyx Onyx™ ™ technology & advantages technology & advantages

• Monolith vs. particle-based technology

VS.

• Individual

silica particles

• Monolithic

porous rod

• High flow

resistance

• High flow

rates

• High

backpressure

• Low

backpressure

• Bed splitting

possible

• No inlet bed

splitting

Onyx Onyx™ ™ technology & advantages technology & advantages

• Versus traditional particle-based columns

VS.

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 1: High sample throughput

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 2: Short run times

min 1 2 3 4 mAU 10 20 1 2 3 4 5 5 10 15 20 25 30 mAU 10 20 30 5 4 3 2 1

Onyx™ C18

• 5 minutes @ 4 mL/min

Particle-based C18

• 35 minutes @ 2 mL/min

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 3: Short equilibration time

– Total run time = separation time + equilibration time

Onyx™ Traditional silica particle-based column

4 runs 4 runs

Equilibration time Separation time

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 4: Very low backpressure

– Equivalent to 10µ particle-based material

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 4 cont.: Very low backpressure

– On average 60% less than particle-based columns

mAU min 1 2 3 4 50 100 150 200 1 2 3 4 min 1 2 3 4 mAU 50 100 150 200 1 2 3 4

Particle-based 5µ C8 Backpressure: 130 bar Onyx™ C8 Backpressure: 75 bar

Dimensions: 100 x 4.6mm Mobile phase: A: 0.1% phosphoric acid B: Acetonitrile Gradient: from (70:30) to (20:80) in 4min. Flow rate: 2.0 ml/min. Temperature: ambient Detection: 254 nm Injection:

• 1. phenol
• 2. 2,4-dinitrophenol
• 3. 3,4-dinitrophenol
• 4. pentachlorophenol

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 5: High efficiency

– On par with 3µ particle-based materials

Onyx Onyx™ ™ technology & advantages technology & advantages

• Advantage # 5: High plate counts with coupling

– Able to separate closely-eluting, complex samples

Plate count (N) Anthracene 10,000 20,000 30,000 40,000

Onyx Onyx™ ™ technology & advantages technology & advantages

• 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 Onyx™ ™ technology & advantages technology & advantages

• Advantage # 6: Minimal sample carryover

– Pore structure reduces contamination issues

min 0.5 1 1.5 2 2.5 mAU

• 0.5

0.5 1 1.5 2 2.5 3 3.5 min 0.5 1 1.5 2 2.5 mAU

• 0.5

0.5 1 1.5 2 2.5 3 3.5

Onyx™ C18 100 x 4.6 10µg on column Almost No Carryover

Mobile phase: 40:60 0.1 % TFA:Acetonitrile Flow rate: 3.0 ml/mi Temperature: 30oC Detection: 254nm Sample:Naproxen

Onyx Onyx™ ™ technology & advantages technology & advantages

• 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 Applications -

• DMPK

DMPK

• Hypertensives in plasma by LC/MS

– Onyx™ C18, 100 x 4.6mm – Post protein precipitation

1 2 3 Time [min] 0. 1.0 2.0 3.0

• Intens. x 105

Mobile phase: A: 0.1% Formic Acid B: Acetonitrile Gradient: From 10-65% B in 4.0min Flow rate: 4.0 ml/min Temperature: Ambient Detection: LC/MS ESI+ Sample:

• 1. Pseudoephedrine (M/Z=166)
• 2. Propranolol (M/Z= 260)
• 3. Diltiazem (M/Z= 415)
• 4. Verapamil (M/Z=455)

Applications Applications -

• DMPK

DMPK

• 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 Gradient: From 5-95% B in 4.0min Flow rate: 4.0 ml/min Temperature: Ambient Detection: LC/MS ESI+ Sample:

• 1. Nordiazepam ( Diazepam metabolite)

(M/Z=272)

• 2. Diazepam (M/Z=286)-

Applications Applications – – food/beverage food/beverage

• Cereal extract

– Onyx™ C18, 100 x 4.6mm (x2)

min 2 4 6 8 10 mAU 2 4 6 8 10 12 14

Multi-Grain Cereal extracted with Methanol: 50 µL Injected Column: Onyx C18 (2 columns connected with column coupler) Dimension: 200 x 4.6 ({2} 100 x 4.6 columns coupled together) Mobile phase A: 0.1% TFA in water B: Acetonitrile/ TFA (95%: 0.08%) Gradient: 5-70% B in 15 min Flow rate: 1.0 ml/min Temperature: Ambient Detection: UV at 280 nm

Applications Applications – – food/beverage food/beverage

• Wine phenolytics

– Onyx™ C18, 100 x 4.6mm

min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50 min 5 10 15 20 25 30 mAU 10 20 30 40 50

Column: Onyx C18 Dimension:100 x 4.6 mm Mobile phase A: 0.1% Phosphoric Acid in Water B: Methanol Gradient: Hold at 3%B from 0-10 minutes, From 3-18%B from 10-22 min, From 18-50% from 22-30 min Flow rate: 2.0 ml/min Temperature: Ambient Detection: UV @ 254 nm Sample: Red Wine: 50 µL Injection

Applications Applications – – proteomics proteomics

• Peptide digest: -amylase

– Onyx™ C18, 150 x 0.1mm

10 20 30 40 50 60 10 20 30 40 mAU min

Column: Onyx C18 Capillary Dimension: 150 mm x 0.1 mm ID Mobile Phase: A: 0.1% TFA/Water B: 0.08% TFA/95% Acetonitrile Gradient: From 5% to 65% in 60 min. Flow: 3 µl/Min Detection: UV @ 210 nm

Applications Applications – – proteomics proteomics

• Peptide digest: Cytochrome C

– Onyx™ C18, 150 x 0.1mm – 10µL/min loading…2nL/min analysis

1 5 10 15 20 25 30 35 40 45

• 2

2 4 6 mAU min

Column: Onyx C18 Capillary Dimension: 150 mm x 0.1 mm ID Loading Pump Mobile Phase: 0.1% TFA/5% Acetonitrile Loading Pump Flow: 10 µL/min for 2 minutes Eluting Pump Mobile Phase: A: 0.1% TFA/Water B: 0.08% TFA/95% Acetonitrile Gradient: 5% to 65% B in 60 minutes Flow Rate: 400 nL/Min Detection: UV @ 210 nm Sample: Cytochrome c digest

Summary Summary Summary Summary

• Onyx is ideal for high speed, high throughput

applications

• Onyx demonstrates high efficiencies with low

backpressures

• Onyx is suited for applications with dirty &

complex samples like serum

• Onyx works for applications in a variety of

industries