Silica Monolith Columns forSilica Monolith Columns forUltra High Speed SeparationsUltra High Speed Separations

OutlineOutline• Do more in less time

• Onyx™technology & advantages

• Product offerings

• Applications

• Summary

Do more with less timeDo 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 timeDo 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 timeDo 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 timeDo more with less time

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 pressurePressuretoo high!!

Do more with less timeDo 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 timeDo 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

OnyxOnyx™™ technology and advantagestechnology and advantages

OnyxOnyx™™ technology & advantagestechnology & 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

OnyxOnyx™™ technology & advantagestechnology & advantages• Structure:

– Monolithic silica rod with bimodal pore structure

OnyxOnyx™™ technology & advantagestechnology & advantages• Structure:

– Bimodal pore structure contains macropores and mesopore

Macropores

Mesopores

OnyxOnyx™™ technology & advantagestechnology & advantages• Macropore structure: Allows rapid flow (up

to 9 mL/min) at low pressuresMacropores

are 2µm in diameter

Dense network of pores through

which mobile phase can

rapidly flow at low pressure

Contaminantseasily pass

and don’t clog column

OnyxOnyx™™ technology & advantagestechnology & advantages• Mesopore structure: Creates large

surface area for adsorption

Mesopores are 130Å (13nm) in

diameter

Form the very porous structure of the column

interior

OnyxOnyx™™ technology & advantagestechnology & advantages• Column housing

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

OnyxOnyx™™ technology & advantagestechnology & advantages• Specifications

– 2.0 – 7.5 pH stability

– 45 C max temperature

– 3000 psi (200 bar) max pressure

– 1 – 9 mL/min flow rate range

OnyxOnyx™™ technology & advantagestechnology & advantages• Monolith vs. particle-based technology

VS.

• Individualsilica particles

•Monolithicporous rod

• High flow resistance

•High flow rates

• Highbackpressure

•Lowbackpressure

• Bed splitting possible

•No inlet bed splitting

OnyxOnyx™™ technology & advantagestechnology & advantages• Versus traditional particle-based columns

VS.

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 1: High sample throughput

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 2: Short run times

min0 1 2 3 4

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mAU

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54321

Onyx™ C18

• 5 minutes @ 4 mL/min

Particle-based C18

• 35 minutes @ 2 mL/min

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 3: Short equilibration time

– Total run time = separation time + equilibration timeOnyx™

Traditional silica particle-based column

4 runs

4 runs

Equilibration time

Separation time

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 4: Very low backpressure

– Equivalent to 10µ particle-based material

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 4 cont.: Very low backpressure

– On average 60% less than particle-based columns

mAU

min1 2 3 4050100150200

1

2 3

4

min1 2 3 4

mAU

050

100150200

1

2 3

4

Particle-based 5µ C8Backpressure: 130 bar

Onyx™ C8Backpressure: 75 bar

Dimensions: 100 x 4.6mmMobile phase:

A: 0.1% phosphoric acidB: Acetonitrile

Gradient: from (70:30) to (20:80) in 4min.Flow rate: 2.0 ml/min.Temperature: ambientDetection: 254 nmInjection: 1. phenol

2. 2,4-dinitrophenol 3. 3,4-dinitrophenol 4. pentachlorophenol

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 5: High efficiency

– On par with 3µ particle-based materials

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 5: High plate counts with coupling

– Able to separate closely-eluting, complex samplesPl

ate

coun

t (N

) Ant

hrac

ene

10,000

20,000

30,000

40,000

OnyxOnyx™™ technology & advantagestechnology & 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

OnyxOnyx™™ technology & advantagestechnology & advantages• Advantage # 6: Minimal sample carryover

– Pore structure reduces contamination issues

min0 0.5 1 1.5 2 2.5

mAU

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0.51

1.52

2.53

3.5

min0 0.5 1 1.5 2 2.5

mAU

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0.51

1.52

2.53

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Onyx™ C18 100 x 4.610µg on column

Almost No Carryover Mobile phase: 40:60 0.1 % TFA:AcetonitrileFlow rate: 3.0 ml/miTemperature: 30oCDetection: 254nmSample:Naproxen

OnyxOnyx™™ technology & advantagestechnology & 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 offeringsCurrent product offerings

Current product offeringsCurrent 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 offeringsCurrent product offerings• Reversed phase (RP) capillary format

– C18, 150 x 0.1mm (100µm)

– For peptide digests & peptides

– Directly couple to MS

Current product offeringsCurrent 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 offeringsCurrent 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 offeringsCurrent product offerings• Normal phase

– Si• 100 x 4.6mm

• For polar, non-ionic interactions

• Can be used for in-situ surface modifications

ApplicationsApplications

ApplicationsApplications• Steroids

– Onyx™ C18, 100 x 4.6mm

ApplicationsApplications• Sulfa drugs

– Onyx™ C18, 100 x 4.6mm

ApplicationsApplications• Vitamins

– Onyx™ C8, 100 x 4.6mm

ApplicationsApplications• Anisoles

– Onyx™ Si, 100 x 4.6mm

ApplicationsApplications• Flow rate flexibility

– Improve separation and run time

ApplicationsApplications -- DMPKDMPK• 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

3.0Intens. x 105

Mobile phase:A: 0.1% Formic AcidB: Acetonitrile

Gradient: From 10-65% B in 4.0minFlow rate: 4.0 ml/minTemperature: AmbientDetection: LC/MS ESI+Sample:

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

ApplicationsApplications -- DMPKDMPK• 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 phaseA: 0.1% Formic H2OB: Acetonitrile

Gradient: From 5-95% B in 4.0minFlow rate: 4.0 ml/minTemperature: AmbientDetection: LC/MS ESI+Sample:

1. Nordiazepam ( Diazepam metabolite)(M/Z=272)

2. Diazepam (M/Z=286)-

ApplicationsApplications –– food/beveragefood/beverage• Cereal extract

– Onyx™ C18, 100 x 4.6mm (x2)

min0 2 4 6 8 10

mAU

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2

4

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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 waterB: Acetonitrile/ TFA (95%: 0.08%)

Gradient: 5-70% B in 15 minFlow rate: 1.0 ml/minTemperature: AmbientDetection: UV at 280 nm

ApplicationsApplications –– food/beveragefood/beverage• Wine phenolytics

– Onyx™ C18, 100 x 4.6mm

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Column: Onyx C18 Dimension:100 x 4.6 mmMobile phase

A: 0.1% Phosphoric Acid in WaterB: 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/minTemperature: AmbientDetection: UV @ 254 nmSample: Red Wine: 50 µL Injection

ApplicationsApplications –– proteomicsproteomics• Peptide digest: -amylase

– Onyx™ C18, 150 x 0.1mm

0 10 20 30 40 50 600

10

20

30

40mAU

min

Column: Onyx C18 CapillaryDimension: 150 mm x 0.1 mm IDMobile Phase:

A: 0.1% TFA/WaterB: 0.08% TFA/95% Acetonitrile

Gradient: From 5% to 65% in 60 min.Flow: 3 µl/MinDetection: UV @ 210 nm

ApplicationsApplications –– proteomicsproteomics• 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

0

2

4

6mAU

min

Column: Onyx C18 Capillary Dimension: 150 mm x 0.1 mm IDLoading Pump Mobile Phase:

0.1% TFA/5% Acetonitrile Loading Pump Flow:

10 µL/min for 2 minutesEluting Pump Mobile Phase:

A: 0.1% TFA/WaterB: 0.08% TFA/95%

AcetonitrileGradient: 5% to 65% B in 60 minutesFlow Rate: 400 nL/MinDetection: UV @ 210 nmSample: Cytochrome c digest

SummarySummary

SummarySummary• 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