silica monolith columns for ultra high speed … monolith columns for ultra high speed separations...
TRANSCRIPT
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
mAU
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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
-0.50
0.51
1.52
2.53
3.5
min0 0.5 1 1.5 2 2.5
mAU
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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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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