Living the LC Dream - Agilent...Living the LC Dream Innovative Agilent Column Designs for Maximum...
Transcript of Living the LC Dream - Agilent...Living the LC Dream Innovative Agilent Column Designs for Maximum...
Living the LC Dream Innovative Agilent Column Designs for Maximum Resolution and Speed
Paul Dinsmoor Technical Specialist, Bio-Columns 2014 ASTS
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History of Commercial HPLC Particle Development
Narrow-Bore
The Path to UHPLC Separation - Column and Instrument Evolution Superficially Porous Particles - 40µ 250 mm length 150 mm length
15 mm length Superficially Porous Particles – 300A, 5µm Monolith Overlapped Injections
Nano HPLC Superficially Porous Particles – 120A, 2.7µm 1200 bar Instruments
10 µm particles
5 µm particles 400 bar Instruments 3.5 µm particles
High Temp with Steric-Protection Non-Porous Particles Micro-Bore Capillary Columns Well-Plate Samplers 1.8 µm particles 1000 bar Instruments THE FUTURE?
New Instrument Design Minimizes Dispersion Effects
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Extracolumn Volume
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b) Radial Diffusion (dispersion)
More Efficient Particles in Smaller Volume Columns Reduces Dispersion Within the LC Column a) Longitudinal Diffusion (dispersion) Direction of Flow
Protein/Peptide Separations by Reversed-phase Larger Molecules = Slower Diffusion So, a need to decrease the diffusion time for macromolecules!
To improve, we can increase the Diffusion Rate by:
elevating operating temperature decreasing solvent viscosity and, or……………..
Decrease the Diffusion Distance!
1. Rapid Resolution High Definition (RRHD) 300 Family (C3, C8, C18, diphenyl) Developed very small (totally porous) particles (<2 um)
RRHD 1.8um UHPLC Superior efficiency Very narrow bands Ultra high resolution Fast flows Short column lengths (pressure constraint)
2. Poroshell 300 and AdvanceBio120 Peptide Mapping Limit diffusion distance into a particle (shell particle)
HPLC Superior efficiency Very narrow bands Ultra high resolution Fast flows Long column lengths (no pressure constraint)
5um Poroshell 300 2.7um AdvanceBio120 Peptide
Reversed-Phase – Product Families
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Particle Porosity Functionalities Particle Sizes
Pore Size Application
ZORBAX Stable Bond Silica Fully Porous C18, C8, C3, CN
3.5um, 5um 300A Low pH
ZORBAX 300Extend
Silica Fully Porous
Extend-C18 3.5um, 5um 300A High pH
ZORBAX RRHD Silica Fully Porous
SB-C18, SB-C8, SB-C3, Diphenyl
1.8um 300A High speed High resolution
ZORBAX Eclipse AAA Silica Fully Porous C18 3.5um, 5um 80A Amino Acids
AdvanceBio Peptide Mapping
Silica Superficially
EC-C18
2.7um 120A Peptide Mapping
Poroshell 300 Silica Superficially SB-C18, SB-C8, SB-C3, Extend-C18
5um 300A High speed High resolution
PLRP-S PS/DVB Fully Porous 3um, 5um, 8um, 10um, 12um, 18um
100A, 300A Peptides, oligos, proteins
PLRP-S PS/DVB Fully Porous 5um, 8um, 10um, 30um
1000A, 4000A
Proteins and DNA
VariTide RPC PS/DVB Fully Porous 6um 200A Synthetic peptides
Page 17 Emphasized column technology
Sub 2um Totally Porous Particle RRHT 1.8um Commercial Intro 2003
Superficially Porous Particle Poroshell 300 Commercial Intro 2001
Agilent Particle Choices for UHPLC Longest History of UHPLC Particle Innovation and Manufacture
SOLID CORE
Agilent Particle Choices for UHPLC Longest History of UHPLC Particle Innovation and Manufacture
Zorbax 300 SB RRHD for Proteins and Peptides
• Stablebond 300 silica
•1.8 um particle size
• 1200 Bar pressure limit for UHPLC
• C-18, C-8, C-3, and diphenyl bonded phase
UHPLC Columns Increase Resolution and Increase Speed
Column Length (mm)
Resolving Power
N(5 µm)
Resolving Power
N(3.5 µm)
Resolving Power
N(1.8µm)
150 12,500 21,000 32,500
100 8,500 14,000 24,000
75 6000 10,500 17,000
50 4,200 7,000 12,000
30 N.A. 4,200 6,500
15 N.A. 2,100 2,500
Analysis Time
Peak Volume
Analysis Time*
-33%
-50%
-67%
-80%
-90% Solvent Usage
* Reduction in analysis time compared to 150 mm column
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1290 Higher Pressure Limits Allow Use of Long Lengths to Achieve Much Higher N and Resolution
150 + 100 mm = 250 mm 1.8um @ 1100 bar
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Rs3,2: 8.76 N6 : 50,200
Rs3,2: 6.42 N6 : 31,400
150 mm (P:582 bar)
150 +100 mm (P:1095 bar)
min 0 0.5 1 1.5 2 2.5 3 3.5
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UHPLC Column Particles > N and > Peak Capacity
• The number of peaks which can be separated with defined resolution (example: R=1.5)
• in a certain period of time • for a given system column length and particle size
5µm
1.8µm
2 Peaks
3 Peaks 50% more!!
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More Peak Capacity with 1.8 um Columns Snapshot of portion of BSA Tryptic Digest
673
502
Peak Capacity
SB-C18, 2.1x150mm, 3.5µ
SB-C18, 2.1x150mm, 1.8µ
min 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19
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Peak Capacity
Res
olut
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Change in Selectivity (Chemistry) Produces Most Change in Resolution 7.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Increase N Increase Alpha Increase k'
Plates: Alpha: k’:
5000 1.10 2.0
10000 1.35 4.5
15000 1.60 7.0
20000 1.85 9.5
25000 2.1 12.0
Selectivity Impacts Resolution Most • Change bonded phase • Change mobile phase
Rs = N½/4 • (α-1)/α • k’/(k’+1)
C3 versus Diphenyl
Large Pore Structure is Important for Improved Peak Shape of Large Biomolecules
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0
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300SB-C18 (300Å) SB-C18 (80Å)
PW
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Decrease the diffusion time for macromolecules and limit the diffusion path! More efficient mass transfer!
Reduces secondary interactions & enables selective separation for a wide range of peptides. .
Superficially Porous Particle Technology
NEW! AdvanceBio Peptide Mapping Column for HPLC and UHPLC: • 2.7um Superficially Porous • 120A pore size • 600 bar pressure limit • 2um frit to reduce clogging
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Save Time: 2 to 3 times faster than fully porous particles
Increased Flexibility: Highly compatible with TFA and formic acid mobile phases for efficient LC/MS analysis
Greater analytical confidence: Each batch is tested with a rigorous peptide mix to ensure suitability and reproducibility
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min 0 2.5 5 7.5 10 12.5 15 17.5 20
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2.1 x 150mm AdvanceBio Peptide Mapping Column Mobile phase: A-water (0.1%TFA), B- ACN (0.08%TFA), 40 C, flow: 0.52mL/min
Hydrophilic peptide retention
Narrow Peaks w baseline resolution
Hydrophobic peptide retention
Reduced and fast analysis time
Critical and desired peptide mapping components to achieve fast, selective and highly efficient peptide separations across a wide dynamic range.
Peptide Mapping AdvanceBio Peptide Mapping Column Highlights
BSA tryptic digest
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Peptide Mapping for LC/MS
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Cpd 1: 12.155: +ESI ECC Scan Frag=170.0V EPOdigest1d0010.d
12 155 Cpd 1: 12 155
Counts vs. Acquisition Time (min)
11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5
Agilent LC/MS 100% sequence coverage
Digested EPO (recombinant humanized) separated on a 2.1 x 250mm AdvanceBio Peptide Mapping Column Mobile phase: A-water (0.1%FA), B- ACN (0.08%FA), temp: 55C, Flow: 0.5mL/min
Excellent separation capability for generating peptide maps of highly desired proteins, i.e., glycopeptide mapping during fast analysis times
Provides compatibility with TFA and Formic acid mobile phases for LC/MS analyses
Pressure maintained below 500bar for 250mm column length
Digested EPO Protein Glycopeptide mapping
TB# 5991-1813EN
Poroshell 300
300 A pore size
Stablebond chemistry
available in C-3, C-8,C-18, and C-18 Extend
5 um particle size
Comparison of Diffusion Distance Totally porous silica vs. superficially porous silica
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0.25 µm
5 µm Totally Porous Particle
2.5 µm Required diffusion distance for a macromolecule reduced 10 fold !
5 µm Superficially Porous Particle
Flow Rates for Poroshell Columns
Column Internal Diameter Porous Particle Flow Rate Range
Poroshell Flow Rate Range
2.1 mm 0.1 – 0.3 mL/min 0.3 – 3 mL/min
1.0 mm 30 – 60 µL/min 0.08 - 0.75 mL/min
• Very high flow rates can be used effectively with Poroshell columns
High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations
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Columns: Poroshell 300SB-C18 2.1 x 75 mm, 5 µm MP: A: 0.1% TFA B: 0.07% TFA in ACN Gradient: 5 – 100% B in 1.0 min. Flow Rate: 3.0 mL/min. Temperature: 70°C Pressure: 250 bar Detection: UV 215 nm
Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbumin
TB# 5989-9899EN
Reversed Phase Heavy and light chain analytical characterization
Use of 1200 LC, Poroshell 300SB columns and UV detection to characterize antibodies
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TB# 5989-0604EN
High Flow Rate, High Sensitivity LCMS
min 0 0.5 1 1.5 2 2.5 3 3.5 0
20000000
40000000
60000000
80000000
1E8
pmoles of protein 0.5
2.5
5
0.75 1
Mobile Phase Gradient: 20 -100% B in 5.5 min. A: water + 0.1% formic acid B: ACN + 0.1% formic acid Flow Rate: 0.6 mL/min Temperature: 80°C Injection volume: 1 uL LC/MS: Pos. Ion ESI Peakwidth: 0.06 min
Sample: Mixture of insulin, lysozyme, cytochrome C, myoglobin, BSA, carbonic anhydrase
Column: Poroshell 300SB-C18, 1.0 x 75 mm
More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution:
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Poroshell SB-C3, 2.1 x 75 mm
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1.8
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Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 70 0C; Flow: 0.5 mL/min; Det: UV 215 nm Mobile Phase: A= 0.1% TFA/H2O, B= 0.07% TFA/ACN; Gradient: 5-100% B in 3.0 min
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Samples: 1. Angiotensin II 2. Neurotensin 3. RNase A 4. Insulin B Chain 5. Insulin 6. Cytochrome C 7. Lysozyme 8. Myoglobin 9. Carbonic Anhydrase
Poroshell 300SB-C18 2.1 x 75 mm
Poroshell 300SB-C3 2.1 x 75 mm
•Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3 min!
Size Exclusion – Product Families
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Particle Porosity Functionalities Particle Sizes
Pore Size Application
Agilent Bio SEC-3 Silica Fully porous N/A 3um 100A, 150A, 300A
High efficiency
Agilent Bio SEC-5 Silica
Fully porous
N/A
5um 100A, 150A, 300A 500A, 1000A, 2000A
More pore sizes
ProSEC 300S Silica
Fully porous N/A
5um 300A Extend linear range broader sample range
ZORBAX GFC Silica
Fully porous
N/A
4um, 6um
150A 300A
Larger column sizes
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1. High efficiency small particles
2. Multiple pore sizes to cover broad range of molecular sizes
3. Single pore size to cover range of globular proteins/antibodies
4. Larger columns for fractionation
NEW Size Exclusion Columns
• 5µm Particle • 100Å, 150Å, 300Å, 500Å, 1000Å,
2000Å pore sizes • High stability and long lifetime • Great reproducibility
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• Unique, 3µm particle • 100Å, 150Å, 300Å pore sizes • Highest resolution • Highest efficiency • Faster SEC separations
Fast, High resolution SEC characterization
Step 1: Improve Resolution
Technology: Small Particle SEC Columns Results: Higher Resolution SEC Separations
Use Higher Resolution to Achieve Faster Separations
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Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mAb (2mg/ml) Injection: 5ul Flow rate: 1.0, 1.5 and 2ml/min (56 bar , 75 bar, 105 bar) Eluent: 150mM sodium phosphate + 100mM Na-sulfate Detection: 220nm
Flow Rate Resolution Monomer/Dimer
Monomer Efficiency
Percentage Dimer
1.0ml/min 1.53 3,510 0.64
1.5ml/min 1.43 2,502 0. 47
2.0ml/min 1.13 1,917 0.64
4 Minutes
monomer dimer
2.0ml/min 1.5ml/min 1.0 ml/min
Increase flow rate and use a shorter column for faster separations.
Technology: Small Particle SEC Columns
Results: Faster SEC separations
Step 2 – Go Faster
Fast SEC – TB5990-8613EN
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Size separations over range of salt concentrations
PERFORM EFFICIENT SIZE SEPARATIONS WITH MINIMAL SECONDARY INTERACTIONS
Ion Exchange – Product Families
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Particle Porosity Functionalities Particle Sizes Pore Size Application
Agilent Bio-IEX Polymer Non-porous SAX, WAX, SCX, WCX
1.7um, 3um, 5um 10um
N/A Peptides, proteins
Agilent Bio MAb Polymer
Non-Porous WCX 1.7um, 3um, 5um 10um
N/A IgG
PL-SAX PS/DVB Fully Porous SAX 5um, 8m, 10um, 30um
1000A, 4000A Peptides, oligos, proteins. Larger column sizes
PL-SCX PS/DVB Fully Porous SCX 5um, 8m, 10um ,30um
1000A, 4000A Peptides, proteins. Larger column sizes
Bio-Monolith IEX Polymer Monolith QA, DEAE, SO3 N/A N/A BioMacromoleucles
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1. Non-porous particles for high efficiency analytical separations
2. Porous particles for scale up to purification
3. Monoliths for high speed separations
Selectivity Comparison
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- SCX - WCX - MAb
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Columns: Agilent Bio SCX, NP 3, 4.6x50 mm, SS Agilent Bio WCX, NP 3, 4.6x50 mm, SS Agilent Bio MAb, NP 3, 4.6x50 mm, SS Eluent A:10 mM NaH2PO4.2H2O, pH 5.75 Eluent B: A + 1 M NaCl Gradient: 0 to 100% b in 25 mins Flow rate: 0.5 mL/min Temperature : Ambient Detector: UV at 220 nm
Sample: 1. Ribonuclease 2. Cytochrome C 3. Lysozyme Sample concentration: 2 mg/mL Injection volume: 2 uL
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Fast, High Resolution IEX Separations
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Bio WCX NP3, 4.6 x 50 mm Bio WCX NP1.7, 4.6 x 50 mm 1.0 mL/min 1.0 mL/min
Technology Used: small particle columns (3um, non porous) Result: 5 minute, high resolution IEX
Standard Protein Separation: Bio WCX
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Conventional HPLC (400 bar)
1. Ovalbumin pI 4.5 2. RNase A pI 9.4 3. Cytochrome C pI 9.8 4. Lysozyme pI 11
Gradient: 20mM sodium phosphate buffer, pH 6.5 0-800mM NaCl (0-20 mins) Flow rate: 1.0mL/min Sample: 10µl inj. Detection: UV, 220nm
Column: Bio WCX NP3 (4.6x50mm SS)
Column: Bio WCX NP5 (4.6x250mm SS) 1 2
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Change a 5um standard to 3um short column: • saves time • maintains resolution