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

.

Extracolumn Volume

.

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

Page 12

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

mAU

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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!!

Page 14

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

mAU

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min 15 16 17 18 19

mAU

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40

60

Peak Capacity

Res

olut

ion

0.00

4.00 3.00 2.00 1.00

6.00 5.00

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

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

300SB-C18 (300Å) SB-C18 (80Å)

PW

1/2

0 min 12 0 min 11

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

mAU

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70

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

min 0 5 10 15 20 25 30

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6x10

0

0.

1

1.

2

2.

3

3.

4

4.

5

5.

6

6.

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

Page 24

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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5

67

8

0 0.5 1.0Time (min)

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:

min0 0.5 1 1.5 2 2.5

mAU

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400

Poroshell SB-C18, 2.1 x 75 mm

1

2 3 4

5, 6

7

8

9 0.9

76

1.0

77

1.2

89

1.3

95

1.4

63

1.5

23

1.7

41

1.8

60

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512

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6

7

8

9

Poroshell SB-C3, 2.1 x 75 mm

0.9

01

1.0

36 1.2

64

1.3

54 1.4

30 1

.449

1.4

97

1.7

39

1.8

37

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

1.5

5

4 6

7

1.5

5

4 6

7

1.5

4

5, 6

7

1.5

4

5, 6

7

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

Page 65

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

Page 46

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

1

2

3

1 2

3

1

3

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

3

4

min 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

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Change a 5um standard to 3um short column: • saves time • maintains resolution