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The Agilent 1260 Infinity BioInert Quaternary

P mpScope of a low-pressure

Pump mixing UHPLC pump with Bio-Inert Capabilities

Patrick Cronan

April 4, 20131

Patrick CronanApplications ScientistAgilent Technologies

Boston, MA

Comparison of pumpsAgilent 1260 Infinity Quaternary and Binary Pump

1260 Infinity Quaternary Pump• 600 bar at 5 mL/min

• Integrated Degasserg g

• 4 solvent channels, mixing of all 4 channels possible

• Flow rate up to 10 mL/min (200 bar) for semi-prep work

• Lower in price (approx. 15 %)*

1260 Infinity Binary Pump• 600 bar at 5 mL/min

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• External Degasser

• 4 solvent channels**, mixing of 2 channels possible

• Better performance

• Delay volume can be reduced down to 120 µL *Incl. Degasser for both pumps**with solvent selection valve

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Differences between the Agilent 1260 Binary & Quaternary Pumps

• Point of mobile phase mixing before pump head

• Point of mobile phase mixing after pump head

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after pump head

1260 Infinity Bio-inert HPLC

What is similar compared to standard1260 Infinity Quaternary LC?

Pumping performance, gradient performance Pumping Specifications Detector, Autosampler Performance and

Specs Overall System Robustness TCC specs

600 bar also for fast or high resolution Bioseparations

Modular flexibility maintained

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1260 Infinity Bio-inert HPLC

What is different compared to standard 1260 Infinity Quaternary LC

Pump titanium based, no SST Autosampler metal-freeCapillaries and connections metal-freeDetector Flow Cell metal-freepH-range 1-13, short term pH 14 Active Seal wash included in pump Low pressure is possible

•Wide pH range•High salt tolerance•Lowest surface activity Bio-Inertness

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New Capillary Design Ensures Bio-inertness

Capillaries:

Metal cladded PEEK capillary

New capillary technolgy enablesNew capillary technolgy enables

600 bar AND is completely metal free !!

Needle:

SST housing

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gfor robustness

PEEK and ceramic for metal free sample path

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Proof of concept: surface activityATP Analysis – 1260 Infinity standard LC system

Chromatographic conditionsFlow rate: 0.5 mL/minIsocratic run with buffer A, B, C, D or EStop time: 5 minutesInjection volume: 0.2 μLTemperature TCC: 40 °C

mAU

300

400

500

6000

% M

eOH

10 %

MeO

H

MeO

H

eOH

OH

Temperature TCC: 40 CDiode array detector: 254 nmNo column, PEEK restrictionSolventsBuffer A: 10 mM ammonium acetateBuffer B: 10 mM ammonium acetate +10% methanolBuffer C: 10 mM ammonium acetate +50% methanolBuffer D: 10 mM ammonium acetate +70% methanolBuffer E: 10 mM ammonium acetate +90% methanol

min0.5 1 1.5 2 2.5

0

100

200

50 %

70 %

Me

90 %

Me

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Proof of concept: low surface activityATP Analysis – 1260 Infinity Bio-inert LC

OH

OH

OH

OH

OH

mAU

300

400

500

600

700

0 %

MeO

10 %

MeO

50 %

MeO

70 %

MeO

90 %

MeO Chromatographic conditions

Flow rate: 0.5 mL/minIsocratic run with buffer A, B, C, D or EStop time: 5 minutesInjection volume: 0.2 μLTemperature TCC: 40 °CDiode array detector: 254 nmNo column, PEEK restrictionSolventsBuffer A: 10 mM ammonium acetateBuffer B: 10 mM ammonium acetate +10% methanol

min0 1 2 3 4 5 6

0

100

200

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Buffer C: 10 mM ammonium acetate +50% methanolBuffer D: 10 mM ammonium acetate +70% methanolBuffer E: 10 mM ammonium acetate +90% methanol

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Analysis of phosphate compoundsResults Tailing Factor

64

4

16

Tail

ing

fac

tor

1260 Infinity LC

1260 Infinity Bio-inert LC

10% 10% 50% 70% 90%

Methanol amount in buffer

Ion Exchange: Salt Gradient

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Positively charged salt Ions compete and displace positively charged proteins

Cation Exchange

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Ion Exchange Chromatography - AIEX

Na+

Na+

Na+

Cl-

Cl-

Cl-

Cl-

N +

Na+

Na+

Na+

Na+

Cl

Cl-

Cl-

Cl-

Cl-

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Na+

Binds at Low Ionic Strength Elute at High Ionic Strength

Separations are based on the accessible protein surface charges and opposite charges on particle

Proteins are eluted using increasing concentration of salt or changing pH

Ion Exchange: pH Gradient

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Chromatofocusing leads to better resolution

Protein charge changes during gradient due to protonation/deprotonation of functional groups

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Cha

rge

+

elec

tirc

Poi

nt (

pI)

pH greater than

Protein Isoelectric Point - ChargeP

rote

in N

et C

_

pH4 6 8 10 12

Isoe

+

----- - - - --

-pH lower thanthe protein pI

pH greater than the protein pI

Cation ExchangeParticle

-----

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Ion Exchange Separation Sequence

Equilibration Loading Gradient Clean-up Equilibration

Sal

t Con

cent

ratio

nUnbound

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Equilibration/clean-up is typically over 10 column volumes – essential for reproducibility

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IEX Technique

Technique requires gradients for elution

Proteins interact with the stationary phase due to charge

Separation based on differences in degree of chargep g g

Sample is injected in a mobile phase buffer with a low salt concentration – this binds proteins to the column

Proteins are typically eluted at constant pH with increasing salt gradients (mobile-phase ionic strength) to displace the proteins from the stationary phase

Higher charge proteins bind more strongly and an increased salt gradient is

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needed to elute them

A typical mobile phase will contain salt, NaCl, KCl,

Technique does not denature protein

Columns

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Learn more:www.agilent.com/chem/biohplc

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Ion Exchange – Product Families

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

A il Bi MAb P l N P WCX 1 7 3 N/A I GAgilent 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

Specifications

Parameter Agilent Bio IEX

Particle Size 1.7, 3, 5, and 10um

Binding Capacity Expressed as mg/mL by PS> 100 ug protein on column

pH Range 2-12

Temp Limit 80°C

Hardware/Pressure 600 bar for SS400 bar for PEEK

Pressure Limit 10um – 275 bar (4000 psi)5um – 413 bar (6000 psi)3um – 551 bar (8000 psi)1 7 m 689 bar (10000 psi)1.7um - 689 bar (10000 psi)

Recommended Flow Rate: 0.1 – 1 mL/min

Mobile phase Compatibility Acetonitrile/acetone/methanol mixtures

April 4, 2013Agilent Restricted

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Buffer Selection

Anionic Buffers (negatively charged) are used for cation exchange & Cationic Buffers (positively charged) are used for anion exchangeanion exchange

If pH of the buffer < pI then net protein charge is positive.

If pH of the buffer > pI then net protein charge is negative.

With salt gradient elution, start at least one pH unit away from the protein’s pI.

Use a buffer concentration that is sufficient to maintain buffer capacity to keep pH constant, usually between 20 and 50 mM.

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Buffer Advisor SoftwareQuaternary mixing

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Buffer Advisor SoftwareWorkflow

1: Select Buffer and Gradient Solution

2: Enter Gradient Timetable

3: Review Composition of Stock Solutions and Adjust

4: Gradient Display

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4: Gradient Display Section: Process and Review Gradient

Experimental Situation

Chromatographic ConditionsFlow Rate: 1.5mL/minGradient: 0 min – 200mM NaCl

2 min – 200mM NaCl

ColumnAgilent Bio Mab, PEEK,4.6 x 250mm, 5um

2 min 200mM NaCl10 min – 450mM NaCl12 min – 700mM NaCl13 min – 200mM NaCl

Injection Volume: 20uL

Temperature TCC: 35˚C

SolventsBuffer A: H20Buffer B: NaCl 3MBuffer C: MES (2-(N-morpholinoethanesulfonic acid monohydrate) 60mMBuffer D: MES-Na (2-(N-morpholino Ethanesulfonic acidsodium salt) 35mM

DAD: 280nm/4nmREF: Offpeak width: >0.013 min(0.25s resp time – 20Hz)

SampleBioRad Cation Exchange Standard:Myoglobin: 17,699 pI 7.4, 6.9Cytochrome C: 12,384 Da pI 10.7, 9Ribonuclease A: 13,700 Da pI 8.88

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Buffer Advisor SoftwareWorkflow

Buffer Advisor SoftwareTimetable import into Chemstation

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Buffer Advisor SoftwarepH scouting

pH 5.4pH 5.8pH 6.0pH 6 4

MMyoglobin: pI 6.9Cytochrome C: pI 10.7, 9Ribonuclease A: pI 8.88

pH 6.4pH 6.8pH 7.0

R

R

C

C

27

M

Buffer Advisor Softwarestable pH

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Conclusion

In General, the LPM, quaternary pump is advantageous for:• Solvent selection in Method Development (limited to 4 solvents)

• Ternary or quaternary gradients

• Blending of buffer/modifier

Using Buffer Advisor with BioInert Quat System:

• Shortens and simplifies the workflow for pH scouting by using dynamically mixed gradients

• Provides a wide range of pre-validated, user-selectable buffer systems

• Results in pH values that are more accurate and precise than manually prepared buffer solutions using pH optimization software

• provides an excellent tool for automated pH scouting and accurate ion exchange chromatography

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