Information, descriptions, and specifications in this publication are subject to change without notice.

© Agilent Technologies, Inc. 2010Printed in the USA April 13, 20105990-5526EN

Our measure is your success.

For more help, please visit www.agilent.com/chem/contactus

for your local Agilent Representative or Agilent Authorized Distributor. For technical support, visit www.agilent.com/chem/techsupport

Agilent BioHPLC Columns NavigatorAnalytical Bio-Molecule Separations

300Extend-C18uses a unique, bidentate bonded phase

providing excellent lifetime and reproducibility at high pH

.

At high pH

, retention and selectivity of peptides and

polypeptides can change dramatically as a result of

changes in charge on molecules.

Rugged, high and low pH

separations of

polypeptides and peptides from pH

2-11.5.

Different selectivity possible at high and

low pH

. High efficiency and good recovery

of hydrophobic peptides at high pH.

Ideal for LC/MS w

ith ammonium

hydroxide-modified m

obile phase.

IDs: 0.3 – 4.6 m

m

Lengths: 50 – 250 mm;

12.5 mm (guard)

Particle size: 3.5, 5 µm

Pore size: 300Å

Max tem

p: 60°C

pH stability: 2.0-11.5

Endcapped: Double

Separations of proteins and peptides

with m

olecular weight <

25 kDa

at mid and high pH

C8 is an excellent choice for intact proteins because

of its moderate hydrophobicity

C18 and C8 are best for peptides and protein digests,

also for proteins

C3 and CN are for larger, hydrophobic polypeptides

and proteins, also for peptides300StableBond columns are unmatched in

their durability at low pH, such as with the

TFA containing mobile phases. For LC/MS

separations at low pH, 300StableBond

columns can also be used with formic

acid and acetic acid mobile phase.

IDs: 0.075 – 4.6 mm

Lengths: 35 – 250 mm;

12.5 -17 mm (guard)

Particle size: 3.5, 5 µm

Pore size: 300Å

Max temp:C18: 90°C

C8, C3, CN: 80°C

pH stability:1.0-8.0

Endcapped: No

Proteins and peptides with molecular

weight < 50 kDa at low pH

Column exclusion limits (in kilo Daltons)100Å MW range: 0.1 – 100 kD150Å MW range: 0.5 – 150 kD300Å MW range: 5 – 1,250 kD

3 µm silica particles coated with ahydrophilic, polymeric layer providing higher

resolution, sharper peaks and fasterseparations than large-particle SEC columns,

compatible with organic modifiers anddenaturants; optimized for use at

low salt concentrations.

IDs: 4.6 mm, 7.8 mmLengths: 150, 300 mm, 50 mm (guard)Particle size: 3 µmPressure limits: 240 barpH stability: 2-8.5

Size-based separations of bio-molecules, aggregation analysis

Colum

n exclusion lim

its (in kilo D

altons)

100Å M

W range: 0

.1 – 100 kD

150Å M

W range: 0

.5 – 150 kD

300Å M

W range: 5

– 1,250 kD

500Å M

W range: 1

5 – 5,0

00 kD

1000Å MW ra

nge: 5

0 – 7,5

00 kD

2000Å MW ra

nge: >

10,000

kD

5 µm silica particles coated with a

hydrophilic polymeric layer that decreases

secondary interactions for maximum

efficiency and stability; compatible with

organic m

odifiers and denaturants;

optimized for use at low

salt co

ncentrations.

IDs: 4.6 m

m, 7.8 mm

Length

s: 150, 300 m

m,

50 mm

(guard)

Partic

le size: 5 µm

Pressu

re lim

its: 240 b

ar

pH sta

bility: 2-8

.5

Size-base

d separations of

bio-molecules, ag

gregation analysis

Column exclusion lim

its (in kilo Daltons)

GF-250(150Å): 4 – 400 kD

GF-450(300Å):10 – 900 kD

Hydrophilic diol bonded phase for high

recovery of proteins and a unique zirconia

modification of the silica, com

patible with

organic modifiers and denaturants;

best performance with moderate to

high salt concentrations.

IDs: 4.6 mm, 9.4 mm

Lengths: 250 mm,

12.5 mm (guard),

15 mm (guard)

Particle size:

4 µm

(GF-250), 6 µm (G

F-450)

Pressure limits: 350 bar

pH stability: 3-8Size-based separations of

bio-molecules, aggregation analysis

Monoclonal antibodies are typically basic and W

CXcolum

ns

are the columns of choice to separate m

inor charge differences.

1.7 µm, 3 µm

, 5 µm, and 10

µm particle sizes

(Bio M

Ab particles have a m

ore dense layer of WCX

than the Bio IEX (W

CX) particles).

Unique non-porous PS/D

VB

particles coated with a hydrophilic

polymeric layer and a highly uniform

,

densely packed, weak cation exchange

(WCX) layer for im

proved selectivity for

monoclonal antibody charge variants.

IDs: 4.6 m

m, 2.1 m

m

Lengths: 50 – 250 mm;

10 mm (stainless guard);

50 mm (PEEK guard)

Particle Sizes: 1.7 µm, 3 µm

,

5µm

, and 10 µm

Pore Size: Non-porous PS/D

VB

Max Tem

p: 80°C

pH Stability: 2.0-12.0

Pressure limits: PEEK 400 bar,

Stainless steel 600 bar

Charge isoform analysis of

monoclonal antibodies

General Rule for choosing a Bio IEX column:

Acidic proteins: SAX orWAX

Basic proteins: SCX orWCX

1.7 µm, 3 µm, 5 µm, and 10 µm particle sizes, with the

smaller particles offering improvements in resolution.

Non-porous PS/DVB particles are

grafted with a hydrophilic, polymeric layer,

eliminating nonspecific binding, with

multiple ion exchange functional groups per

anchoring providing excellent capacity.

IDs: 4.6 mm, 2.1 mm

Lengths: 50 – 250 mm;

10 mm (stainless guard);

50 mm (PEEK guard)

Particle Sizes: 1.7 µm, 3 µm,

5 µm, and 10 µm

Pore Size: Non-porous PS/DVB

Max Temp: 80°C

pH Stability: 2.0-12.0

Pressure limits: PEEK 400 bar,

Stainless steel 600 bar

Charge-based separations of proteins,

peptides, oligonucleotides, etc.

Bio-Monolith QA (SAX): Adenovirus process monitoring, DNA impurity removal monitoring

Bio-Monolith DEAE (WAX): Process monitoring and quality control of bacteriophage manufacturing, process monitoring

of plasmid DNA purificationBio-Monolith SO3 (SCX): Fast analytical separations of large basic bio-molecules, hemoglobin A1c fast analytics

Polymer-based, spherical monolith HPLCcolumn (100 �µL column volume), with

continuous channels, eliminating diffusionmass transfer of plasmid DNA, viruses,phages and other macro bio-molecules.

Dimensions: 5.2 mm x 4.95 mmMax Temp: 40°CpH Stability: 2.0-13.0Pressure limit: 150 bar

Charge-based separations of macro bio-molecules, process analytics

The h

igh resolution ob

tained from

the superficia

lly

porou

s particle

allow

s better ide

ntifica

tion o

f dige

sted

proteins, faste

r. 1.7 µ

m solid

core wit

h 0.5 µ

m

porou

s oute

r layer

– for a 2.7 µ

m particle

.

Availa

ble in EC-C1

8 and SB-C1

8 phases

for

metho

d deve

lopme

nt flexibility

.

Poroshell 120 reinvents the tech

nology

introduce

d in Poroshell 300 with a unique

single-step porous shell layering process

that further minim

izes molecular

diffusion effects and provides

exceptional perform

ance for

ultra-high-definition LC.

IDs: 2.1 –

4.6 mm

Length

s: 30 –

150 mm

Partic

le Size: 2.7 µ

m

Pore Size: 1

20Å

Max Tem

p: 60° C (EC-C1

8);

90° C (SB

-C18)

pH sta

bility: 2.0-8.0 (EC-C1

8);

1.0-8.0 (SB-C1

8)

Endca

pped: D

ouble

(EC-C

18);

No (SB-C

18)

Optimized for rapid peptide mapping

of antibodies and proteins

Poroshell 300StableBond C18, C8, C3phases provide

excellent stability and selectivity cho

ices with

TFA

and

form

ic acid mobile phases.

The Po

roshell 300Extend-C18

column can be used fro

m

pH 2-10 for a

lternate or unique selectivity for identificatio

n

of proteins/peptides th

at co-elute at low pH.

Superficially porous 300Å particles

made with a thin layer of porous silica

on a solid core of silica, reducing the

diffusion distance for faster m

ass transfer

which results in rapid, high resolution

HPLC separations of peptides and

proteins up to 500-1,000 kDa.

IDs: 0.5 – 2.1

Lengths: 75 mm;

12.5mm (g

uard),

17mm (g

uard)

Particle size: 5 µm

Pore size: 300Å

Max te

mp:

300S

tableB

ond: 90°C

300Extend:

40°C above pH 8,

60°C below

pH 8

pH Stability:

300S

tableB

ond: 1.0-8.0

300Extend: 2.0-11.0

Separations of proteins and

peptides with molecular weight

up to 1000 kDa at low pH

min0 14 16 18 20 22 24 26 28 30 32 34 36 38 4012 10 8 6 4 2

A

B

C

D

E

Optimization of method conditions for the isoform characterization of a monoclonal antibody. Changes in the gradient conditions sharpen peaks and increase resolution of acidic and basic isoforms.

Agilent BioMAbNP10, 4.6 x 250 mm, p/n 5190-2415

Mobile phase: A, 10 mM phosphate, pH 7.5B, A + 0.1M NaCl

Gradient: 25-45% B in 10 min

Flow rate: 0.8 mL/min

Sample: Monoclonal Antibody

Injection: 10 µL (1.5 mg/mL)

Temperature: 25°C

Detection: UV 214 nm

min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -.005

.000

.005

.010

.015

.020

.025

.030

.035

AU

Aggregation analysis of a monoclonal antibody(MAb), the Agilent Bio SEC-3 HPLC columnsprovides baseline separation of the antibodyaggregate and monomer peaks in 15 minutes.

Agilent Bio SEC-3300Å, 7.8 x 300 mm, p/n 5190-2511

Mobile phase: 150 mM Phosphate, pH 7

Flow rate: 1.0 mL/min

Temperature: Ambient

Sample: Monoclonal antibody (10 µL, 5 mg/mL)

min 0 1 2 3 4

1

2

3

4

5 6 7 8 9 10 11 12 13 14 15

Agilent Bio SEC-5300Å, 7.8 x 300 mm, p/n 5190-2526

Buffer: 150 mM Phosphate buffer, pH 7.0

Flow Rate: 1.0 mL/min

Detector: 214 nm

Injection: 10 µL

Even after 300 injections of a five-proteinmixture, the Agilent Bio SEC-5 HPLCcolumns provide reproducible results withminimal peak tailing over time.

Dimer

Monomer

Excipients

Injection No. Efficiency Tailing Line1 26512 1.1922 25959 1.1833 26182 1.18010 25682 1.19020 25872 1.19464 25811 1.187102 25776 1.170140 25910 1.174175 25177 1.208192 24300 1.196250 25707 1.172300 25720 1.166

1. Thyroglobulin

2. BSA

3. Ribonuclease A

4. Uracil

LCBP015Time (min)

420 6 8 10

420 6 8 10

420 6 8 10

Light Chains

Glycosylated Heavy Chains

Treated with DTT

Treated with DTT Peptide-N-Glycosidase F

Treated with DTT Peptide-N-Glycosidase F Carboxypeptidase -B

Courtesy of:Novartis Pharma,Biotechnology, BaselDr. Kurt ForrerPatrik Roethlisberger

0

5

10

15

20

25

30

35

40

45

50

Rel

ativ

e A

bsor

banc

e (m

AU

)

0

20

40

60

80

100

120

% G

radi

ent

1 3

2

0 2 4 6 8 10 12

Time (min)

Time 36 min

Time 158 min

Time 191 min

% Buffer B

Phages andother components

In the late stages of phage particle fermentation, genomic DNA (gDNA) from lysed cells begins todegrade into fragments. These gDNA fragments cannot be removed during purification. Thechromatogram above represents three samples taken from the bio-reactor at 36 min, 158 min and191 min. Peak 1 represents phage, media and host cells, peak 2 the intact gDNA and peak 3 thefragmented gDNA. The fermentation would need to be stopped at 158 min, before the gDNAdegradation begins.

Bio-MonolithDEAE, 5.2 x 4.95 mm, p/n 5069-3636

Mobile phase: A: 125 mM Phosphate buffer, pH 7.0B: 125 mM Phosphate buffer + 1M NaCl, pH 7.0

Gradient: 100% buffer A in 2.5 min,

0-100% buffer B in 10 min,

100% buffer A in 2 min

Flow rate: 1.0 mL/min

Detector: UV at 280 nm

Instrument: High pressure gradient HPLC system, Agilent 1200

Poroshell 300SB-C8, 2.1 x 75 mm, 5 µm, p/n 660750-906

Mobile phase: A: 90% water: 10% ACN + 3 mL/L of MW 300 PEGB: 10% water: 90% ACN + 3 mL/L of MW 300 PEG

Gradient: 0 min 25% B10 min 40% B10.1 min 25% B12 min 25% B

Flow rate: 1.0 mL/min

Temperature: 70° C

Sample: Monoclonal IgG1

min0 2 4 6 8 10 12 14 2216 18 20

1.7 µm

3 µm

5 µm

10 µm

Peak N

Peak N

Agilent Bio IEXWCX-NP, 4.6 x 50 mm, p/n 5190-2441 (WCX-NP1.7), 5190-2443 (WCX-NP3), 5190-2448 (WCX-NP5), 5190-2456 (WCX-NP10)

Buffer A: 20 mM PBS

Buffer B: A+1.0 M NaCl

Gradient: 0-100%B (20 min)

Flow rate: 1.0 mL/min for NP10, NP5, NP3 0.75 mL/min for NP1.7

Sample: 1. Ribonuclease A

2. Cytochrome C

3. Lysozyme

Concentration: 1.0 mg/mL

Detector: 280 nm

Average N~80,000 for WCX-NP1.7

min0 2 4 6 8 10 12 14 2216 18 20 24 26 3428 30 32

Bio SAX, NP104.6 x 50 mmPeak Capacity > 40

Bio SAX, NP54.6 x 50 mmPeak Capacity > 60

Bio SAX, NP34.6 x 50 mmPeak Capacity > 70

The separations above demonstrate how a smaller particle size gives you the flexibility to push for sharper peaks, improved resolutionand higher peak capacities. Smaller ion exchange particles are able to significantly improve the separation of an impurity of lysozyme(top) and provide higher peak capacities when separating a complex lysate sample (bottom).

SAX-NP, 4.6 x 50 mm, p/n 5190-2476 (SAX-NP10), 5190-2468 (SAX-NP5), 5190-2463 (SAX-NP3)

Buffer A: 20 mM Tris, pH 9.0

Buffer B: A + 0.5 M NaCl

Gradient: 0-100%B (30 min)

Flow Rate: 0.5 mL/min

Injection: 10 µL/min (2.5 mg/mL)

Sample: E. coli lysate

Agilent Bio SEC-3

AgilentBio

SEC-5

ZORBAXGF-250/450

Agilent B

ioMAb

Agilent Bio IEX

Bio-Monolith

Poroshell 300

Porosh

ell120

ZORBAX300Å

Extend-C18

ZORBAX 300Å StableBond

SI Z

EEXCL U

S ION

C HR O

M A T O G R A P H Y

I ON

EXCHA

NGE

C H R O M A T O G R A P H Y

REVERSE

PHASE

CHROM

ATOGRAPHY