-
HPLC2006Presentation �HPLC2006Presentation
K. Flook, S. Rao, J. Thayer, S. Xie, and C. A. Pohl, Dionex
Corporation, Sunnyvale, CA, USA
High Throughput Liquid Chromatography UsingPolymeric ProSwift
Monolithic Bioseparation ColumnsHigh Throughput Liquid
Chromatography UsingPolymeric ProSwift Monolithic Bioseparation
ColumnsK. Flook, S. Rao, J. Thayer, S. Xie, and C. A. Pohl, Dionex
Corporation, Sunnyvale, CA, USA
INTRODUCTIONSpeed and resolution are two competing performance
factors in chro-matography using conventional porous separation
media. One feature is often achieved while the other is sacrificed.
This is often the case during separation of proteins and peptides,
since these large molecules exhibit low diffusivity which
translates into higher mass transfer resistance. The net result is
significant peak broadening especially at higher flow rates.
A new generation of styrene-based reversed-phase separation
media from Dionex, ProSwift™ polymer monoliths, is designed to
address the above problem. They contain a network of large pores in
which separation is achieved primarily by convective flow rather
than diffusive flow. Highly improved mass transfer rates allow high
speed analysis without band broadening or significantly sacrificing
resolution thereby improving throughput and productivity. The new
ProSwift phases use a patented process to produce a well defined
polymer monolith. An in-situ polym-erization process allows
excellent batch-to-batch reproducibility and column stability.
Separations can be achieved at high flow rates without loss of
resolu-tion. These columns have an excellent longevity and are
stable over a wide pH range.
We discuss the performance of three columns; two recently
introduced phases, RP-2H and RP-3U, as well as our newest
introduction, RP-1S. Ex-cellent separation of a wide variety of
proteins and peptides is shown here.
OBJECTIVES• Characterize the morphology and pore structure of
the ProSwift
monoliths.• Investigate the chromatographic properties of the
ProSwift monoliths.• Develop applications for proteomic
research.
Eluents: (A) DI H2O / CH3CN (95:5 v/v) + 0.1% TFA (B) DI H2O /
CH3CN (5:95 v/v) + 0.1% TFAInjection: 10 µL (unless otherwise
stated)
Figure 1. Manufacturing Process
• Column efficiency of the monolith base support is controlled
by a one-step polymerization process.• The simplicity of a one-step
process results in highly reproducible products.
Monomers
Porogen
InitiatorPolymerization Wash
23133
Surface Modification
Reversed-PhaseColumn
Ion-Exchange Column
Figure 2. SEM Images of ProSwift Monoliths
Through Pores
ProSwift RP-1S
23138
ProSwift RP-2H
ProSwift RP-3U
High permeability Low backpressure High flow rate
Flow through pores Fast mass transfer High resolution
Figure 3. Peptide Separation on ProSwift RP-1S
0 1 2 3 4 5 6 7 8 9 10 11 12–10
600
mAU
Minutes
• ProSwift RP-1S allows separation of a wide range of molecular
weights.
23217
4 mL/min
1 mL/min
Sample: Standard Peptide Mix (Sigma # H2016) 1. Gly-Tyr (MW
238.2) 2. Val-Tyr-Val (MW 379.5) 3. Methionine Enkephalin Acetate
(Tyr-Gly-Gly-Phe-Met) (MW 573.7) 4. Leucine Enkephalin
(Tyr-Gly-Gly-Phe-Leu) (MW 555.6) 5. Angiotensin II acetate
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (MW 1046.2)Injection: 5 µL
Gradient: 1 - 25% B in 6 min at 1 mL/min, 1 - 25% B in 1.5 min
at 4 mL/min
AnutaNew Stamp
-
2
HighThroughputLiquidChromatographyUsingPolymericProSwiftMonolithicBioseparationColumns
Figure 5. Protein Separation on ProSwift RP-2H
0 1 2 3 4 5 6 7 8 9 10 11–300
600
mAU
Minutes
• ProSwift RP-2H column’s faster separation enables higher
productivity.
22903
Protein standards
8 mL/min4 mL/min
2 mL/min1 mL/min
Sample: 1. Ribonuclease A (1.5 mg/mL) 2. Cytochrome c (0.5
mg/mL) 3. BSA (1.5 mg/mL) 4. Carbonic Anhydrase (0.9 mg/mL) 5.
Ovalbumin (1.5 mg/mL)Injection: 5 µL
Gradient: 1% B for 2 min, 1 - 75% B in 6 min at 1 mL/min
1% B for 1 min, 1 - 75% B in 3 min at 2 mL/min
1% B for 0.5 min, 1 - 75% B in 1.5 min at 4 mL/min
1% B for 0.25 min, 1 - 75% B in 0.75 min at 8 mL/min
0 2 4 6 8 10 12 14–10
500
mAU
Minutes23134
1
2
34
56
7
8
9
10
11
12
13
14
Figure 6. Cytochrome c Tryptic Digest Separation on ProSwift
RP-1S
Flow: 1.0 mL/min
Sample: Cytochrome c Tryptic Digest (2 mg Cytochrome c, 20 µg
trypsin per mL) Injection: 20 µL
Gradient: 1% B for 2 min, 1 - 50% B in 12 min
• High resolution allows easy separation of digest peptides.
Minutes0 2 4 6 8 10 12 14 16.1
–100
300
mAU
Flow = 1 mL/minFlow = 4 mL/min
22909
Figure 7. Separation of Snake Venom Proteins/Peptides• The
ProSwift RP-2H column’s uniquely engineered pore size allows
separation of samples with a wide molecular weight range.
Gradient: 1 - 75% B in 12 min at 1 mL/min 1 - 75% B in 3 min at
4 mL/min
Minutes0 2 4 6 8 10 12 14 16
–200
1,200
mAU
BSA
IgG
22908
Figure 8. Separation of IgG and Albumin from Bovine Serum• The
ProSwift RP-2H column has large flow through channels that result
in high resolving power for large proteins.
Flow = 1 mL/minFlow = 4 mL/min
Sample: 1 part Bovine Serum, 4 parts Eluent A
Gradient: 1 - 75% B in 12 min at 1 mL/min 1 - 75% B in 3 min at
4 mL/min
Figure 9. Batch-To-Batch Reproducibility of ProSwift RP-2H
0 1 2 3–10
mAU
Minutes23135
5
2
1
4
ProSwift reversed-phase media are manufactured by a patented in
situ manufacturing process which has the least number of variables
affecting the reproducibility among all technologies. It does not
require additional sieving, coating, multiple surface modification,
and packing processes.
Flow: 2.5 mL/min
Sample: 1. Angiotensin II (30 µg/mL) 2. Substance P (30 µg/mL)
3. Ribonuclease A (30 µg/mL) 4. Cytochrome C (30 µg/mL) 5. BSA (30
µg/mL)
Gradient: 1 - 60% B in 3 min
100
3
Figure 4. Peptide and Protein Separation
RP-3U
RP-2H
RP-1S
0 2 4 6 8 10 12–5
250
mAU
Minutes23132
Flow: 1.5 mL/min
Sample: 1. Methionine Enkephalin acetate (Tyr-Gly-Gly-Phe-Met)
(MW 573.7) 2. Leucine Enkephalin (Tyr-Gly-Gly-Phe-Leu) (MW 555.6)
3. Angiotensin II acetate (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (MW
1046.2) 4. Physalaemin (Glu-Ala-Asp-Pro-Asn-Lys-
Phe-Tyr-Gly-Leu-Met) (MW 1265.4) 5. Substance P acetate
(Arg-Pro-Lys-Pro- Gln-Gln-Phe-Phe-Gly-Leu-Met) (MW 1347.6) 6.
Ribonuclease A 7. Cytochrome C 8. Carbonic Anhydrase 9. Bovine
Serum Albumin 30 µg/mL each
Gradient: 1 - 64% B in 17 min
-
HPLC2006Presentation �
Figure 10. Robustness of the ProSwift RP-1S
Injection 6
Injection 506
Injection 206
Injection 106
Injection 306
Flow: 4 mL/min
Sample*: 1. Ribonuclease A (1.5 mg/mL) 2. Cytochrome c (0.5
mg/mL) 3. BSA (1.5 mg/mL) 4. Carbonic Anhydrase (0.9 mg/mL) 5.
Ovalbumin (1.5 mg/mL)Injection: 5 µL
Gradient: 1% B for 1 min, 1 - 75% B in 2.5 min
*Sample is injected every 50 gradient cycles
2,000
–20
mAU
3210 4Minutes
23136
• Durability and robustness are ProSwift characteristics. The
ProSwift monoliths exhibit stability and reproducibility over many
runs.
Figure 11. pH Stability of the ProSwift RP-3U
No treatment
0.1M HCl48 hrs
1M NaOH48 hrs
The ProSwift RP monolith column is stable with treatment of 1 M
NaOH and 0.1 M HCl in the regeneration process. It is shown to be
stable at pH 2 to 12 at normal temperature and flow for long
periods. This figure shows the resolution of four protein standards
before and after base and acid regeneration treatments.
Flow: 4 mL/min
Sample: 1. Ribonuclease A (0.1 mg/mL) 2. Cytochrome c (0.05
mg/mL) 3. BSA (0.1 mg/mL) 4. Carbonic Anhydrase (0.1 mg/mL)
Gradient: 15 - 80% B in 3 min
325
0.6 0.90.3 1.2Minutes
23137
0–5
mAU
1.5
CONCLUSIONCharacteristics of Polymeric Monolithic Columns • High
resolution • High flow velocity and low backpressure • High
capacity • Highly reproducibility • Much more chemically stable
than silica-based media
Applications • High-throughput, high-resolution separation of
proteins,
peptides, and other biomolecules
REFERENCEhttp://www1.dionex.com/en-us/columns_accessories/life_science_pharma/cons35517.html
ProSwift is a trademark of Dionex Corporation.
0
500
1000
1500
2000
2500
3000
3500
0 2 4 6 8 10
Colu
mn
Back
pres
sure
(psi)
ProSwift RP-1S
ProSwift RP-2H
ProSwift RP-3U
Polymer Beads, 8 µm
Figure 12. Column Backpressures
• The backpressures of ProSwift RP-2H and RP-3U columns are
significantly lower than columns packed with beads. This allows the
use of a higher flow rate for a much faster separation.
• The ProSwift RP-1S column can be operated at higher flow rates
than columns packed with polymer beds without compromising the
column.
Eluent: 30% B
Flow Rate (mL/min)23131
North America
Sunnyvale, CA (408) 737-8522 Westmont, IL (630) 789-3660
Houston, TX (281) 847-5652 Atlanta, GA (770) 432-8100 Marlton, NJ
(856) 596-0600 Canada (905) 844-9650
Europe
Austria (43) 1 616 51 25 Belgium (32) 3 353 4294 Denmark (45) 36
36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 210 Italy
(39) 06 66 51 5052 The Netherlands (31) 161 43 43 03 Switzerland
(41) 62 205 99 66 United Kingdom (44) 1276 691722
Asia Pacific
Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22
28475235 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580
LPN 1862-01 06/06©2006 Dionex Corporation
Dionex Corporation
1228 Titan Way P.O. Box 3603 Sunnyvale, CA 94088-3603 (408)
737-0700
Passion. Power. Productivity.
www.dionex.com