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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

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

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www.dionex.com