2009 Column Guide

-Protein Columns -Peptide Columns -Small Molecule Columns -SCX for 2D-Mudpit -Halo Fused-Core Columns -SEC for Removal of Proteins -Specialty Columns Available -Nanoscale -Capillary -Microbore -High Throughput -Various ID’s & Lengths Available

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Table of Contents Contents

Page #

General Information & Tips

3-4

Hardware Types

5

Stationary Phase Types (Summary)

6

Magic Packings

7

Magic Peptide Columns

8-10

Magic Small Molecule Columns

11-13

Magic Protein Columns

14-15

Using Magic Columns

16

Halo Packings

17

Halo Columns

18

Using Halo Columns

19

PLRP-S Packing

20

PLRP-S Protein Columns

21

Using PLRP-S Columns

22

SCX Poly-SEA Columns

23-25

Alternative Ion Exchange Columns

26

Size Exclusion (SEC) Columns

27-28

HILIC Columns

29-30

Trouble-shooting Guide

31

Please note that Michrom offers a large variety of columns (packing materials, ID’s, Lengths, Hardware formats, etc. Please call Michrom at (530) 888-6498 if you are unable to find ordering information for a particular column within this guide.

Magic is a trademark of Michrom BioResources, Inc. PLRP-S, PL-SAX and PL-SCX are trademarks of Polymer Laboratories, Ltd. Poly CAT-A, Poly WAX LP, Poly HEA and Poly SEA are trademarks of PolyLC, Inc. Halo is a trademark for Advanced Material Technologies.

General Information & Tips for Michrom Columns

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The purpose of this guide is to help you successfully use a Michrom column. General information and instructions are included within this guide, as well as detailed information on hardware types, phase types, instructions for proper use, maintenance and trouble-shooting techniques. Column Protection Column lifetime can be extended by following a few simple guidelines: 1. 2. 3. 4.

Use HPLC Grade Solvents, Reagents and Buffers. Filter solvents prior to use with a 0.45µ filter. Change solvents on a regular basis. Do not use larger m.w. analytes than recommended for your columns pore size 5. Do not over-load the column 6. Store your column properly. Pressure and Temperature Limits Michrom columns are packed at high pressures which varies depending on packing material used. Columns packed with polymeric materials (PLRP-S) are designed to withstand pressures to 4000 PSI. Columns packed with silica materials are designed to withstand pressures to 6500 PSI. The recommended temperature range is from 0-50ºC. Chemical Stability The chemical stability of a column is dependant on the phase packed within the column. Since each phase is chemically different, details on the chemical stability of each phase type will be detailed within this manual. Particle Size The particle (bead) size of the stationary phase directly affects the resolution of peaks. Smaller diameter particles generally produce sharper peaks and better resolution. Typically, particles range from 3-10µ for most HPLC applications.

Pore Size Versus Molecular Weight The lifespan of a column is also dependant on the size of analytes loaded onto a column. If too large of an analyte is loaded onto a column, the pores on the column beads can become blocked. If the pores are blocked, the column back pressure will increase causing chromatography problems. Eventually, the column will become completely plugged. To avoid your column from plugging, please use the following guidelines: Pore Size (Å)

Molecular Weight

100Å

100-5000 m.w.

200Å

100-20,000 m.w

300Å

5,000-50,000 m.w

1000Å

10,000-100,000 m.w.

4000Å

20,000-500,000 m.w.

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General Information & Tips for Michrom Columns Column Capacity and Flow Rate Range

Overloading a column will cause poor resolution, poor peak symmetry, and reduce the lifespan of the column. Use the appropriate column ID for sample size and complexity (see chart below for guidelines). Complex sample mixtures require higher capacity columns. Single analyte capacity is much less than total analyte capacity. Column ID (mm)

Flow Rate Range* (µl/min)

Optimal Flow Rate (µl/min)

Capacity Range For Complex Mixtures (ug)

Capacity Range Of a Single Analyte (pmol)

0.075

0.125-1.25

0.25

0.5-2

0.05-0.125

0.1

0.25-2.5

0.5

1-4

0.1-0.25

0.15

0.5-5.0

1.0

2-8

0.2-0.5

0.2

1.0-10.0

2.0

4-16

0.4-1

0.3

2.0-20.0

5.0

10-40

1-2.5

0.5

5.0-50.0

12.0

25-100

2.5-10

1.0

20.0-200

50.0

100-400

10-40

2.0

100-1000

200

400-1600

40-160

Micro Bullet

100-1000

500

400-1600

40-160

Macro Bullet

500-5000

2000

1600-6400

160-640

*Note: Flow rate range of a column is dependant on column size and back pressure limits.

Column Storage Proper column storage will increase the lifespan of a column. Whereas, improper column storage will reduce the lifespan dramatically. Prior to storing your column, flush it for approximately 30 minutes with the storage chemical recommended for that phase type. After removing the column from the instrument, screw the provided plugs into both ends of the column to insure the column will not dry out during the storage period. Performance Testing and Shipping A column test certificate and a column test mixture are supplied with each Reversed-Phase (RP) and Strong Cation Exchange (SCX —Poly-SEA Only) Column. Columns packed for other modes of HPLC such as Normal Phase (NP), Ion-exchange (IE), Hydrophilic Interaction (HI) are tested for proper back pressure and screened for packing efficiency but are not provided with a test certificate. Technical Assistance It is our intent to include the answers to the most commonly asked questions regarding columns within this user guide. If you have a question that is not covered or you simply need further details, please do not hesitate to contact technical support. Michrom has a staff of qualified and experienced HPLC and LC/MS specialists that are available Monday through Friday to help you with any questions that you may have regarding the use of our products. Technical Support Phone: (530) 888-6498 Fax: (530) 888-8295 Email: [email protected]

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Column Hardware Types MS Certified Capillary & Nano Columns for Any HPLC (CP Series) Capillary HPLC columns made of PEEKSIL with titanium screens to ensure biocompatibility and durability. For use in any HPLC or LC/MS systems with conventional 1/16” 10-32 inlet and outlet Upchurch connectors. −Available in 0.5 mm, 0.3 mm, 0.2 mm, 0.15mm, 0.1mm, and 0.075mm ID and lengths of 50, 100 and 150mm

Nano & Capillary Columns for Any HPLC (CN or CC Series) Nanoscale HPLC columns made of fused silica sheathed with PEEK with titanium screens to ensure biocompatability. For use in any HPLC or LC/MS systems with conventional 1/16” 10-32 inlet and outlet Upchurch connectors. −Available in 0.5 mm, 0.3 mm, 0.2 mm, 0.15mm, 0.1mm, and 0.075mm ID and lengths of 50, 100 and 150mm

Nano Columns for Michrom NanoTrap Platform (CL Series) Nanoscale HPLC columns made of fused silica sheathed with PEEK with titanium screens adhered to each end to ensure biocompatability. For use on Michrom’s NanoTrap Platform Only (Valco Nuts & Ferrules at each end). −Available in 0.1 and 0.075 mm ID and lengths of 50, 100, and 150mm Note: No end-fittings are present on CL Series Columns.

Nano Spray Tip Packed Columns (ST Series) Nanoscale HPLC columns made of fused silica with a pulled-tip end for direct nanospray LC/MS. For use in LC/MS systems with a conventional 1/16” 10-32 inlet connector and nanospray source. −Available in 0.1 and 0.075 mm ID and bed lengths of 150, 200 and 250mm

Microbore Columns for Any HPLC (CM Series) Microbore HPLC columns made of glass lined stainless steel with titanium frits to ensure biocompatability. For use in HPLC systems with a conventional 1/16” 10-32 inlet and outlet connectors. -Available in 2.0, 1.0 and 0.5 mm ID and lengths of 50 and 150mm

Magic Bullet Columns for Any HPLC (CB Series) The Michrom Magic BulletΤΜ is made from injection molded PEEK (with a continuously tapered bore over its 25 mm length) and titanium frits. The Magic BulletΤΜ fits into a 316SS holder with Holder standard 10-32 female end fittings for use in any HPLC or LC/MS system. -Available in 2 sizes:

Micro (ID 2mm→0.5mm) or Macro (ID 4mm→1mm) Bullet

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Stationary Phase Types

Michrom offers a wide range of premium HPLC column packings as detailed on the following pages. These materials are packed in Michrom’s custom column hardware under license from the packing manufacturers. Reversed-Phase—Proteins, Peptides, and Small Molecules Reversed-Phase (RP) chromatography is by far the most widely used technique in HPLC. It is popular because it is applicable to most nonpolar analytes and to many ionizable and ionic compounds, such as peptides and proteins. Most of the RP stationary phases are hydrophobic in nature. Analytes are separated by their degree of hydrophobic interaction using an increasing gradient of organic solvent (typically Acetonitrile). Michrom offers a variety of RP materials to meet your separation needs. Analyte Type Recommended Page # Stationary Phase Protein

PLRP-S

21

Peptide

Magic C18 AQ

8

Magic C18

11

Halo

18

Small Molecule All

Ion Exchange—For 2D Applications (i.e. MUDPIT) Ion exchange chromatography is designed for the separation of ionic substances (positively or negatively charged). There are 2 types of ion exchange: cation exchange and anion exchange. Cation exchangers have acidic groups attached to the stationary phase with H+ exchangers. Anion exchangers have basic groups attached to the stationary phase with X– exchangers. Ion Exchange Chromatography is commonly used as the 1st dimension in a 2D application. Analyte Type

Peptides Misc.

Recommended Stationary Phase

Page #

Poly-SEA (SCX)

23

Alternative Phases

26

Size Exclusion—Removal of Proteins from Plasma/Serum/Urine Size exclusion chromatography is designed for the separation of analytes based on size and shape. Phases are typically gels or controlled pore materials. Molecules exceeding the “critical size” are not retained. Larger molecules elute first. Size exclusion chromatography can be used as a 1st dimension in a 2D analysis to exclude unwanted analytes based on molecular weight, such as the removal of proteins from a urine sample. Analytes to Exclude Proteins

Analytes of Choice

Recommended Stationary Phase

Page #

Small Molecules or Peptides

Poly-HEA (DSEC)

27

Hydrophilic Interaction Chromatography—HILIC Hydrophilic interaction chromatography is a variant of Normal Phase Chromatography. It depends on the stationary phase having very polar properties while the mobile phase is mostly organic. Analytes stick to the stationary phase at high organic (200°C) superheated water as an eluent without fear of damage to the stationary phase. Outstanding Chemical Stability PS/DVB macroporous packings, such as PLRP-S, are inherently and uniformly hydrophobic, and do not necessitate bonded ligands, unlike alkyl-functionalized silica reversed phase materials. Polymeric PLRP-S materials do not possess residual surface functionalities, and do not therefore suffer from the typical silica problems of acidic silanol groups or other ionic species, which can interfere with the separation performance of the matrix and are difficult to remove entirely and reproducibly by endcapping. Unsurpassed pH Stability of Polymeric Media PLRP-S columns are designed for easy use across the pH spectrum from 1 to 14, with none of the restrictions associated with silica packings. Polymeric packings offer the convenience of separations at the extremes of pH. • • • • •

available in available in available in available in available in

3µ 100 Å (for Small Molecules) 5µ 100 Å (for Small Molecules) 5µ 300 Å (for Peptides or Small Proteins) 5µ or 8µ 1000 Å (for Small-Medium Proteins) 5µ or 8µ 4000 Å (for Proteins of all sizes)

+ESI & -ESI High Throughput Screening of Lead Drug Candidates On a Magic Bullet Column packed with 5µ 100 Å PLRP-S These LC-TOF/MSD traces show separation of intact proteins from two EDGE fractions of rat liver lysate on a 0.1x50mm column run at 1 ul/min.

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Reversed-Phase: PLRP-S Protein Columns

PLRP-S 8µ 4000Å is our most popular material for protein separations. The 4000Å material has proven to be very robust due to less clogging as compared to smaller pore size materials. Proteins as small as 5kD and as large as 500kD can be separated with these columns. For smaller bore columns (