Thermo Fisher Scientific Packing and Testing Conditions Instruction Sheet

Brand: Thermo Fisher Scientific

Model: Packing and Testing Conditions

Type: Instruction Sheet

Instruction Sheet

Read Me Before You Read the

Packing Device Document!

Use this instruction sheet in conjunction with the document titled

POROS Self Pack Packing Device for High Performance Perfusion

Chromatography Columns (shipped with the packing device).

Note: Section 2 of this instruction sheet replaces Section 4.3 of the

POROS Self Pack Packing Device instruction sheet.

Begin by reading the POROS Self Pack Packing Device instruction

sheet. When you reach Section 4.3, switch to Section 2 of this

instruction sheet.

Instructions at the end of Section 2 direct you back to the appropriate

section of the POROS Self Pack Packing Device instruction sheet.

Contents of this Instruction Sheet

For the media you have purchased, this instruction sheet tells you:

•The specific slurry conditions, packing conditions, and packing

instructions for Perfusion Chromatography® systems

(VISION™ or BioCAD® Workstation or BioCAD® SPRINT™

System) or FPLC® systems.

•The specific slurry conditions, all packing conditions except

flow rate, and packing instructions for other HPLC systems.

•The expected performance of a properly packed column.

1 Preparing the Slurry

Note: POROS 20 HE media is packaged in slurry form, using

20% ethanol as a preservative. This slurry is not ready for use in the

packing device. You must prepare the slurry before using it in the

packing device.

This section tells you what solvents to use and how to slurry the media.

1.1 Solvents

Warning: Always wear eye protection when working with solvents.

Use these solvents:

•Slurry solvent (to slurry the media): 0.5M NaCl

•Packing solvent: 0.1M NaCl

1.2 Slurrying the Media

The bottle of POROS media slurry contains enough slurry, when

prepared, to pack the following number of columns:

Table 1 Number of Columns

To prepare the media:

1. Swirl the media bottle gently until the beads are well suspended.

2. Pour the required amount of slurry into a scintered glass funnel:

•For a 4.6 mmD/50 mmL column: 3 ml

•For a 4.6 mmD/100 mmL column: 6 ml

3. Store any slurry remaining in the original media bottle

at 4°C.

4. Wash the media in the scintered glass funnel once with water to

remove the ethanol.

5. Wash the media three times with the new slurry solvent.

6. Resuspend the media into the required volume of new slurry

solvent:

•For a 4.6 mmD/50 mmL column: 6 ml

•For a 4.6 mmD/100 mmL column: 12 ml

Media Quantity

Number of Columns

4.6 mmD/50 mmL 4.6 mmD/100 mmL

(0.8 g) 2 1

(2.7 g) 6 3

Packing and Testing Conditions

for Self Pack® POROS® 20 HE Media

2 Packing Conditions

If you are packing a column using a Perfusion Chromatography system

or a conventional HPLC system, install a backpressure regulator on

your column. See Using the Backpressure Regulator, in the POROS

Self Pack Packing Device instruction sheet for information.

See Table 2 for packing conditions for your system.

Note: Set system pressure before you pack your column. See

Preparing Your LC System, in the POROS Self Pack Packing Device

instruction sheet for information.

*After you add this media slurry to the device, top the device off with slurry

solvent as described in Filling the Packing Device, in the POROS Self Pack

Packing Device instruction sheet.

**To keep within the pressure specifications of FPLC pumps, program your FPLC

system to deliver the flow rate with pump A and B (set “Conc % B” to 50 and

prime both pumps with packing solvent).

***See Packing a Column Using Other Systems in the POROS Self Pack Packing

Device instruction sheet to determine the packing flow rate.

Note: You may need to reduce the flow rate because of variations in frit

permeability or system backpressure. See Packing a Column Using

Other Systems in the POROS Self Pack Packing Device instruction

sheet for more information.

3 Packed Column Performance

This section describes:

•The maximum recommended flow rate for your packed column

•How to do a permeability test on your packed column

•The conditions necessary to run a protein separation test on

your packed column

You can verify your success each time you pack a fresh column and

monitor column performance over time. See Section 4.6, Testing the

Column, in the POROS Self Pack Packing Device instruction sheet for

information about periodic testing.

3.1 Recommended Maximum Flow Rate

The maximum recommended flow rate for the column during normal

operation is 85% of the packing flow rate. This flow rate keeps the

pressure within the operating limit you recorded in Packing the

Column, in the POROS Self Pack Packing Device instruction sheet.

When you work with viscous solvents, lower the operating flow rate to

account for the greater pressures generated by the greater viscosity.

3.2 Permeability

Column pressure/flow characteristics are called column permeability.

Test column permeability at the recommended maximum flow rate to

establish a baseline.

To test column permeability:

1. Run 50 mM phosphate pH 7.0/0.15 M NaCl buffer through the

column at the recommended maximum flow rate.

2. Record the generated pressure (permeability baseline).

Whenever you re-test column permeability, do so under solvent and

flow rate conditions identical to those of this initial test.

3.3 Protein Separation

Before you run the protein separation test, equilibrate the column with

10 to 15 column volumes of buffer (see Table 3). Column bed volumes

are:

•For a 4.6 mmD/ 50 mmL column: 0.8 ml

•For a 4.6 mmD/100 mmL column: 1.66 ml

Then run the protein test standard under these conditions:

To prepare the protein test standard:

1. Create the test standard by dissolving 5 mg bovine serum albumin

and 5 mg lysozyme in 1 ml of buffer.

2. Filter the test standard using a 0.22 µ filter.

3. Store unused reconstituted test mix frozen.

Table 2 Packing Conditions

Volume

of media

slurry to

add to

device

(ml)*

Recom-

mended

initial

packing

flow rate

(ml/min)

Volume of

packing

solvent to pass

through

column during

packing

(ml)

Perfusion Chromatography systems

4.6 mmD/50 mmL 6 20 35

4.6 mmD/100 mmL 12 20 35

FPLC® systems**

4.6 mmD/50 mmL 6 10 35

4.6 mmD/100 mmL 12 10 35

Other systems

4.6 mmD/50 mmL 6 *** 35

4.6 mmD/100 mmL 12 *** 35 Table 3 Protocol

These conditions are common to all column sizes:

Eluent A 20 mM MES, pH 5.5

Eluent B 1 M NaCl in A

Flow rate 5 ml/min for FPLC system

10 ml/min for BioCAD Workstation

Sample 20 µl

Gradient 0–100% B in 10 column volumes

Detection 280 nm

3.4 Results

Compare your chromatogram with Figure 1 (generated on a

4.6 mmD/100 mmL column packed with a Perfusion Chromatography

system). To verify that your column is operating properly, confirm that

the two proteins on your chromatogram are separated and the peaks

are symmetrical. The areas of the two peaks may change according to

the purity of the proteins in your standard, but variation in area is not

important in measuring the resolving power of the column. Retention

times and bandspreading on your separation may be different but the

general profile should be similar.

If the general profile is not similar, make sure that your system is

functioning properly. If your system is functioning properly, unpack your

column and start again (see Unpacking a Column, in the POROS Self

Pack Packing Device instruction sheet).

Figure 1 A POROS 20 HE Chromatogram

Headquarters

850 Lincoln Centre Drive

Foster City, CA 94404 USA

Phone: +1 650.638.5800

Toll Free (In North America): +1 800.345.5224

Fax: +1 650.638.5884

Worldwide Sales and Support

Applied Biosystems vast distribution and

service network, composed of highly trained

support and applications personnel, reaches

into 150 countries on six continents. For sales

office locations and technical support, please

call our local office or refer to our web site at

www.appliedbiosystems.com.

www.appliedbiosystems.com

Applera Corporation is committed to providing

the world’s leading technology and information

for life scientists. Applera Corporation consists of

the Applied Biosystems and Celera Genomics

businesses.

Printed in the USA, 08/2001

Part Number 8-0147-40-1195 Rev. A

For Research Use Only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice.

Applied Biosystems assumes no responsibility for any errors that may

appear in this document. This document is believed to be complete and

accurate at the time of publication. In no event shall Applied Biosystems

be liable for incidental, special, multiple, or consequential damages in

connection with or arising from the use of this document.

POROS products and perfusive chromatography are covered by U.S.

patents 5,552,041; 5,605,623; and 5,833,861; foreign patents pending.

Chromatography materials of certain pore geometries, including certain

wide-pore supports, can permit perfusive chromatography, that is,

separations in which the rate of intraparticle convection exceeds the

rate of intraparticle diffusion. Use of any such chromatography media at

sufficiently high linear velocity, without license, may constitute patent

infringement. A limited license to use the patented perfusive

chromatography process is granted with the purchase of POROS

products from Applied Biosystems. The license terminates upon

expiration of the useful life of the product.

Subtractive Assay technology, enabled by the use of ImmunoDetection

(ID) Sensor Cartridges and the INTEGRAL Micro-Analytical

Workstation, is covered by U.S. patent 5,234,586. Other patents

pending.

Applied Biosystems, BioCAD, ImmunoDetection, INTEGRAL, Perfusion

Chromatography, POROS, and Self Pack,are registered trademarks of

Applera Corporation or its subsidiaries in the U.S. and certain other

countries.

AB (Design), Applera, SPRINT, and VISION are trademarks of

Applera Corporation or its subsidiaries in the U.S. and certain other

countries.

FPLC is a registered trademark of Pharmacia LKB Biotechnology AB.

All other trademarks are the sole property of their respective owners.

Thermo Fisher Scientific Packing and Testing Conditions Instruction Sheet

Brand: Thermo Fisher Scientific

Model: Packing and Testing Conditions

Type: Instruction Sheet

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Thermo Fisher Scientific Packing and Testing Conditions Instruction Sheet

Thermo Fisher Scientific Packing and Testing Conditions Instruction Sheet

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