Thermo Fisher Scientific Packing and Testing Conditions Instruction Sheet

Type
Instruction Sheet
Instruction Sheet
1
Read Me Second!
Before you read this document, please read the document entitled
POROS Self Pack® Packing Device for High Performance Perfusion
Chromatography Columns (shipped with the packing device). Then
read these instructions, which are a supplement to the instructions
provided with the packing device.
For the media you have purchased, this instruction sheet tells you:
The specific slurry conditions and packing conditions to use on
Perfusion Chromatography® systems (BioCAD® Workstation
or BioCAD SPRINT System) or FPLC® systems.
The specific slurry conditions and all packing conditions,
except flow rate, to use on other HPLC systems. (The POROS
Self Pack Packing Device instruction sheet explains how to
determine the packing flow rate.)
The expected performance of a properly packed column.
1 Preparing the Slurry
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): 0.5 M NaCl
Packing solvent: 0.1 M NaCl
1.2 Slurrying the Media
The bottle of dry POROS media contains enough media to give the
correct slurry density to pack the number of columns shown in Table 1.
Table 1 Number of Columns/Solvent
Requirements
To guarantee proper results, slurry the entire bottle even if you will not
use all the slurry at once. Do not divide the powder into portions.
To slurry the media:
1. Pour the required volume (see Table 1) of slurry solvent into the
media bottle.
2. Swirl the bottle gently until the material is well suspended.
When you tilt the bottle, no powder should remain on the bottom.
The slurry should have a milky appearance and consistency.
3. Store unused media slurry in the refrigerator.
Note: Add to the media bottle 20 µl of a 1.0% sodium azide stock
solution (a preservative) per 1 ml of unused slurry (final azide
concentration is 0.02%).
CHEMICAL HAZARD. 1% Sodium azide in water is a
poison. It may be fatal if inhaled, swallowed, or absorbed through the
skin. Exposure may cause nerve and heart damage. Contact with
acids liberates toxic gases. DO NOT ADD acids to any liquid wastes
containing sodium azide. Sodium azide may react with lead and
copper plumbing to form highly explosive metal azides. Read the
MSDS, and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
Media
Quantity
Number of Columns Required
Volume (ml)
4.6 mmD/
50 mmL 4.6 mmD/
100 mmL Slurry
Solvent
(0.8 g) 2 1 12
(2.7 g) 6 3 36
DANGER
!
Packing and Testing Conditions for
Self Pack® POROS® 20 S Media
2
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 the section on 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 the
section on 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 the section on
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 the section on 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 the section on 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 the section on 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 the section on
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 the packing solvent 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 Eluent A (see Table 3). (Bed volume of a
4.6 mmD/50 mmL column is 0.8 ml; bed volume of a 4.6 mmD/
100 mmL column is 1.66 ml.) Then run the protein test standard under
the conditions shown in Table 3.
To prepare the protein test standard:
1. Dissolve the test standard in 1 ml of Eluent A (concentration
4 mg/ml bovine pancreas ribonuclease A, 1 mg/ml chicken egg
lysozyme).
2. Filter the test standard using a 0.22 µ filter.
3. Store unused reconstituted test mix frozen.
3.4 Results
Compare your chromatogram with Figure 1 (generated on a
4.6mmD/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 the 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.
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 9 35
Other systems
4.6 mmD/50 mmL 6 *** 35
4.6 mmD/100 mmL 12 *** 35
Table 3 Protein Test Conditions
Test Standards Cation Exchange Protein Test Standards
(PN 1-9003-00)
Sample Size 20 µl
Eluent A 20 mM MES pH 6.2
Eluent B 0.5 M NaCl in A
Gradient 0100% B in 5 minutes
Flow rate 5 ml/min
Detection 280 nm
3
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 the section on unpacking a column, in the
POROS Self Pack Packing Device instruction sheet).
Figure 1 POROS 20 S Chromatogram
Ribonuclease
Lysozyme
Headquarters
850 Lincoln Centre Drive
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Phone: +1 650.638.5800
Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884
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support and applications personnel, reaches
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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 8-0055-40-1293 Rev. A
© Copyright 1995, 2001, Applied Biosystems. All rights reserved.
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.
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Thermo Fisher Scientific Packing and Testing Conditions Instruction Sheet

Type
Instruction Sheet

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