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APPENDIX
APPENDIX
Guarding your column
GLP procedures demand that the separation column be protected by
a guard column. Tosoh Bioscience supplies an assortment of packed
guard columns and guardgel kits. Guardgel kits contain the hardware
and the gel packing material to fill a guard column using an aspirator.
For those columns where a guard column is not available, Tosoh
Bioscience recommends the use of an in-line filter with a 0.5 µm cut-
off to avoid frequent plugging of the 1.0 µm pores in the column frit. A
pre-injector membrane filter is also recommended to prevent particles
generated by pump seal wear from reaching the column.
Rehydration
Dehydration of TSKgel liquid chromatography columns can occur during
long-term storage or from improper use. Dehydration can also occur
if the plugs are not tightened or if air inadvertently is pumped into the
column during use. It is easier to detect dehydration in glass columns
because the dry packing will appear to pull away from the column walls.
This condition can be remedied by using the following procedure:
1.
Connect the column to your LC system in the reverse flow
direction.
2.
Do not connect the column to the detector.
3.
Pump a filtered mobile phase of 20 % methanol in ultrapure
water over the column at half of the recommended
maximum flow rate.
Note: reversed phase columns require 60 % methanol.
4.
Continue this procedure until the column has been
rehydrated. Rehydration can take several hours, depending
on the column size.
5.
Connect the column to the LC system in the proper flow
direction.
6.
Rinse with 3 column volumes (CV) of ultra pure water to remove
the organic if it is not part of the normal mobile phase.
7.
Equilibrate with loading buffer (usually 3-5 CV).
8.
Perform the recommended QC tests to ensure that the column
is performing properly. Evaluation methods are available from
the Technical Service Department of Tosoh Bioscience.
Column Storage
When the column will be used the next day, allow it to run overnight at
a low flow rate in a buffer that does not contain a halide salt. When the
column will not be used for more than a day, clean it first, then flush salt
from the column and store in 0.05 % sodium azide or 20 % ethanol. Seal
tightly to prevent the column from drying out.
Scaling Up
for Size Exclusion Chromatography
Tosoh Bioscience offers semi-preparative (21.5 mm ID), preparative
(55 mm ID), and larger ID stainless steel columns packed with TSKgel
SW-type or PW-type resin for seamless scale-up to commercial
production of therapeutic proteins and other biopharmaceuticals.
These packing materials have a larger particle size that is appropriate
for use in process scale equipment. The packing materials, however,
have the same pore size and provide the same selectivity as the
corresponding TSKgel analytical column. The column volume (CV) of
the preparative column that is needed to produce the required amount
of product (per injection) is given by the relationship:
(CV)pc / (CV)ac = (mg product)pc / (mg product)ac
in which pc and ac refer to the preparative and analytical column
respectively. The volume of a column is equal to 1 /4
π
(ID)
2
L, in which
ID is the internal diameter and L the length of the column. In scaling up,
column length (L) is usually kept constant. If so, to achieve a 100-fold
increase in product per run, the ID of the prep column should be 10
times larger than that of the analytical column. As noted, the particle
size in the preparative column is usually larger, and one should select
a larger ID column than predicted by the above equation. As a rule of
thumb, a 2-fold increase in particle size reduces resolution and thus
output by the square root of 2.
Since scale-up from analytical columns is relatively straightforward,
preparative TSKgel SW columns may be an economical route for the
rapid production of biomolecules for clinical testing. See the SEC
section of this catalog for more information and request a copy of the
process media catalog. For more detailed analysis of your scale-up
requirements, please contact Tosoh Bioscience’s Technical Service
Specialists .
Scaling Up
for Hydrophobic Interaction and Ion Exchange
Chromatography
Tosoh Bioscience provides various ID preparative columns for
hydrophobic interaction (HIC) and ion exchange (IEC) chromatography.
As shown above, to calculate the sample capacity of a larger column,
multiply the capacity obtained on a 7.5 mm ID column by the ratio of the
column volumes. The table below lists the column volumes for TSKgel
HIC and IEC columns and their ratios relative to the 7.5 mm ID x 7.5 cm L
column.
Dimensions
(mm ID x cm L)
Volume (mL)
Volume ratio*
5 x 5
1.0
0.3
7.5 x 7.5
3.3
1.0
8.0 x 7.5
3.8
1.2
20 x 15
47.1
14.3
21.5 x 15
54.4
16.4
55 x 20
474.9
143.6
108 x 20
1831.2
554.8
* Relative to 7.5 mm ID x 7.5 cm L column
Based on a 1 mg capacity for a 7.5 mm ID x 7.5 cm L column, the capacity
for a 55 mm ID x 20 cm L column is expected to be about 150 mg. Much
larger amounts of crude sample can be injected as long as impurities do
not co-elute from the column with the compound of interest.