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APPENDIX
APPENDIX
APPENDIX A
About TSKgel Columns, their Maintenance and Scale Up
Tosoh Corporation closely monitors all stages of the manufacturing
process for chromatographicmedia that is used to pack TSKgel columns.
Packing materials are produced in large gel batches which must pass
stringent quality control specifications for particle size distribution, pore
size distribution, pore volume, and surface area. After producing the
particles, each lot is then used to prepare multiple batches of bonded
phase by attaching the appropriate ligand. Each gel lot is again tested
to ensure that it meets the specifications for parameters such as ligand
density, retention, selectivity, etc.
TSKgel columns are designed for general purpose HPLC or FPLC
applications. They are not guaranteed to work for specific customer
applications. Suitability of a column has to be determined by the end
user. Good Laboratory Practice (GLP) demands that a rugged method
must be developed by testing at least three different gel lots to
understand the type of variability in retention and selectivity that may
be encountered with future columns.
Tosoh Bioscience recommends that shipments are inspected for
the presence of the Inspection Data sheet, Operating Conditions and
Specifications (OCS) sheet, and column appearance. After review of the
shipping contents, the column should be tested within 30 days according
to the conditions listed in the Inspection Data sheet to confirm that the
column meets the specifications listed in the OCS sheet.
Troubleshooting column problems
Listed below are the five most common causes of poor column
performance and the precautions that must be taken to prevent
these problems:
1. Void or dead space at the column inlet or channeling of
the packing
Sudden pressure surges and higher than recommended flow rates can
compress the column packing, which can result in a void or a channel,
especially with large pore size columns such as TSKgel G4000SW and
TSKgel G4000SW
XL
. We recommend using an injector that ensures
continuous flow onto the column during injection, i.e., no pressure
pulse due to interrupted flow, and installation of a pulse dampener to
suppress the sudden pressure surges encountered with quick-return
pumps.
Bulk packing material is available to refill voids in some of the analytical
and semi-preparative columns. We highly recommend the use of a
guard column to protect your analytical column from pressure surges
and to prevent irreversibly binding impurities from reaching the
analytical column. A guard column also helps to neutralize the pH of the
sample solvent if it is different from that of the mobile phase. The pH of
the sample will be equilibrated with the mobile phase before it reaches
the analytical column. This is particularly important in the silica-based
SW-type columns because this silica-type is not stable at a pH higher
than 7.5.
2. Air in Column
The column should be tightly capped when not in use to prevent air
from entering it. Air dissolved in the mobile phase must be removed
before it can enter the column. This is particularly important for
polymer-based columns. Air can be removed by sparging with helium,
mobile phase filtration or other degassing procedures. If air does enter
the column, follow the rehydration procedure described on page 107.
3. Column contamination or incomplete sample recovery
Cleaning conditions for all column types are provided on the OCS sheets
that are shipped with each column. Cleaning solvents are discussed in
the cleaning section below.
4. Frit plugging and high pressure
Solvents and samples should be filtered through at least a 0.45 µm
filter to prevent clogging the column frits. If the frit becomes partially
plugged, the result may be split peaks or high pressure. The entire end-
fitting can be removed and sonicated in 6 M nitric acid. Rinse the end-
fitting thoroughly after cleaning. (Be careful not to disturb the packing.)
Alternatively, this end-fitting can be replaced. Installing a membrane
filter prior to the injector is recommended to prevent particles created
by pump seal wear from reaching the analytical column. Consult the
price list for these and other hardware products.
5. Peak splitting
Column overload, whether in volume or concentration, can cause peak
splitting and poor resolution. Consult the sample capacity information
for each column type to determine the appropriate concentration and
volume of analyte.
Cleaning
Columns should be cleaned at regular intervals. The frequency
depends on the purity of the samples. Occasionally, samples are run
which adsorb onto the packing material. If one of the performance
characteristics (asymmetry factor, retention time, theoretical plates, or
resolution) changes by 10% or more, it is prudent to clean the column.
A Data Inspection sheet and an Operating Conditions and Specifications
(OCS) sheet accompanies all TSKgel columns. The Data Inspection
sheet identifies the testing method that was used to verify the column’s
performance. The column’s specifications are listed on the OCS sheet.
However, a well resolved sample component could be used to monitor
the column. Establish that the column is performing properly using the
standard test probes listed on the Data Inspection sheet. Calculate the
asymmetry factor, theoretical plates and resolution of one or more of
the sample components. Note the retention time. This becomes the
baseline test mix which provides a basis for comparison.
