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APPENDIX
APPENDIX
Guarding your column
GLP procedures often specify that the separation column be protected
by a guard column. The guard column is installed between the injector
and the analytical column. It is designed to protect the analytical
column from unwanted materials, such as highly retained or irreversibly
adsorbed compounds and particulate matter. Tosoh Bioscience
supplies an assortment of packed guard columns, guardgel kits, guard
cartridges, and guardfilters.
Guardgel kits contain the hardware and the gel packing material to fill a
guard column using an aspirator. In addition, step-by-step instructions
are avaible on the Tosoh Bioscience YouTube channel
com/tosohbiosciencellc). Figure 1 is an example of a guardgel kit, in this
case for a TSKgel DEAE-5PW column.
Guard cartridges (Figure 2) are pre-packed, small replaceable columns
easily inserted into a hand-tight guard cartridge holder (Figure 3).
Guardfilters (Figure 4) are pre-packed, small replaceable columns easily
inserted into a hand-tight guardfilter holder (Figure 5).
For those columns where a guard product is not available, Tosoh
Bioscience recommends the use of an in-line filter with a 0.5 µm cutoff
to avoid frequent plugging of the 1.0 µm pores in the column frit of TSKgel
ODS-140HTP, Super-ODS, Super-Octyl, and Super-Phenyl columns. 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.
figure 1
Guardgel kit
figure 3
Guard cartridge holder
figure 2
Guard cartridges
figure 5
Guardfilter holder
figure 4
Guard filter
