Waters BioSuite Columns - Manual (Page 2)

[ Care and Use ManUal ]
BioSuite Columns 2
a. Mobile Phase
Prevent air bubbles from entering the column during its installation, use,
and storage since this may cause degradation of column performance
through the formation of channels in the packed bed. Mobile phases must be
thoroughly degassed before use. This can be accomplished by vacuum filtra-
tion, helium sparging or by in-line vacuum degassing. In addition to degas-
sing the solvent, vacuum filtration of the solvent will also prevent small
particles from plugging the column frit. You can use 0.20 or 0.45 µm mem-
brane to filter aqueous and aqueous/organic mobile phases. (Note: Consult
with filtration membrane manufacturer for details on solvent compatibility.)
Note: Use high quality reagents, HPLC grade water, and HPLC grade solvents
for preparing buffers to maximize column efficiency especially when using
Waters BioSuite Non-Porous (NP) columns.
The useful column lifetime is a function of numerous factors including: the
cleanliness and composition of the mobile phase and the sample; the flow
rate and pressure used; and the temperature. Refer to the section below
about “Cleaning” for information on extending column life.
Note: Cleaning, however, is not effective when the column is damaged by
irreversible sample adsorption, channeling, or exposure of the packing mate-
rial to excessive heat or shock.
b. Flow Direction
The recommended flow direction through the column is indicated by the
arrow on the tag. Operating the column with the flow in the reverse direc-
tion is only recommended as part of a cleaning procedure when removing
particulates from a clogged frit.
II. connectIng your column to the hPlc system
Due to the absence of an industry standard, various column manufacturers
have employed different styles of chromatographic column connectors. The
chromatographic performance of your separation can be negatively affected
if the style of column end fitting does not match the existing instrumenta-
tion tubing ferrule setting. Waters BioSuite columns require end fittings
that have a 0.175” depth between the ferrule and the end of the extending
stainless steel tubing (Figure A) that must perfectly seat to the end of the
BioSuite column. Connecting a non-BioSuite style connector to a Waters
BioSuite column will leave a gap between the end of the stainless steel
tubing and the column that will result in undesired peak broadening (Figure
B). To correct this problem, Cut the tubing, place a new ferrule on it and re-
make the connection (i.e., 0.175” depth) to the Waters BioSuite column.
In a proper tubing/column connection, the tubing touches the bottom of the
column end fitting with no void between them. (Figure C)
After manufacturing and quality control, the column has been flushed with
storage solvent and closed with caps to prevent solvent evaporation. When
installing the column to the Liquid Chromatograph, it is important to prevent
air from entering the column. The following steps can be used to minimize
this potential problem:
• Remove the cap from the column’s inlet side. Solvent should be visible at
the inlet fitting (if not, see below).
• Start solvent flow through the liquid chromatograph BEFORE connecting
the Waters BioSuite column to the system. Turn off the pump after a
steady flow of solvent from the system is observed. You can now connect
the waters BioSuite column to the liquid chromatograph and slowly begin
delivering solvent to the column (Note: If the Waters BioSuite column
inlet fitting appears dry, we recommend that you first disconnect the
bottom cap and hook up the column exit to the injector. Then slowly start
Figure A
0.175 inches
Figure B
Figure C
Void