Waters Protein-Pak Hi Res HIC Column and HIC Protein Standard - Manual (Page 2)

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[ CARE AND USE MANUAL ]
Protein-Pak Hi Res HIC Column and HIC Protein Standard Test Mix Care & Use
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 filtration, helium
sparging, or in-line vacuum degassing. In addition to degassing
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 membrane 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 Protein-Pak Hi Res HIC,
non-porous 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 is not effective when the column is damaged by
irreversible sample adsorption, channeling, or packing material
exposure 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 direction is only recommended as part of a cleaning
procedure when removing particulates from a clogged frit.
II. CONNECTING COLUMN TO LC SYST EM
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 endfitting does not match the existing
instrumentation tubing ferrule setting. Waters Protein-Pak Hi
Res HIC Columns require endfittings 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
Protein-Pak Hi Res HIC Column. Connecting a non-Protein-Pak Hi
Res HIC style connector to a Protein-Pak Hi Res HIC 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
Protein-Pak Hi Res HIC Column.
Figure A.
Figure B.
Figure C.
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: