Waters Protein-Pak Hi Res HIC Column and HIC Protein Standard - Manual (Page 3)

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[ CARE AND USE MANUAL ]
Protein-Pak Hi Res HIC Column and HIC Protein Standard Test Mix Care & Use
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Remove the cap from the column’s inlet side. Solvent should be visible at the inlet fitting (if not, see below).
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Start solvent flow through the liquid chromatograph before connecting the Protein-Pak Hi Res HIC Column to the system. Turn off the
pump after observing steady flow of solvent from the system. You can now connect the Protein-Pak Hi Res HIC Column to the liquid
chromatograph and slowly begin delivering solvent to the column
Note: If the Waters Protein-Pak Hi Res HIC Column inlet fitting appears dry, we recommend that you first disconnect the bottom cap and
hook up the flow in this reversed flow direction until a few drops of mobile phase exit from the column. Turn off the flow, let the pressure
go to zero, and disconnect the column from the system. Turn the column around and hook it up so that the flow is now in the direction of
the arrow. Start the flow at a low setting and stop it as soon as the mobile phase exits from the bottom fitting. Now you can hook up the
column to the detector inlet and increase the flow to the desired setting.
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Be sure to set the flow within the recommended range shown in the following tables.
III. COLUMN SPECIFICATIONS AND USE
Shipping solvent: Distilled Water
Max. flow rate: 1.2 mL/min: Protein-Pak Hi Res HIC , 4.5 x 35 mm
1.5 mL/min: Protein-Pak Hi Res HIC , 4.5 x 100 mm
When a buffer with high viscosity is used, the maximum flow rate may have to be reduced so as not to exceed the
maximum pressure drop.
Max. pressure: 3000 psi
pH range: 2–12 (pH above 12 or below 2 can only be used for a short time)
Salt conc.: < 4 Molar
Organic conc.: < 50% (salt precipitation should be avoided when adding salts to the mobile phase containing organic solvents)
Temperature: 10–60 °C. Reduce flow rate when operating at low temperatures (e.g. 10 °C) to avoid excessive column pressure.
Column washing
and regeneration: Repeated 100–250 µL injections of 0.1–0.2M NaOH are recommended for cleaning or regenerating the column.
When this procedure is not effective, we recommend repeated injections of 100–250 µL of 20% aq. acetic acid.
Note: The above cleaning step using 0.1–0.2M NaOH is best performed after each day of column use.
Following use of cleaning solvent, rinse the column with 5–10 column volumes of HPLC-grade water before storage.
Column storage: Store the column in shipping solvent at the end of each day of use. For overnight storage, continuously flush the column
with the mobile phase at 10–20% of the maximum recommended flow rate. Store the column in the HPLC-grade water
when it will be used within 24 hrs or in either 20% ethanol or methanol for long term storage. (Caution: Rinse column
in with 5–10 column volumes of HPLC-grade water before switching to 20% organic solvent to avoid salt precipitation
in column.
Column No guard column is available for the Protein-Pak Hi Res HIC Column. Be sure to use a filter after the injector with 0.5 µm
protection: pores to avoid plugging of the one micron pore size column frit. We also recommend a pre-injector membrane filter
to prevent particles from pump seal wear to reach the column. Also, use high quality reagents, water, and solvents for
preparing buffers. Fouling of the resin, leading to a loss in retention and/or efficiency, occurs faster due to the small
surface area of non-porous resin particles.