Good column care can extend the lifetime of HPLC columns, and in general requires little

additional time. Column care involves operating and storing a column in a suitable mobile phase,
following the manufacturer or test method guidelines for column operation. The guidelines listed
in this job aid will answer many column care and storage questions and will serve as a general
guideline for HPLC column care.

1.0 Procedure

1.1 Precautions

HPLC columns should always be stored with the inlet and outlet openings
capped or sealed. Additionally, exposing the column to shock or wide variations
in temperature will decrease the lifetime.

For the purpose of this job aid, equilibration is defined as approximately 20

column volumes of mobile phase being pumped through the column at the
method specified flow rate. This amount can vary depending on the type of
analysis or the individual test method.

1.2 First Time Use Columns

Initially, only pump solvents onto the column that are compatible with the
shipping solvent. For reversed-phase separations, do not pump buffer onto a
column shipped or stored in 100% organic. This will prevent buffer precipitation in
the column. First equilibrate the column with the mobile phase without the buffer.
Second equilibrate the column with buffered mobile phase. Some columns, such
as CN or NH2, are used with normal phase and reversed-phase solvents. Check
that your solvents are miscible with the shipping solvents before equilibration.
The shipping solvent can be found by consulting the column care leaflet shipped
with each new column. Converting a normal phase column for use with a
reverse phase analysis may require flushing with a mutually miscible solvent,
such as isopropanol, before equilibrating with mobile phase.

1.3 Storage Solvents

Do not store HPLC columns in buffers. A buffer may precipitate inside the
column, resulting in plugged hits and packing material. Buffers may also
encourage bacterial growth within the column, potentially plugging both the
column frit and packing material. This is more likely to occur with mobile phases
with higher concentrations of buffer. Bacteria may also affect your analytes, and
organic products from the dead bacteria may cause "ghost peaks" in
chromatograms.
1.4 Short Term Storage

Flush the column with your mobile phase, substituting water for the buffer.
For Example:

Mobile Phase: 65% Methanol & 35% Buffer

Flush with: 65% Methanol & 35% Water

This will minimize the risk of buffer salts precipitating in the column and reduce
equilibration time when the column is used next.

If the column is left on the instrument for extended periods of inactivity it is

recommended to keep the mobile phase flowing at a reduced rate; i.e. 0.1 - 0.2
mL/min. This is very helpful when using complex mobile phases (buffers and ion-
pairing reagents), since it reduces equilibration time to a few minutes. Reducing
the flow rate to zero when a mobile phase contains a high concentration of buffer
is not advisable. If the organic solvent evaporates when the system is
standing idle, it can leave buffer residues inside the column as well as on
the pump pistons and other exposed parts. This will shorten pump seal
lifetimes and cause premature failure of other parts.

1.5 Long Term Storage

After flushing the column as described in section 2.3 the column can be returned
to the solvent that it was shipped in. If you are using a buffered mobile phase,
first wash the column with the same mobile phase composition without the buffer,
next flush with the shipping solvent. It is not necessary (or desirable) to flush the
column with 100% water just to wash out the buffer salts.

1.6 Specialty Columns

Every column is shipped with a list of care-and-use instructions. If you

cannot find them, they should be posted on the column manufacturer's web
site. These generally contain suggested conditions of use, pH limits, any
prohibited solvents, and a cleaning procedure. The procedures discussed
previously should be applicable to nearly all silica-based reversed-phase
LC columns. Normal phase columns and certain specialty columns can
have restrictions in terms of solvents or other suggested cleaning
procedures. In all instances, consult your test method first.

2.7 Column Regeneration