HPLC Analysis of Biomolecules Technical Guide - Interscience

36
HPLC Analysis of Biomolecules
Column Care and Maintenance
General Recommendations for Usage
pH: For maximum lifetime, the recommended pH range for silicabased
HPLC columns is 2 – 7. The stability of various columns
outside this pH range will differ considerably. However, for all
columns, lifetime will be progressively shorter as operating pH
deviates further from the 2 – 7 range.
Temperature: The typical operating temperature range for
silica-based HPLC columns is 5° to 60°C. Higher temperatures
may shorten column lifetime.
Buffers: Biological samples often require the chromatographer to
include a buffer in the mobile phase to control pH. Care must be
taken to ensure that no precipitation occurs in the mobile phase
when buffer solutions and organic solvents are mixed. The column
and/or HPLC system should never be stored in a mobile phase
containing buffer or salt.
Pressure: Column lifetime will generally decrease with increasing
operating pressure. A general recommendation is to keep operating
pressure below 3000 psi. Pressure is directly proportional to flow
rate and column length and inversely proportional to the square of
the particle size. Note that a 3 µm column will have approximately
twice the pressure of a 5 µm column of the same dimensions.
Choice of mobile phase can also have a large effect on pressure. For
example, 60/40 methanol/water will generate approximately twice
the back pressure of 60/40 acetonitrile/water. A gradual increase in
pressure with extended usage is common. A sudden increase in
pressure often indicates that the column or connecting tubing has
become plugged.
COLUMNPLUS Filter (CPF)
UNIFILTER
Javelin Filter
ColumnSaver
UNIGUARD Guard
Cartridge Holder
Stand-Alone Guard
Cartridge Holder
Drop-In Guard Cartridges
Column Protection
Using in-line filters and guard columns can extend the lifetime of
your column by preventing particles and other impurities from
accumulating on the column. For maximum protection install an
in-line filter followed by a guard column between the injector and
analytical column.
Filters: An in-line filter installed between the injector and analytical
column will protect the column from particles that could originate
from the mobile phase, pump seals, or sample. For maximum protection
the porosity of the in-line frit should be equal or less than that
of the inlet frit of the analytical column. A common frit porosity for
an in-line filter is 0.5 µm. An increase in system pressure often
indicates that it is time to change the in-line filter.
Guards: A guard column is generally a short version of the analytical
column it is protecting. The guard protects the analytical column by
adsorbing strongly retained impurities that would otherwise foul the
analytical column. The frits of the guard can also retain particles as
an in-line filter. For maximum protection, and to avoid altering the
separation, the guard column should contain packing having the
same or similar bonded phase as the analytical column (i.e. a C18
guard for a C18 analytical column). The guard packing should also
be no more retentive than the packing in the analytical column to
avoid significantly increasing overall retention. To minimize the
contribution of the guard column to the separation, use a guard
packing that is less retentive than that of the analytical column. For
example, a wide pore (e.g. 300Å) C18 having less surface area, and
thus less retention, could be used as a guard for a smaller pore (e.g.
150Å) C18 analytical column. A noticeable deterioration of the
separation or a significant increase in back pressure can signal that
it is time to change the guard column.