Waters SunFire Columns - Manual (Page 2)

[ Care and Use ManUal ]
2
I. GettInG Started
Each SunFire column comes with a Performance Test Chromatogram.
This Performance Test Chromatogram is specific to each individual
column and contains the following information: gel batch number,
column serial number, USP plate count, USP tailing factor, capacity
factor, and chromatographic conditions. The performance test chro-
matogram should be stored for future reference.
a. Column Installation
Note: Flow rates given in the procedure below are for a typical 4.6
mm i.d. column. Scale the flow rate up or down accordingly based
upon the column i.d., length, particle size, and backpressure of the
SunFire column being installed. See “Scaling Up/Down Isocratic
Separations” for calculating flow rates when changing column i.d.
and/or length. See “Connecting the Column to the HPLC System” for a
more detailed discussion on HPLC connections.
reversed-phase Columns (SunFire C
18
and SunFire C
8
)
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the
injector outlet.
2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 1 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column outlet,
stop the flow and attach the column outlet to the detector. This
prevents entry of air into the detection system and gives more
rapid baseline equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.
normal-phase Columns (SunFire Silica)
Note: It is assumed that your system has been used for reversed-phase
chromatography. If this is not the case, you can start with step 3.
1. Purge the pumping system of any buffer-containing mobile phases.
2. Flush the system thoroughly with acetonitrile.
3. Switch the system over to the mobile phase that you are planning
to use in normal-phase chromatography.
4. Connect the column and equilibrate it with the mobile phase.
Note: Equilibration with the mobile phase may require a larger amount
of solvent than in reversed-phase chromatography.
b. Column equilibration
SunFire columns are shipped in 100% acetonitrile. It is important
to ensure mobile phase compatibility before changing to a different
mobile-phase system. Equilibrate the column with a minimum of 10
column volumes of the mobile phase to be used (refer to Table 1 for
a listing of empty column volumes).
reversed-phase (SunFire C
18
or SunFire C
8
) Columns
To avoid precipitating out mobile-phase buffers on your column or in
your system, flush the column with five column volumes of a water/
organic solvent mixture, using the same or lower solvent content as in
the desired buffered mobile phase. (For example, flush the column and
HPLC system with 60% methanol in water prior to introducing 60%
methanol/40% buffer mobile phase.)
Note: If mobile-phase additives are present in low concentrations (e.g.,
ion-pairing reagents), 100 to 200 column volumes may be required
for complete equilibration.
normal-phase Columns (SunFire Silica)
SunFire normal-phase (NP) columns are delivered in 96% heptane/
4% isopropyl alcohol. Care should be taken not to pass any mobile phase
through the column that might cause a precipitate (see above). SunFire
Silica normal-phase columns are compatible with water and all common
organic solvents, provided that solvent miscibility is accounted for.
Equilibrate normal-phase silica columns in the mobile phase. Very
small quantities of water in the mobile phase can dramatically affect
the activity of normal-phase packings. For good reproducibility,
ensure that the mobile phase always has the same water content.
It is difficult and usually unnecessary to completely eliminate the
water from the mobile phase. Dry mobile phases can take a very long
time to equilibrate the column. A water content of 50 percent of
saturation is recommended for most applications.
To equilibrate your column:
1. Starting at 0.0 mL/min, increase the flow rate in
0.1 mL/min increments to 1.0 min.