Waters XSelect HSS HPLC Columns - Manual (Page 7)

[ Care and Use ManUal ]
XSelect HSS HPLC Columns 7
e. Pressure
XSelect HSS HPLC columns can tolerate pressures of up to 6,000 psi
(400 bar or 40 Mpa) although pressures greater than 4,000 - 5,000 psi
should be avoided in order to maximize column and system lifetimes,
and the risk of system shutdowns and leaking.
f. Temperature
Temperatures between 20 ˚C - 45 ˚C are recommended for operat-
ing XSelect HSS HPLC columns in order to enhance selectivity, lower
solvent viscosity and increase mass transfer rates. However, any
temperature rise above ambient will have a negative effect on life-
time which will vary depending on the pH and buffer conditions used.
The combination of operating at elevated temperatures and at pH
extremes should be avoided.
g. Scaling Up/Down Isocratic Methods
The following formulas will allow scale up or scale down, while maintaining
the same linear velocity (retention time), and provide new sample
loading values:
If column i.d. and length are altered: F
2
= F
1
(r
2
/r
1
) 2
or: Load
2
= Load
1
(r
2
/r
1
) 2 (L
2
/L
1
)
or: Inj vol
1
= Inj vol
2
(r
2
/r
1
) 2 (L
2
/L
1
)
Where: r = Radius of the column, in mm
F = Flow rate, in mL/min
L = Length of column, in mm
1 = Original, or reference column
2 = New column
XIII. CoLUMn CLeanInG, ReGeneRatInG anD stoRaGe
a. Cleaning and Regenerating
A sudden increase in pressure or shift in retention or resolution may
indicate contamination of the column.
Flush with a neat organic solvent to remove the non-polar
contaminant(s). If this flushing procedure does not solve the problem,
purge the column with a sequence of progressively more non-polar
solvents. For example, switch from water to tetrahydrofuran to methylene
chloride. Return to the standard mobile-phase conditions by reversing
the sequence.
For Normal-Phase Conditions:
The XSelect HSS Cyano column can be used for both reversed-phase
separations as well as normal-phase separations. The column is
originally shipped in acetonitrile and is ready to use for reversed-
phase conditions. If you intend to use the column for normal-phase
applications you will need to condition the column with the following
procedure:
1. Flush the column with a minimum of 20 column volumes of
100% methanol using a low flow rate to avoid overpressuring
the LC system. Refer to Table 2 for minimum solvent volume.
2. Flush the column with a minimum of 20 column volumes of
100% isopropanol using a low flow rate to avoid overpressuring
the LC system. Refer to Table 2 for the minimum solvent volume.
3. Flush the column with a minimum of 20 column volumes of
100% dichloromethane using a low flow rate to avoid overpressuring
the LC system. Refer to Table 2 for the minimum solvent volume.
4. Flush the column with the intended mobile-phase conditions until
a stable baseline is achieved.
b. Storage
For periods longer than four days, store the column in 100% acetonitrile.
Do not store columns in buffered eluents. If the mobile phase
contained a buffer salt, flush the column with 10 column volumes
of HPLC grade water (see Table 2 for common column volumes) and
replace with 100% acetonitrile for storage. Failure to perform this
intermediate step could result in precipitation of the buffer salt in the
column when 100% acetonitrile is introduced.
Completely seal column to avoid evaporation and drying out of the bed.
Note: If a column has been run with a formate-containing mobile
phase (e.g., ammonium formate, formic acid, etc.) and is flushed to
remove the buffer, slightly longer equilibration times may be required
after the column is re-installed and run again with a formate-containing
mobile phase.
For Normal-Phase Use:
For rapid equilibration upon start-up, it is recommended that you
store the XSelect HSS Cyano column in the mobile phase that is com-
monly used for your normal-phase separation. Completely seal the
column to avoid evaporation and drying out of the bed.