Waters XTerra and XTerra Prep Columns - Manual (Page 7)

[ Care and Use ManUal ]
XTerra Columns 7
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all buffers before use. Pall
Gelman Laboratory Acrodisc
® filters are recommended. Solvents
containing suspended particulate materials will generally clog the
outside surface of the inlet distribution frit of the column. This will
result in higher operating pressure and poorer performance. Degas
all solvents thoroughly before use to prevent bubble formation in
the pump and detector.
d. Pressure
All XTerra columns, regardless of dimension, can be operated at
pressures up to 6000 psi, 400 bar or 40 Mpa.
e. Temperature
Temperatures between 20 – 60˚C are recommended for
operating Waters XTerra columns to enhance selectivity, lower
solvent viscosity and increase mass transfer rates. However, any
temperature rise above ambient will have a negative effect on
lifetime which will vary dependingon the pH and buffer conditions
used.
IV. scALInG uP/doWn
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 only column i.d. is changed:
X = (r
2
/r
1
) 2
If both column i.d. and length are altered: F
2
= F
1
(r 2 /r
1
) 2
Load
2
= Load
1
(r
2
/r
1
) 2 (L
2
/L
1
)
Where: X = Factor by which original flow must be modified (also adjusts
sample load)
L = Length of column, in mm
r = Radius of the column, in mm
F = Flow rate, in mL/min.
1 designates the original, or reference column
2 designates the new dimension column.
V. coLuMn cLeAnInG, reGenerAtInG And storAGe
a. Cleaning and Regeneration
A shift in retention or resolution may indicate contamination of the
column. Flushing with a neat organic solvent is usually sufficient to
remove the contaminant. If the flushing procedure does not
solve the problem, purge the column with a sequence of
progressively more nonpolar or hydrophobic solvents. For example,
switch from water to tetrahydrofuran (THF) to methylene chloride.
Return to the standard mobile phase conditions by reversing the
sequence.
Guard columns need to be replaced at regular intervals as
determined by sample contamination. When system backpressure
steadily increases above a set pressure limit, it is usually an
indication that the guard column should be replaced.
b. Storage
For periods longer than four days store the column in 100%
acetonitrile. Do not store columns in buffered, acidic or basic
eluents. If the mobile phase contained a buffer salt flush the column
with 10 column volumes of HPLC grade water (see Table 2) and
replace with 100% acetonitrile. Completely seal column to avoid
evaporation and drying out of the bed.
VI. TROUBLESHOOTING
Changes in retention time, resolution, or backpressure are often due
to column contamination. See the Column Cleaning, Regeneration
and Storage section of this instruction sheet. Information on column
troubleshooting problems may be found in HPLC Columns Theory,
Technology and Practice, U.D. Neue, (Wiley-VCH, 1997) or the Waters
HPLC Troubleshooting Guide (Literature code # 720000181EN).