Waters PAH Columns - Manual (Page 6)

[ Care and Use ManUal ]
Name of Document 6
Note: If mobile phase additives are present in low concentrations (such as
ion-pairing reagents, at 5 to 10 mmol/L) 100 to 200 column volumes may
be required for complete equilibration.
Table 1. Volume of Standard Column (mL), Multiply by 10 for Flush
Solvent Volume
IV. COLUMN USAGE
To ensure the continued high performance of your columns and cartridges,
follow these guidelines:
a. Guard Columns
Sample impurities very often contribute to column contamination.
Two ways to avoid this are:
a. Use of Waters Oasis® solid-phase extraction sample clean-up car-
tridges or columns or Sep-Pak® cartridges of the appropriate chemistry
to clean up your sample before analysis.
b. Use of a Waters guard cartridge of matching chemistry and particle
size between the injector and main column. It is important to use a
highperformance matching guard column to protect the main column
while not compromising analytical resolution.
b. pH Range
Recommended pH ranges for solvent and buffer combinations for Waters PAH
columns are between 2.0 and 8.0. A pH less than 2 may cause hydrolysis of
the bonded phase. At a pH greater than 7.0, the alkaline solvent buffers will
attack the silica substrate resulting in void formation in the column as the
silica solubilizes.
c. Solvents
To maintain maximum column performance, use high quality chromatography
grade solvents. Filter all buffers before use. Pall Gelman Laboratory Acro-
disc® 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 perfor-
mance. Degas all solvents thoroughly before use to prevent bubble formation
in the pump and detector.
d. Pressure
All Waters PAH columns, regardless of dimension, can be operated at pres-
sures up to 6000 psi, 400 bar or 40 Mpa.
e. Temperature
Temperatures between 20 – 50 °C are recommended for operating Waters
PAH 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 depending on the pH and buffer
conditions used.
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 meth-
ylene 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 1) and replace with 100% acetonitrile. Completely
seal column to avoid evaporation and drying out of the bed.
Column Length
Column Internal Diameter (mm)
2.1 3.0 4.6
50 mm 0.2 0.3 0.8
100 mm 0.4 0.7 1.7
150 mm 0.5 1.0 2.5
250 mm 0.9 1.8 4