196 / 413
• 22 •
Tech Tip
Under Pressure?
Reduce System Stress by Backflushing Your HPLC Column
By Tim Herring, Technical Service
Experiencing a higher pump pressure than usual? Or perhaps a complete pressure shut-down of the system has occurred, even after
replacing the in-line frit and guard column. High pump pressures can be caused by heavily retained impurities building up within
the head of the analytical column. Such contamination can cause poor chromatography, usually in the form of broad, split, or mis-
shapen peaks, and ultimately can compromise results. Backflushing a contaminated analytical column using the following procedure
can help restore column performance and reduce pump pressure and system strain.
If back pressure is abnormally high, first take the column out of the equation by disconnecting it from the system altogether. Install
a union and run the pumps to verify that the back pressure problem is due to the column, and not to the HPLC system. If the pres-
sure is normal, then the column is most likely the cause of the high back pressure. To address this, reverse the column flow and rinse
(backflush) the column to remove the contaminants from the inlet frit and column head. This will move the contaminants down the
path of least resistance, instead of forcing them further into the analytical column. Reverse rinse into a waste beaker at low flow (e.g.
0.5mL/min. for a 4.6mm ID column) for 10-15 minutes initially, and then increase the flow to 1.5-2 times the optimal flow (1.5 to
2.0mL/min. for a 4.6mm ID column). Do not reconnect to the detector when backflushing the column. Rather, flush the waste
stream into a beaker so that the detector cell is not contaminated by impurities or obstructed by particulate build-up.
Solubility is a key issue when backflushing columns, so remember the old adage, “like
dissolves like”. For example, if the contaminants are suspected to be oily or
hydrophobic in nature, then backflush with a strong, nonpolar solvent such as
hexane. If the contamination is polar (a salt for instance), then use a polar
solvent, such as water or methanol. Solvent miscibility also needs to be
considered, so be sure to use solvents that are miscible with one anoth-
er. If in doubt, use isopropanol (IPA) as an intermediary solvent
between solvent wash steps, as it is miscible with all common sol-
vents. This is particularly true when switching from typical nor-
mal phase solvents (such as hexane) to reverse phase solvents
(such methanol, acetonitrile, or water) and vice versa. Note that
10 to15 column volumes are generally necessary at each step to
remove all traces of immiscible solvents prior to the next step.
If the contaminants are unknown, or vary in chemistry, a series
of solvent washes will provide an array of differing chemical
interactions and maximize the removal most types of contami-
nation. The solvent order presented in Table I considers miscibil-
ity, polarity, and eluotropic strength and is a very effective series
for removing most contaminants. Column backflushing, with
proper solvent selection, is a simple way to regenerate analytical
columns, improving column performance and reducing system stress.
Contact Restek Technical Service
at
support@restek.comor 800-356-1688
with questions on backflushing, or any other
technical area. At Restek, we are here to help you!
Reversed phase series:
Normal phase:
A. 1% glacial acetic acid in methanol and water (50:50)
A. isopropanol
B. methanol
C. chloroform
D. hexane (or heptane)
E. methylene chloride (dichloromethane)
F. methanol
Table I
Restore column performance by backflushing with
recommended solvent washes.