the Column from Clogging
1. Insoluble material from the solvent:
-Use HPLC grade solvents! Membrane filter buffers (0.45 ~m or finer)!
2. Insoluble material from the sample:
-Membrane filter sample (0.45 ~m or finer)!
3. Insoluble material from the pump, injector, etc:
-Set line-filter between the pump and injector, pre-filter between
the injector and column!
4. The formation of insoluble material inside the column
4-1. Precipitation caused by solvent immiscibility.
- Wash the column
with a solvent, which can dissolve the precipitation!
caused by injecting the sample in immiscible solvents.
- Check whether
the solvent of the sample is miscible with the mobile phase!
caused by temperature change, or due to the drying up of
- Seal the column
tightly and keep it at constant room temperature!
for Increased Operational Pressure
of the HPLC system: Suction-filter >> pump >> line-filter
>> pre-filter >> guard column >> main column >>
detector and drain
parts starting from the end of the flow-chart, i.e., starting with
Parts most likely to clog are the pre-filters, guard columns and
NOTE! The operational pressure of the new columns under routine analysis
conditions should be recorded and documented!
Manual for Clogged Columns
1. After using salt-containing buffers (e.g., ion-pair reagents)
flush a solvent of high solubility against salts through the column
2. After disconnecting the detector, flush through the column using
solvent of high solubility against materials that might originate
from the samples. For reversed phase column methanol, acetonitrile,
THF, chloroform, heptane, etc., can be used.
In any case, avoid solvents below pH2 and above pH8!
If after Step 1 and Step 2 there is no drop in the pressure, move
to Step 3.
3. Disconnect the detector, reverse the column the other way
around and check the pressure.
If the pressure decreases steadily, move to Step 4
If there is no pressure decrease, move to Step 5.
4. Keep the column connected the other way around and wash with
pressure bellow 0.5ml/min for about 1 hour.
If Step 4 has not brought decrease of pressure, move to Step s.
5. If the inlet-filter is clogged it should be washed or replaced.
The removal and reinstallation of the end fitting of the column
can cause decrease in the column's efficiency (decreased effective
plate number, asymmetric peak). If Step 5 has not brought decrease
of pressure, move to Step 6.
6. If there is a clogging in the packing material at the inlet-side,
real recovery is not possible. Limited improvement can be achieved
by taking off some of the inlet-side's packing material and replacing
it with fresh packing material. (2-5 mm).
Note: in many case the column's efficiency
is declining heavily.
7. If Step 6 has not brought improvement, then repair is impossible.
Please note that guarantee will be lost after taking off the end
Efficiency of the Columns
main reason for the decreasing effective plate number and decreasing
retention capacity is deterioration of the stationary phase and
loss of the active chains from the surface of the silica gel and
in these cases recovery is not possible.
additional Help Please visit the HPLC
Troubleshooter web page
Research, 5999 West 34th, Suite 106 Houston, TX 77092 USA
Tel: (713) 686-9662 • Fax: (713) 686-9635 • Toll Free: 1 (800) 275-0082