Cause 1. Insoluble material from the solvent:
-Use HPLC grade solvents! Membrane filter buffers (0.45 ~m or finer).
Cause 2. Insoluble material from the sample:
-Membrane filter sample (0.45 ~m or finer).
Cause 3. Insoluble material from the pump, injector, etc:
-Set line-filter between the pump and injector, pre-filter between the injector and column!
Cause 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!
4-2. Precipitation caused by injecting the sample in immiscible solvents.
– Check whether the solvent of the sample is miscible with the mobile phase.
4-3. Precipitation caused by temperature change, or due to the drying up of the stationary phase.
– Seal the column tightly and keep it at constant room temperature.
Trouble-shooting for Increased Operational Pressure
Flow-chart of the HPLC system: Suction-filter >> pump >> line-filter >> injector
>> pre-filter >> guard column >> main column >> detector and drain
Disconnect parts starting from the end of the flow-chart, i.e., starting with the detector.
Parts most likely to clog are the pre-filters, guard columns and main columns in that order.
NOTE! The operational pressure of the new columns under routine analysis conditions should be recorded and documented.
Repair Manual for Clogged Columns
Step 1. After using salt-containing buffers (e.g., ion-pair reagents) flush a solvent of high solubility against salts through the column (e.g., water).
Step 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.
Step 3. Disconnect the detector, reverse the column the other way around and check the pressure.
(1) If the pressure decreases steadily, move to Step 4
(2) If there is no pressure decrease, move to Step 5.
Step 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.
Step 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.
Step 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.
Step 7. If Step 6 has not brought improvement, then repair is impossible. Please note that guarantee will be lost after taking off the end fittings.
Decreased Efficiency of the Columns
The 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.
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