Resolution problems

Nico Vonk, Avans+, Breda, The Netherlands

LevelBasic
It is very important to locate the problem before installing a new column. Keep in mind that use of guard columns will prolong column lifetime and avoid many problems of contamination.

Reduced resolution / separation.

Reduced resolution / separation
Possible cause	Solution
Extra column band broadening.	Make sure the tubing length and tubing id are kept to a minimum. Check if the detector flow cell volume is smaller than 0.1 x peak volume. Finally check the influence of the detector's time constant; keep this as low as possible (< 0.5 s.)
Improper column temperature stability.	In such cases the use of a column thermostat is strongly advised.
Improper eluent flow rate.	The best eluent flow rate is found just above the optimum of the h-u curve. The use of smaller particles usually results in a shallower slope of the h-u curve. Check the system for solvent leakage and check whether the pump is delivering the eluent with a constant flow.
Insufficient equilibration time with mobile phase.	Use longer equilibration time for the column. Check baseline and retentions stability before starting calibration and injection of samples.
Improper ph or ionic strength of eluent.	The ph strongly influences the concentration of the neutral acidic compounds in the eluent and therefore strongly affects retention behaviour.
Unstable gradient or mixing.	Check proportional valves or mixing chamber of the gradient pump.
Packing bed contamination.	High column pressure accompanying resolution loss. Particulate accumulation on column inlet frit or top of the stationary phase Sample: filter or centrifuge samples Eluent: filter eluent, cap off eluent réservoirs System: flush all lines and pumps, install in-line system filter
Accumulation of proteins in biological samples	Microbial growth in samples . Refresh samples or improve stockage conditions. Microbial growth in eluent. Prepare fresh mobile phases or use 0.1 w-% sodium azide in the aqueous part of the solvent.
Eluent too aggressive (ph too low or too high)	Adjust the mobile phase pH within the column pH limits.
Organic contamination	Fats, oils, lipids in samples Non-specific organics from sample or improperly prepared eluents Bed compression (voids), especially when using polymer based columns, caused by either excessive eluent flow rate or use of inappropriate organic modifier type or concentration.