How to Troubleshoot High Performance Liquid Chromatography?

Chromatography

High performance liquid chromatography plays an important role in pharmaceutical, chemical, biological, genetic engineering and other fields, with the advantages of high separation efficiency, good selectivity, sensitiveness, quickness and less sample. However, some troubles may inevitably appear during the operation, which can influence its working performance. It’s necessary to rapidly and accurately solve these troubles that may appear in the operation.

There are some common failures, possible causes and solutions concluded as follows.

No.

FailureCause

Solution

1The detector can’t set to zero.a. The sample cell is dirty.

b. It is beyond the solvent limit.

c. The solvent contains impurities that absorb ultraviolet light.

d. The lamp energy is low.

e. The equilibrium time of the detection cell is short.

a. Wash the cell.

b. Change the detection wavelength or choose a different solvent.

c. Use the solvent without impurities.

d. Change the lamp.

e. Wash the cell.

2Baseline noisea. The sample cell or reference cell is polluted.

b. The lamp is aged.

c. There are small bubbles in the sample cell.

d. There is something wrong with the recorder or the ground wire of the instrument.

e. The solvent is dirty or impure.

a. Wash the cell.

b. Change the lamp.

c. Do degassing for the solvent.

d. Check the ground wire.

e. Use high purity solvent.

3Baseline drifta. The column is polluted.

b. Leakage of the reference cell or sample cell.

c. Change of the column temperature.

d. Change of the solvent compositions.

a. Column regeneration or replacement.

b. Check the leakage.

c. Control the temperature.

d. Control the solvent compositions.

4No liquid flows out of the chromatography column or the column pressure is zero.No solvent or the solvent leaks.Add the solvent or check the leakage.
5The column pressure or the flow rate is wrong.a. Leakage of the mobile phase.

b. There are bubbles in the pump.

c. There are foreign contaminants.

a. Check the leakage.

b. Do degassing for the solvent and wash the pump.

c. Filter or use high purity solvent.

6The column pressure is too high.a. Chemical adsorption.

b. Residue of the sample or compositions.

a. Wash the column or replace the column.

b. Filter or use high purity solvent.

7The retention time is changed.a. Change of the solvent compositions.

b. Change of the column temperature.

a. Control the solvent compositions.

b. Control the column temperature.

8The column head pressure is too high.a. The column head is blocked.

b. The detection cell is dirty.

a. Wash the cell reversely or dismount the column head for ultrasonic treatment.

b. Wash the cell.

9The retention time is extended.a. Leakage of pipelines.

b. Use the wrong solvent.

c. Low flow rate.

d. Too low column temperature.

a. Check the leakage.

b. Check and correct the solvent.

c. Improve the flow rate.

d. Enhance the column temperature.

10The retention time is shortened.a. Loss of the stationary phase.

b. The mobile phase isn’t fully balanced after being changed.

a. Change the column.

b. Balance the column.

11Bad separation.a. The column is overloaded.

b. There is large dead volume in the column.

c. The sintered filter in the column is blocked.

d. The column head collapses.

e. The strongly retained material retains on the column.

f. Wrong solvent compositions.

g. Too high flow rate.

h. Too high or too low pH value of the mobile phase.

i. The column doesn’t match the solvent.

a. Reduce the sample amount.

b. Fill the column and reduce the dead volume.

c. Wash the column reversely or replace the sintered filter.

d. Replenish the column packing and use the column in the reverse direction or replace the column.

e. Wash the column.

f. Change the solvent compositions.

g. Lower the flow rate.

h. Choose suitable buffer solution and adjust pH value.

i. Replace the solvent.

12Flat peak.a. The column is overloaded.

b. The detector is overloaded.

a. Reduce the sample amount.

b. Reduce the sample amount or change the detection wavelength.

13Skew peak or bifurcation peak.a. There is large dead volume in the column.

b. Bad column.

a. Fill the column.

b. Replace the column.

14The signal of the detector is pulsed oscillation.a. There are bubbles in the mobile phase system.

b. The lamp doesn’t work.

a. Do degassing for the solvent.

b. Replace the lamp.

15The detector has a low background.a. The reference cell is dirty or has foreign materials.

b. The reference cell leaks or has moisture.

a. Wash the cell.

b. Repair the cell.

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