Troubleshooting of HPLC Analysis

Introduction :

HPLC having an large application in pharmaceutical, biology and chemical industries. Analytical method developed in HPLC gives accurate results. Also this instrumentation is quite simple to understand.

Still we are facing many problems during the analysis, commonly faced issues are listed out below;

                1. Over pressure/Pressure Fluctuation

                2. Tailing of peak. 

                3. Fronting of peak. 

                4. Abnormal peak shape.

                5. Retention Time Shifting. 

                6. Baseline Noise. 

                7. Peak not eluting. 

                The above mentioned problems, we are facing day to day work. Let's see the possible cause for these problems and troubleshooting of these problems. 

1. Over Pressure/Pressure Fluctuations:

• There are few things related to this problem. First thing is filtration of your mobile phase. If you are not filtered the mobile phase, it will leads to the deposition of salt and minute particles in the instrument and column. Not filtering the mobile phase won't show the problem immediately but later it will be a problem. It will leads to damage of your instrument and as well as column life will go bad soon.

• So first thing to verify when you face high pressure, need to check mobile phase filtration

• Next cause may be the column. If a same column will be continuously used for next analysis without washing, immediately pressure will raise. So consider Washing the system and column after every analysis is must. It will helpful to improve the column and instrument life.

• Sample filtration also affect the pressure. Column will be blocked by the sample particles. So filtering the sample before injecting into the instrument is must.

• When the column temperature is too low, the pressure will remains high.

• Leakage in the column or seal will give pressure fluctuation.

• Check for the air trap in the mobile phase lines.

2. Tailing of Peak:

There are only few chance for the high tailing. One important factor is sample concentration. When the sample concentration more, tailing will get increase. So optimize the sample concentration while developing the method.

Second thing is your column performance. When the column getting older, tailing and plate count getting reduced, compared to newer. 

3. Fronting of Peak:

Possible cause for fronting of peak is Low Column Temperature. Another few causes are,

       1. Wrong sample solvent,

       2. Sample Overload,

       3. Bad Column Performance.

4. Abnormal Peak Shape:

There are many possible reasons behind this problem.

                 1. Improper washing of the column

                3. Sample solvent may be incompatible with mobile phase

                2. Contamination of Guard column or inlet of the column

Normal peak shape                                                  

5. Retention Time Shifting:

Possible causes are,

                    1. Improper column temperature control.

                    2. Air bubble in the mobile phase

                    3. Improper mobile phase mixing

                    4. Insufficient column equilibration will leads to the retention time shifting.

6. Baseline Noise:

                1. Column temperature fluctuation. Even small changes cause cyclic baseline raise and fall, most often affects RI, UV and conductivity detectors at high sensitivity.

                2. Contamination or air bubble in the detector flow cell.

                3. Mobile phase contamination or used low grade chemicals in mobile phase.

                4. Mobile phase mixing problem, especially when buffer mixed with organic solvent.

                5. Check for pump Pulsation.

7. Peak not Eluting

                    1. Check for the mobile phase preparation and sample preparation.

                    2. Leakage of column or in other area in the instrument.

                    3. Blockage in the injector.

           4. Check for the connectivity of detector, when there is communication error between the detector peak elution will not captured by detector. If above everything if fine, then try restart the detector.

Conclusion: 

HPLC is an very sensitive instrument when compared to other analytical equipment used in quality control department. So more care must be taken while handling the instrument. The life of instrument and column will be depended on the proper handling the instrument. The root cause for each problems should be identified correctly and need to take corrective action.