high performance liquid chromatography, broad peaks

HPLC Troubleshooting

If any problem persists during HPLC analysis, don’t scared about it, think of little things there are the basic possible source of chromatographic troubles are Mobile Phase, Column, Guard Column, Pump, Injector, Detector, In-Line Filter, Tubing and fittings. At the beginning of HPLC analysis to follow basic practices like Filter the mobile phase, Sonication, preparation of buffer properly, Purge the system,  wash the column and equilibrate it with sufficient time, then start the analysis.

If you pay attention to these things, then you will have low problems during the analysis.Here are mentioned. some troubles and possible reasons and solutions.

TROUBLE   POSSIBLE CAUSES   SOLUTIONS
No solvent flow
 
Empty Reservoirs
Leakage,
Air bubbles
 
Refill the solvent bottles,
Find the leak and repair it,
Purge the system
Noisy baseline or spiking on the baseline
 
 
Aging of the column
Air bubbles
Detectors problem
 
Replace the column
Degas the mobile phase, Purge the
system
Check the detector
Mobile phase leaking
 
Loose inlet or outlet fittings
Loose valve
 
Tighten the fittings
Tightening the check valve
Pressure fluctuation
 
Dirty check valve
Air entrap in the pump
The issue with piston seal
 
Clean the valve
Purge the system
Check/Replace the seal
Inconsistent Retention time
 
Flow rate change
Problem with column
Leakage
Air bubble
 
Check the flow rate
Change the column
Find and repair the leakage
Remove air bubbles
Detector not response
 
Issue with wavelength
Issue with lamp
Dirty-flow cell
Leakage
Mobile phase contaminate
 
Select the correct wavelength
Check the lamp
Clean the flow cell
Find and repair the leakage
Replace the mobile phase
Results are not Reproducible
 
The issue with column efficiency
An issue with the mobile phase
An issue with injector valve
 
Wash and equilibrate the column properly.
Prepare fresh mobile phase
Check the rotor seal
Broad Peaks
 
Contaminated guard column
Mobile phase composition
Buffer pH
An issue with column efficiency
 
Replace/Remove guard column
Select the proper composition of Mobile phase
Adjust the proper pH of a Buffer
Check the column performance
Baseline Drift
 
Unstable mobile phase
Dirty column
Issue with lamp
 
Change with the stable mobile phase
Wash/replace the column
Check the lamp
High Backpressure
 
High Flow rate
Guard column blocked
Improper column
Viscous buffer
Buffer Precipitate
Blocked Injector
In line filter clog
 
Use the proper flow rate
Replace/Remove guard column
Wash/Replace column
Use the correct Mobile phase
System wash with warm water
Wash blockage
Wash with warm water
Low back pressure
 
Low flow rate
Leakage
High column temperature
Improper column
 
Use the proper flow rate
Find and repair the leakage
Set correct the Temperature
Wash/Replace column
Peak Tailing
 
pH of Mobile phase
Mobile phase composition
Column inefficiency
 
Use Adjust the proper pH of the mobile phase
Select the proper composition of Mobile phase
Wash/replace the column
Split peaks
 
 
Incompatible Mobile phase
Column inefficiency
 
Change the mobile phase or MP composition
Wash/replace the column
Impurities in sample or mobile phase
 
Impurities in samples or mobile phase
Impurities in column
 
Prepare fresh mobile phase
Wash and equilibrate the column
 

👩‍🔬 If you want to know other articles similar to HPLC Troubleshooting you can visit the HPLC BASICS

Jose Hullgren (Laboratory Analist)

Hello to all readers, my name is Jose Hullgren, it is a pleasure to present you this website of my authorship, I am currently working as a laboratory analyst and for the last 10 years I have been working in the pharmaceutical industry. The main idea of this page is to provide relevant information in the field of the pharmaceutical industry above all. We also cover different areas of chemistry and sciences in general that we find interesting. Perfil Linkedin

You May Be Interested in:

Go up