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
|
You may also like this Column care and maintenance HPLC Column Regeneration Factor Affecting Retention Time Mobile Phase of HPLC Advantages of HPLC Resolution in Chromatography |
No comments:
Post a Comment