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Simple Tips On HPLC Column Equilibration: In Less Time
HPLC Column Equilibration: Introduction and Outcome
HPLC column equilibration plays an important role in HPLC analysis. Most problems during HPLC analysis arise due to column equilibration. So I decided to share my skill-based knowledge on this topic. In this article, you will learn, what is column equilibration, what are effects of the column equilibration, how column can be equilibrated in less time and interview questions.
HPLC Column Equilibration
HPLC column is packed with the stationary phase. In between the stationary phase there is empty space. This empty space is called void space and volume occupied by this space is called void volume. This void volume is occupied by the mobile phase during column equilibration and helps analyte to interact seamlessly with the stationary phase and the mobile phase. Due to this sooth interaction separation of different analyte take place.
In short, we can say that Column Equilibrations is the process of stabilizing the HPLC column with mobile phase before the injection for sooth interaction of analytes with stationary phase and mobile phase.
Effects of HPLC Column Equilibrations on Chromatographic Analysis
The following problems may arise if column is not equilibrated properly:
- Shift in retention time during analysis
- Elution problem
- Variation in analysis result
- Variation in area response
- Failure of system suitability test (SST)
- Wasting unnecessary time troubleshooting
HPLC Column Equilibration in Isocratic mode (RPC)
Column is easily equilibrated in the Isocratic mode. Use the following tips to equilibrate the column:
- Install the column and flush 5 minute with water at the flow rate of 0.7ml/minute
- Flush the column with mobile phase for 45 minutes (if mobile phase does not contain any modifier like triethylamine, diethylamine etc)
- Flush the column with mobile phase for at least 120 minutes if mobile phase contains modifiers like triethylamine, diethylamine etc
- Start the analysis as per method
HPLC Column Equilibration in Gradient mode (RPC)
To equilibrate the column in RPC gradient mode is the challenging task for any chromatographers and hence it needs special attention. Column equilibration depends upon the following factors:
- Nature of the molecule (neutral, acidic, basic and mixture of neutral, acidic and basic compounds)
- Mobile phase (without modifier and with modifier) and
- Gradient
Use the following tips while equilibrating the HPLC column:
- Install the column and flush 5 minutes with water at the flow rate of 0.7ml/minute
- Make a short gradient preferably of 4 to 5 minutes
- Give at least 5 injections of diluent or mobile phase (if mobile phase does not contain any modifier like triethylamine, diethylamine etc)
- Give at least 9 injections of diluent or mobile phase (if mobile phase contains modifier like triethylamine, diethylamine etc)
- Set the chromatographic condition as per method
- Then inject at least 1 or two blanks
- Start the analysis as per method
Case study
Let us consider a sample mixture containing the following compounds of different polarities:
The following mobile phase gradient is used for the analysis:
| Time | A( 0.3MK2HPO4) and pH 10 with Triethylamine) | B(Acetonitrile) |
| 0 | 80 | 20 |
| 20 | 60 | 40 |
| 40 | 20 | 80 |
| 55 | 20 | 80 |
| 56 | 80 | 20 |
| 66 | 80 | 20 |
This is a longer gradient of 66 minutes and problem may arise during analysis due to column equilibration. Now we have to equilibrate the column fast. Use the following steps to equilibrate the column:
- Make a smaller gradient of preferably 4 to 5 minutes
| Time | A( 0.3MK2HPO4) and pH 10 with Triethylamine) | B(Acetonitrile) |
| 0 | 80 | 20 |
| 5 | 20 | 80 |
- Give at least 9 injections of diluent or mobile phase since mobile phase contains triethylamine
- Set the chromatographic condition as per method
- Then inject at least 1 or two blanks
- Start the analysis as per method
HPLC Column Equilibration in Normal phase chromatography (NPC)
Column equilibration takes longer time (at least 6 to 12 hours) in NPC depending upon mobile phase and column chemistry. In this case column should be equilibrated overnight at lower flow rate (0.1 to 0.3ml/minute) and then analysis should be started.
Advantages
- Fast column equilibration
- Prevent analysis error due to column equilibration
- Reduction in analysis cost
Conclusion
I hope this article has helped you understand HPLC column equilibration and its importance. Now you can independently equilibrate the column effectively. You may also want to check out other articles on my blog, such as how to clean and regenerate HPLC column and HPLC column.
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Top Interview Questions on HPLC Column Equilibration
What is the column equilibration?
Column Equilibrations is the process of stabilizing the HPLC column with mobile phase before the injection for sooth interaction of analytes with stationary phase and mobile phase
How to equilibrate the column in Isocratic mode?
In reverse phase isocratic, first column should flush with water for five minutes and then with mobile phase for at least 60 minutes
How to equilibrate the column in gradient mode?
A smaller gradient (up to 5 to 7 minutes) should be prepared and 5 to 7 injections of diluent/mobile should be given. 1 to 2 injections of diluent/mobile phase should be given as per method and then analysis should be started
What are the various effects of column equilibration on analysis?
There may be retention time shifting, variation in result and variation in area response if column is no equilibrated properly
What is the purpose of column equilibration?
To avoid analysis error which may arise due to column equilibration
How many column volume is required to equilibrate the column?
At least 10 column volume
How to equilibrate the column in Normal phase chromatographic mode?
Column should be equilibrated at least 6 hours
References
- https://www.chromatographyonline.com/view/column-re-equilibration-following-gradient-elution-how-long-long-enough-part-i-reversed-phase-and-hi
- https://www.scribd.com/document/583071621/What-is-HPLC-Column-Conditioning-How-Long-to-Equilibrate
Abbreviations
- HPLC: High pressure liquid chromatography
- RPC: Reverse Phase chromatography mode
- NPC: Normal phase chromatography mode
Disclaimer
The numerical data used in the tables or calculations are not actual data. It is designed to explain the topic.