How to Clean and Regenerate HPLC Columns?

Introduction and Outcome

HPLC column cleaning and regeneration determines column performance, column life and accuracy of the pharmaceuticals result. Overtime solute accumulates on the stationary phase of the HPLC column and leads to high pressure and poor column performance. This puts chromatographers in a frustrating situation. That is why I decided to share my skill-based knowledge on this topic. In this article, I will share its solution by implementing right cleaning and regeneration procedure. After reading this post, you will be able to implement a column cleaning and regeneration procedure that will maintain the column in its original condition

Table of Contents

• Introduction and Outcome
• Need of Column cleaning and regeneration
• Column Cleaning and Column Regeneration
• Column cleaning
• C18, C8, C4, Phenyl and other RPC columns cleaning
• Silica, Amine, Diol and other NPC column cleaning
• SCX and SAX – Ion-exchange column cleaning
• Column regeneration
• C18, C8, C4, Phenyl and other RPC columns regeneration
• Silica, Amine, Diol and other NPC column regeneration
• SCX and SAX – Ion-exchange column re-generation
• Difference between column cleaning and column regeneration
• How to Extend the Column life?
• Advantages of column cleaning and column regeneration
• Verification of Column Performance
• Conclusion
• FAQs

Need of Column cleaning and regeneration

Due to accumulation of solute on the stationary phase, performance of the column is affected in the following ways:

• High back pressure in the column
• Poor separation between the peaks
• Peak tailing and peak fronting
• Low column efficiency or theoretical plate
• HETP (H)
• Late peak elution
• Baseline noise
• Ghost peak appearance
• Void formation (at end on position of the column)

Column Cleaning and Column Regeneration

Column cleaning

Column cleaning is the process of removing the mobile phase buffer and other contaminants after HPLC analysis. Column cleaning reduces column damage and increases column life. the column cleaning process is designed as per column chemistry and mobile phase/inorganic buffer used in the analysis.

C18, C8, C4, Phenyl and other RPC columns cleaning

Use the following steps for C18, C8 and other RPC columns cleaning

1. First flush the column with water for 5 to 10 minutes to remove all Inorganic salts from the column.
2. Mix water and organic solvent in the same ratio as these solvents were mixed in mobile phase. Then wash the column with this mixture for 30 to 60 minutes
3. Then flush the column with Methanol for 10 minutes
4. Remove the column from the system, close both ends of the column with end fitting and store the same in designated place.

Silica, Amine, Diol and other NPC column cleaning

1. First flush the column with Isopropyl alcohol for 5 to 10 minutes to remove all contaminate from the column.
2. Mix Isopropyl alcohol and Hexane in ration of 50:50. Then wash the column with this mixture for 30 to 60 minutes
3. Then flush the column with Hexane for 10 minutes
4. Remove the column from the system, close both ends of the column with end fitting and store the same in designated place.

Note: For any special column manufacturer’s recommended procedure should be followed.

SCX and SAX-Ion-exchange column cleaning

Use the following steps for SCX and SAX – Ion-exchange column cleaning:

1. First, flush the column with 50/50 water-methanol mixture for 60 minutes to remove all buffer from the column.
2. Then flush the column with methanol for 20 to 30 minutes
3. Equilibrate the column with mobile phase and perform the analysis

Column regeneration

Column regeneration is the process of removing the inorganic buffer, dissolved stationary phase and other contaminants from the column. Column regeneration restore column performance in original state. Regeneration procedure is performed only for those column that have lost their performance.

C18, C8, C4, Phenyl and other RPC columns regeneration

Use the following steps for C18, C8, C4, Phenyl and other RPC columns regeneration

1. Reverse the HPLC column and reconnect in reverse direction. The back flush the column with water for 5 to 10 minutes to remove all Inorganic salts from the column.
2. Mix water and organic solvent in the same ratio as these solvents were mixed in mobile phase. Then back-flush the column with this mixture for 30 to 60 minutes
3. Flush the column with a 50/50 water-methanol mixture for 20 to 30 minutes
4. Then back flush the column for 10 minutes with each Methanol and Isopropyl alcohol
5. Remove the column and reconnect in the normal direction. Repeat the step-2
6. Equilibrate the column with mobile phase and check the performance

Silica, Amine, Diol and other NPC column regeneration

1. First flush the column with Isopropyl alcohol for 5 to 10 minutes to remove all contaminate from the column.
2. Then flush with Etanal for 10 minutes
3. Mix Isopropyl alcohol and Hexane in ration of 50:50. Then wash the column with this mixture for 30 to 60 minutes
4. Then flush the column with Hexane for 10 minutes
5. Equilibrate the column with mobile phase and check the column performance

Note: For any special column follow the manufacturer’s guidelines

SCX and SAX – Ion-exchange column regeneration

Use the following steps for SCX and SAX – Ion-exchange column regeneration:

1. Double the concentration of buffer in the mobile phase ( keep in mind there should not be any precipitation in the mobile phase) and then flush the column 30 to 60 minutes using this mobile phase.
2. The flush the column with 90/10 water-methanol mixture for 40 to 60 minutes to remove all buffer from the column.
3. Then flush the column for 10 minutes first with methanol then with isopropyl alcohol and then again with methanol
4. Equilibrate the column with mobile phase and perform the analysis

Difference between column cleaning and column regeneration

Verification of column performance

Manufacturer’s COA or In-house SOP can be considered to acceptance criteria of the column. Generally, system suitability parameters like column efficiency (N), tailing factor(T) and resolution (R) are considered for verification of the column performance.

How to Extend the Column life?

Adopt the following procedure to increase the column life:

• Proper sample preparation: Proper sample preparation like filtration and centrifugation be adopted.
• Proper column washing: It increases the column life and keep the column in performing state
• Use Guard column: It removes impurities from the mobile phase buffer and keep the column in performing state.
• Regular system flush: It removes all deposited solute from the system (like injection valve and tubings) and keep the column in a performing state:
• Use HPLC grade solvents and chemicals: It reduces the column contamination due to solvent or solid buffer
• Column storage: Keep the column as per manufacturer recommendation
• Use of column log book: It takes case of column performance like number of injections, column-pressure and SST status

Advantages of column cleaning and column regeneration

The following are the advantages of the column cleaning and column regeneration:

• High column efficiency or theoretical plate (of the peak)
• Low HETP
• High resolution (between the peaks)
• Acceptable tailing factor
• Fast Analytical method development
• Fast routine analysis

Conclusion

The result of Pharmaceuticals depends on the performance of the HPLC column. Therefore a standard operating procedure should be implemented and followed. It will be very helpful in keeping the column in performing state and it will also avoid result failure related to analysis. I hope, this article has clarified all your doubt related to column cleaning and regeneration. Write your learning and suggestions related to this post in the comment section.

FAQs

How to regenerate the HPLC column?

Column regeneration depends upon the type of Column. For reverse column like C18 and C8, flush the column in reverse direction first with water for 5 to 10 minutes, Then with mixture of 50/50 water-methanol for 50 to 60 minutes. Then flush for 10 minutes with each methanol and isopropyl alcohol. The equilibrate the column with mobile phase and perform the analysis as per method of analysis

How to clean a HPLC column?

Column cleaning depends upon the type of column. For reverse column like C18 and C8; after analysis first flush with water for 5 to 10 minutes and then flush with the mixture of water and organic solvent in the same ratio as in the mobile phase for 30 minutes. Then flush the column with Methanol for 10 minutes.

What is the purpose of Regeneration?

The purpose of regeneration is to keep the column in performing state.

How to wash a C18 HPLC column?

For C18; first flush with water for 5 to 10 minutes and then flush with the mixture of water and organic solvent in the same ratio as in the mobile phase for 30 minutes. Then flush the column with Methanol for 10 .

What is cleaning procedure for HPLC column for normal phase?

Normal phase column like Silica; first should be flush with Isopropyl alcohol for ten minutes. Then it should be flush with 50/50 hexane-IPA for 30 minutes. Then it should be flushed with hexane.

How do I use a column for a longer time?

Abbreviations:

• HPLC: High-performance liquid chromatography
• HETP: Height equivalent to theoretical plate
• NPC: Normal phase chromatography
• RPC: Reverse phase chromatography
• SOP: Standard operating procedure
• SST: System suitability

References:

• Principles of instrumental analysis; Douglas A. Skoog, F. James Holler, Stanley R, Crouch