• Created : 
  • Modified : 
  • Category :  Agilent Knowledge Portal
  • Entry Type :  Article
  • Product Type :  Analytical LC Systems
  • Component :  LC Column Compartments
  • Product Name :  1100 Series LC System 1120 Compact LC 1200 Series LC System 1220 infinity II LC System 1260 Infinity II Bio-Inert LC System 1260 Infinity II LC System 1260 Infinity II Prime Bio LC System 1260 Infinity III Bio-Inert LC System 1260 Infinity III LC System 1260 Infinity III Prime Bio LC System 1260 Infinity III Prime LC System 1260 Infinity LC System 1260 Infinity II Prime LC System 1290 Infinity II Bio LC System 1290 Infinity II LC System 1290 Infinity III Bio LC System 1290 Infinity III LC System 1290 Infinity LC System
  • Task :  Best Practices Maintenance Operation
Related Articles

  • Troubleshooting Agilent 5975 and 5977 GC/MS Air Leaks

    This Information Applies To: 5975 and 5977 GC/MS systems with MassHunter Acquisition software Issue: High-quality GC/MS data requires a low, and stable background of air and water present in the MS analyzer. This article demonstrates a systematic procedure to diagnose and troubleshoot a higher than normal air background. Introduction: Air and Water background will always be present in the GC/MS vacuum system. Air background is at m/z 28 (Nitrogen) and m/z 32 (Oxygen). The ratio of...

  • How to Replace the Electron Multiplier Horn - 5977 Series GC/MSD

    This Information Applies To: Agilent 5977A/B/C GC/MSD Issue The Electron Multiplier Horn must be replaced when it is contaminated or the EMV value reaches 3000 V with a clean ion source. Items Required Electron multiplier horn (5977B/C: p/n G7002-80103 , 5977A: p/n G3170-80103 ) Gloves, clean, lint-free Large (p/n 8650-0030 ) Small (p/n 8650-0029 ) Long nose pliers or tweezers Steps to Follow Warning : The analyzer, GC/MS interface, and other components in the analyzer...

  • How to Replace the Gold Seal on the Split/Splitless (S/SL) Inlet for an Agilent GC

    This Information Applies To: Agilent 6890, 7890, 7820, 8890 and 8860 GC Systems with Split/Splitless (S/SL) inlet Issue Replacement of the Gold Seal is required as part of inlet maintenance. Replacement is required when the performance of the inlet degrades over time due to accumulated contamination (e.g., baseline noise, ghost / artifact peaks, loss of reproducibility, carry over, loss of compound response or no compound response). Items Required The following Gold seals are available...

  • Agilent AA Atomizer Disassembly and Re-assembly

    This Information Applies To: All Agilent Atomic Absorption Spectrometry Systems (AA55, AA240, AA280) Issue: How to disassemble and re-assemble the atomizer (burner, spray chamber, nebulizer) Steps to follow: Warning : The burner operates at high temperature. Allow sufficient cooling time before handling. There may be traces of acid on surfaces of the atomizer. Wear appropriate personal protective equipment. 1. Ensure that the flame is extinguished and the burner...

  • How to Bake-out the Split/Splitless Inlet (S/SL) on Agilent GC Systems

    This Information Applies To: Agilent GC Systems with Split/Splitless Inlet (S/SL) Issue The Split/Splitless Inlet (S/SL) can accumulate contaminants when high boiling point and/or high molecular weight compounds are present in the injected sample. Often these compounds are present from the sample matrix. The accumulated contamination is not effectively removed using the analysis method parameters. These accumulated contaminants may appear in the chromatogram as ghost or artifact peaks. They...

General Column Regeneration Procedure

Answer

This Information Applies To: Agilent LC Systems

Issue

After analyzing dirty or concentrated samples, some sample compounds may retain in the column and other parts of the HPLC system. If the column re-equilibration after each run is not enough to elute the retained compounds, you will notice several performance problems. 

Tailing or wider peaks, higher background signal, carry over, or ghost peaks can be a sign of a contaminated column. The use of low-quality solvents can also be the cause of those symptoms. 

As you use your column and expose it to various mobile phases, analytes, and sample matrices, these interactions will subtly affect it. Over time, there may be changes in its resolution. It’s important to keep a test chromatogram, from when your column was new, to compare and understand these changes in performance over time. If resolution degrades beyond an acceptable value for good quantitation, the column should be discarded and replaced with a new one.

Sometimes you can alleviate a high pressure or peak tailing issue by cleaning your column while back flushing it. Before cleaning your column, we recommend that you disconnect it from your detector and run your wash solvents into a beaker or other container. 

Check with your column manufacturer or review the instructions that came with your column before applying the following procedure. Start with the recommended cleaning or regeneration procedure from your column manufacturer. If it is not successful, you can use the procedure in this article as an alternative.

Steps to follow

 Caution 
Caution: Always use chromatographic grade solvents and wear the proper Personal Protection Equipment.

 

 Caution 
Caution: Methylene chloride, tetrahydrofuran, or dimethyl sulfoxide can swell PEEK components of the LC flow path. Confirm the material of the capillaries, fittings, or rotor seals before performing the following procedures. If any of the flow path components is made of PEEK, replace it or bypass it.

 

 Caution 
Caution: Check with your column supplier that one of the following regeneration procedures is suitable for your column and will not cause column damage before performing the following steps.

Regeneration Procedure for Reversed-Phase Silica Columns

  1. Disconnect the column and reconnect it to the system, with the flow through the column in the reversed direction. Do not connect the column to the detector to avoid contamination or clogging.
  2. Flush out any salts/buffers with HPLC grade water. Pump 25 mL of water through the column at 1 mL/min. For the following flushing solvents, use 1ml/min also.
  3. Flush column with 25 mL of isopropanol.
  4. Flush column with 25 mL of methylene chloride.
  5. Flush column with 25 mL of hexane.
  6. Flush column again with 25 mL of methylene chloride.
  7. Flush column again with 25 mL of isopropanol.
  8. Reconnect the column to the system, with the flow in the usual direction. Flush the column with the mobile phase without the buffer, then re-introduce the buffer.
  9. Equilibrate the column with 25 to 50 mL of mobile phase.
  10. To see if performance is restored, inject a standard or a sample.

Note: For some retained compounds that have fouled the column, dimethylformamide may be a better "cleaning" solvent than methylene chloride and hexane.

Regeneration Procedure for Normal-Phase Silica Column

  1. Disconnect the column and reconnect it to the system, with the flow through the column in the reversed direction. Do not connect the column to the detector to avoid contamination or clogging.
  2. Set the flow at 1 mL/min. Flush column with 50 mL of 50:50 methanol:chloroform.
  3. Flush column with 50 mL of ethyl acetate.
  4. Reconnect the column in the proper flow direction.
  5. Equilibrate the column with 50 mL of mobile phase.
  6. To see if performance is restored, inject a standard or a sample.
 Tip 
Tip: Follow the evolution of the signal and the background noise during the flush-out, especially before and after the regeneration procedure.

 

 Tip 
Learn how to effectively troubleshoot the Agilent LC Column:
HPLC-0GEN-2201e - Chromatographic Troubleshooting for HPLC e-learning course available from Agilent Education

 

 
Was this helpful?
Privacy Statement
Terms of Use
Contact Us
Site Help