Hi I'm Gerard Sharp. Welcome to the July 2011 issue of the monthly newsletter. This month the Tech Tip is on HPLC Column Care.

Dr Gerard Sharp...

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Tech Tip - HPLC Column Care

All HPLC columns have a definite lifetime. The better the column is treated, the longer it will last. Besides monitoring chromatograms of standards, it is also good practice to monitor performance through theoretical plates and retention factors. These relationships are defined here. There are standard procedures which you should follow to enhance column lifetime. We will focus on bonded silica reversed-phase columns as these are by far the most popular.

Filter the mobile phase

If the mobile phase is not filtered and particulates from the solvent enter the system, these will block up the frit on the inlet of the column and the resulting symptom will be an increase in system backpressure. Ensure the mobile phase is filtered through a 0.5μm filter. If you use HPLC-grade solvents, these are usually filtered in any case. Click on the icon on the left to view a degassing and filtering animation.

Prevent buffer precipitation

Ensure that the buffer is completely soluble in the mobile phase. Buffer precipitation will cause problems both for the pump and the column. If you are using a gradient method, ensure the buffer is soluble at all mobile phase ratios. This can be easily be done off-line by adding the buffer to the highest organic modifier amount (the mix at the end of the gradient run) and observing whether precipitation occurs. To extend this, you could even filter the mobile phase and weigh the filter both before and after to see if any precipitation has occured.

Don't allow the column to dry out

If the system is not being used for some time, remove the column and quickly plug the ends. Never store the column in 100% water as this encourages microbial growth which will block the pores. Have at least 10-20% organic modifier (e.g. acetontrile or methanol) in the column during storage.

 Use a guard column 

 In reversed-phase HPLC, the guard column will collect the very hydrophobic material and prevent this depositing on the analytical column. The guard column contains a small amount of phase of the same type as the analytical column and has a few hundred plates. To prevent contaminant material eventually spilling on to the analytyical column, the guard column should be exchanged frequently. It can itself be backflushed with 90-100% organic solvent but is usually discarded.

In-line filter

 An in-line filter will protect the column by collecting particulates generated from the pump seals and injector valve rotor seal wear and tear. The in-line filter (usually 0.5μm porosity) will capture smaller particles that could fit through the frits of both the guard column and the analytical column. Therefore the in-line filter should be placed ahead of the guard column and analytical column and after the autosampler.

Column Care Dos and Dont's - Summary:

  • Do filter the mobile phase
  • Do filter the sample
  • Do use an in-line filter
  • Do use a guard column
  • Do plug the column ends when not in use or in storage
  • Do remove components from the sample which will not elute from the column
  • Do use the correct column fitting
  • Don't let the column dry out
  • Don't pressure shock the column
  • Don't use immiscible solvents
  • Don't place excessive backpressure on the column
  • Don't open the column ends - most columns these days are packed at very high pressure 



Chromatography eLearning is yet another way to enhance your chromatography knowledge. While not ever being able to replace a classroom course, eLearning does offer advantages on cost and rich interactive content.  

Also, unlike a classrom course you can learn at your own pace and return to the content time after time.

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