Thursday, 6 February 2014

Help on: For how long should I equilibrate my HPLC column?

MTS HELPDESK
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Question:
"While operating an HPLC system my manufacturer told me to equilibrate my system for approx. 10 minutes. Though I came to know from available literature from various sources that equilibration time depends on column dimensions (which is coming out to be quite large). So what is the purpose of equilibration and how do we determine the time of equilibration? And also do I need to equilibrate every time I run my system?"

Answer:
"Equilibration of an HPLC system is necessary to achieve reproducible chromatography and therefore quantitative results. It would seem that equilibration results in a stable and even distribution of the mobile phase within the stationary phase in the column and thus it is required every time a system is set up for analysis. It has been found that for best results, when an HPLC system is first set up, a minimum of 20 column volumes is required. We measure it in column volumes because the time taken will vary depending on the dimensions of the column (as you have discovered). There is a formula that you can use to determine how long a time will be required for 20 column volumes and I refer you to a blog that I have written on this subject: Equilibration of HPLC columns

I also provide a calculator that will work it out for you on my website which you may find useful: HPLC Calculator

Typical symptoms of incomplete equilibration are drifting baselines and retention times. You will also need to equilibrate your system between each injection if you use a gradient method, 10 column volumes is usually recommended."


If you would like more advice on equilibration and other HPLC-related issues, then why not come along to our HPLC courses on the 10th to 13th June 2014 in Dublin? You can choose from: 
  • How to Run HPLC Methods (10th June)
  • How to Troubleshoot HPLC (11th June)
  • How to Develop HPLC Methods - Part 1 (12th June)
  • How to Develop HPLC Methods - Part 2 (13th June)
Visit the MTS Course List for more information including course descriptions and course fees.

   

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