A lot of people ask the question, “How long does it take a column to equilibrate?”
There is an exact answer. You don’t need to guess.
It takes 5 column volumes to equilibrate a column. Now, you ask yourself the question, “What is a column volume?” That’s how long it takes to flush the column one time. That’s your t0.
Example – 5 centimeter column
So, in this column, my t0 is 0.5 mL. I know that it’s a 5-centimeter column. It’s a 0.5 mL void. So, I need five times that volume, which means I need 2.5 mL to equilibrate this column.
If I’m running 1 mL per minute, I need 2.5 minutes. If I’m running 2 mL per minute, I need 1.25 minutes to equilibrate the column. So, you should do the math every time you’re running and decide what your equilibration time should be, based on the volume of your column.
Example – 25 centimeter column
So, a 25-centimeter column, 2.5 mL void volume, you need five times that, 12.5 mL. You’re running 1 mL per minute, that’s a 12.5-minute equilibration time for those long columns.
So, do the math, make sure you’re using the right number for your columns.
Equation for calculating equilibration time:
Equilibration Time = (Void Volume x 5) / Flow Rate
where, Void Volume is the volume of the empty space in the column and Flow Rate is the rate at which the sample is being pumped through the column.
Using this equation, you can calculate the equilibration time for any column by plugging in the appropriate values for Void Volume and Flow Rate.