Chromatography Forum

Hi!
I have a Q Fast Flow (anionic exchange) column in the lab that was packed by a former colleague. I would like to know:
1-a quick method to find out the column volume?
2-if in this type of chromatography it is more important to know the column volume or the void volume?

Also, any ideas on a protocol to test if the column/resin is working ok? Maybe with a commonly used protein like BSA?

Thanks!

1-a quick method to find out the column volume?

"column volume" is a bit ambiguous. The bed volume (volume of resin in the column) is easy: V = 1/4 * (pi * dc^2 * L) where dc is the radius of the column (half the diameter) and L is the length. The "void volume" (Vm = the amount of mobile phase in the column) is a bit more complex. For silica-based materials commonly used in HPLC, that can be estimated as Vm ≈ 0.5 * (dc^2 * L), but that assumes that the pore volume is around 30 - 35% of the packing particle volume, which is not necessarily true for other types of packings. When all is said and done, if you want an accurate value, you have to measure it by injecting a non-retained solute to find the dead time.

2-if in this type of chromatography it is more important to know the column volume or the void volume?

They serve different purposes. If you want to know how much resin it will take to fill your column, then the bed volume is more important. If you want to characterize elution time, then you need the void volume.

Hi, apotolicchio,

I have a Q Fast Flow (anionic exchange) column in the lab that was packed by a former colleague. I would like to know:
1-a quick method to find out the column volume?
2-if in this type of chromatography it is more important to know the column volume or the void volume?

Also, any ideas on a protocol to test if the column/resin is working ok? Maybe with a commonly used protein like BSA?

1-The equation for the volume of a column is the radius of the column squared in mm multiplied by pi (3.1416) multiplied by the column length in mm, quantity divided by 1000 [corrected formula for units]. This affords the mm^3 or mL unit.

2-The column void volume is more important to know, generally. It depends on what you want to do...see Tom Jupille's response above, he types faster than me!)

These sites may help:

https://www.sigmaaldrich.com/content/da ... 100pis.pdf

https://www.auburn.edu/~duinedu/manuals/QSepharose.pdf

http://www.bio-rad.com/en-us/applicatio ... matography

https://www.waters.com/webassets/cms/su ... 003347.pdf

This last link suggests a QC protein mix (or 3 individual proteins) to try out--this needs a bit of review to see if it will be useful to you and your application. Refer to pg. 4.

Thnk you all!