When we calculate the packed column void volume, we take into account the space occupied by the beads and their internal pore volume. The values for pore volume and total bead volume will vary for different supports (the manufacturer can provide you with this data or you can approximate it). The packed column value will be less than that of just the empty cylinder's volume.

For basic low-pressure systems where glass columns are used, these values are usually approximated (gravity powered or similar systems using a small pump at low pressure).

For true high-pressure liquid chromatography columns (stainless steel columns packed with much smaller supports) we often multiple the cylinder volume by 0.7 to obtain an approx actual volume when it is full of support. In all cases, the true value can then be determined by actual measurement.

Thanks for the answer, Bill!

Just to be sure that I got what you said: in a glass column, pressure not greater than 0.5 MPa, the void volume is approximately equal to the empty cylinder volume?

What would be the justification of that? Aren't the beads still there?

Thanks

Apologies, I got ahead of myself.

In LP chromatography applications they simply calculate the bed volume of the empty cylinder. The beads have volume and of course change the actual volume, but they calculate the media volume separately. So, in this case, the void volume is NOT equal to the column volume because they calculate it w/o any media. Measurements will show you what it actually is.

I see...

Just one last point: as I said, the cylinder-calculated volume of my column is 500mL. However, if I have this column in some buffer, and then pass water through it, I can observe the conductivity changing in a sigmoidal way around the 500mL. I find it strange, since the void volume should be a smaller number.

The only justification that I can tell myself is that, since the sigmoidal change includes some volume range, the smaller volume that I'm talking about should be inside this sigmoidal change too. If this is it, then in my case (glass column, 0.5 MPa), the void volume should be no lesser than 90% of the column volume.

Thanks for you attention, Bill

Why estimate when you could measure it using an unretained sample?

What would be this unretained sample, in a size exclusion chromatography?

My best guess would be the buffer-water system I told you about, since any biomolecule would have some delay due to its size

For SEC, a molecule which does not (as in, can not) enter the beads because it is too large would not be retained. Select the compound based on your column specs. What is the Mw range of your column? How about a very high Mw compound? You can also monitor the baseline at low UV for the pressure pulse that will appear when the injector is switched. This mechanical artifact is very useful too.

The easiest way to determine the void volume of your column is to use Blue Dextran 2000, which is suitable for any kind of size exclusion columns. You should actually determine the void volume with every new column, because you need this value to calibrate the column properly.

However, when you just change the buffer, it will enter the pores but ALSO pass outside, where bigger molecules would pass through. So what you see is actually that the layer between the two buffers runs down homogeneously. So, after 1 column volume your column is filled with the new buffer.

Again, the void volume is important to establish the earliest possible retention time/volume. The (total) column volume is equal to the latest possible retention time (except for stuff that will stick on top because of precipitation etc).