size resolution in gel filtration

[dl318]

Hi,

I am trying to separate two proteins, 1 is ~600kDa the other ~450kDa. Is there a gel filtration column in existence for this type of resolution? Ion exchange has been attempted but is not successful.

Thanks,

Dan

[Uwe Neue]

The Protein KW-804 from Waters/Shodex is recommended for this molecular weight range.

In addition, the way SEC works is that you get the separation by increasing the plate count. If one column does not do the separation, you use two, or three or .... Old-fashioned GPC was done with 6 columns.

[Bill Tindall]

Uwe, these columns seem to have low pressure limits. Do yout soon hit a limit on adding columns because the first column can't stand the pressure?

[Uwe Neue]

Not at all, Bill. The issue is the friction on the column, not the pressure. Or with other words, it is the pressure per column. You can even do UPLC at 15 000 psi using 15 columns with a pressure limit of 1000 psi each (or any number of the same columns as long as your pressure per column is less than the specified limit).

[tom jupille]

Uwe, I see your point as far as the packing is concerned (it's the pressure differential across the column that matters) but what about the end fittings? My understanding was that the Aquity system requires special column hardware to operate at the ultra-high pressure.

[HW Mueller]

The Tosoh TSKgel Super SW 3000 (4.6 X 300 mm) might be the only column to do this with a single column. Careful, though, this is a very touchy column.

Uwe, what about diffusion when you use many columns in series?

On pressure, if you have material which swells on contact with solvents (sephadex?) wouldn´t pressure change the equilibrium, and thus size?

[Uwe Neue]

Tom,

endfittings and hardware all need to be reconsidered when going to UPLC. I was only jokingly suggesting to put 15 30 cm columns in series. However, in the old times, the common practice was to put 6 30 cm GPC columns in series.

[tom jupille]

Sorry, Uwe; my humor detector must have been malfunctioning.

However, that brings up a point. How high can "conventional" end fittings go? For years, the standard instrument rating has been 5,000 psi or so (350 bar), so they should go at least that high. But, how much higher will a nut-and-ferrule tube end go?

[Uwe Neue]

The old nut and ferrule steel tubes also reach pressures beyond 6000 psi. I believe that they can be used to over 1.5 times this pressure, but don't quote me on this (we have special column hardware since the 1970's). There is of course a limit. In addition, one can also get failures at lower pressures. I have seen several column packing labs where they proudly showed me a hole in the floor or the ceiling....