small 3ml Superdex column performance

From: Alexandra Deaconescu
Date: 15 October 2011 23:04


Hi everyone:

I was hoping I could get your opinion on the performance of the small approx. 3ml gel filtration columns from GE Healthcare. We currently have an Akta fplc and we would like to do small runs using small volumes of sample. As far as I know we have two options:

1. use the Superdex 15/150 columns
these appear to have a slightly lower resolution (the no. of theoretical plates is about 25000 m-1)
these apparently can be directly hooked up to our Akta fplc

2. use Superdex PC 3.2
these have somewhat higher resolution (the no of theoretical plates is 30000 m-1)
these can only be hooked up using a special "Precision Holder" equipped with titanium end fittings (which I have heard clog easily?)

What is your experience with these columns?
Which option of the two do you recommend (cost is not a huge issue, but resolution and overall performance is)?
Have you seen significant band broadening when using these small columns with a regular fplc (rather than Akta's microFPLC)?

I would greatly appreciate your comments! Many thanks...

Bests,
Alex

.

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From: Artem Evdokimov

Hi,

 

You're probably referring to the Superdex 5/150 Tricorn column, with working volume of 3ml, and not the 15/150?

 

Those columns work quite nicely for small sample volumes. For analytical runs 15-25ul injection is pretty nice. The PC columns are originally designed to be used with the SMART system, which by the way used to be one of the best analytical products for macromolecules -- optimized path length, one-volume pumps suitable for directly running typical protocols, etc. etc. and for its time the OS was also damn good (OS/2). Sadly, GE did not come up with any direct replacement for this machine, as far as I can tell. AKTA is not optimal for analytical runs - tubing is too long, etc.

 

If you have an old HPLC system moldering in a corner I recommend either of these columns mounted directly in front of the detector. In our current setup the entire portion of the HPLC that is responsible for column selection and heating and so on is bypassed, so there are literally ~4-5 mm of (the thinnest available PPEK) tubing in between the injection valve and the column, and in between the column and the detector. Autosampler is a very helpful feature, esp. when analyzing fractions output from previous step, and as long as the column isn't clogged the run is 8-12 minutes (depending on buffer composition). Now, Agilent software for HPLC is absolutely horrible for this kind of work but it suffices.

 

Artem

P.S. for lower protein quantities don't forget to record the A210, in addition to A280 and A260.

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From: Tommi Kajander


Hi,

if you really want it SMART was replaced be Äkta Ettan and nowaays Äkta micro. The nice thing is the easy of use (same software).

However i other think HPLCs might be more flexible and cheaper (e.g. we have schimadzu - cant complain about anything,
interface is more complex, but you dont really need to care about all that... you use just a few options - and there are several other
manufacturers..) --- just as a comment on the analytical systems.  you can inject few microliters...

And the ca. 20 ml S-200/S-75 10/300 works fine for small volumes by the way. at least on our HPLC. why would you need to go smaller??
20-50 ug. in 20 ul should be fine.... did you try? you could try just chaning the tubing on your current Äktä purifier and injecting
via Hamilto syringe?? Connected to HPLC certainly will work.
Of couse if you want faster runs, thats another thing (i think these smaller columns are mainly good for fast screening of quality)

HTH,
tommi

Tommi Kajander, Ph.D.
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1
(P.O. Box 65)
00014 Helsinki
Finland


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From: Artem Evdokimov

Hi Tommi :)

I like AKTA systems a lot, and it's uncharacteristic of me to say this but the Micro is kind of a copout  - it's a repurposing of the general AKTA system towards smaller volumes and not a completely specific design like the SMART was. Now, I think I understand why they're doing this -- cost is lower when you recycle components.

 

Principal advantage of small columns on an HPLC-like device is the speed of run. At 10 minutes per cycle (and that's not counting any time one loses on changing buffers!) the speed is adequate to make a reasonable judgment regarding e.g. pooling fractions from a run, in order to decide what to do next, or figuring out if the purification is sufficient to stop and set up crystals, etc. (one hour for 4-6 fractions is not bad). Most other analytical methods are quicker (LC-MS at 4-7 minutes/sample is the next slowest option, everything else is faster) but sizing offers unique insight that's hard to match - SLS/DLS does not give the entire picture (unfortunately).

Of course this only applies to people who, like me, believe that purification should be done as quickly as possible and in as few steps as needed (ideally 1-3 steps in one day) and who also have to screen large-ish numbers of samples. In other situations a larger sample size on a larger column is better since one can collect the good fractions and do something useful with them :)

Artem