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CCP4BB  October 2011

CCP4BB October 2011

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Subject:

Re: small 3ml Superdex column performance

From:

Tommi Kajander <[log in to unmask]>

Reply-To:

Tommi Kajander <[log in to unmask]>

Date:

Sun, 16 Oct 2011 10:55:08 +0300

Content-Type:

text/plain

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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




On Oct 16, 2011, at 4:14 AM, Artem Evdokimov wrote:

> 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.
>
> On Sat, Oct 15, 2011 at 5:04 PM, Alexandra Deaconescu <[log in to unmask] 
> > wrote:
> 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
>
> .
>

Tommi Kajander, Ph.D.
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1
(P.O. Box 65)
00014 Helsinki
Finland
p. +358-9-19158903
[log in to unmask]

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