size exclusion column eq. to Superdex 200?

[miro2009]

Hello,
I'm dealing with protein analysis using size exclusion chromatography and would like some help in finding suitable column with a narrow separation range that could allow separation of 60, 40, 19 KD protein mixtures with acceptable resolution, anyone have a good suggestion or recommendation of websites to search in?
I'm currently using Superdex 200 10/300 GL with excellent resolution, but it is not suitable for "heavy duty" analysis of 40 samples/day, in addition, run time is ~2 hours due to usage of slow flow rate (max allowed pressure in the column is 15 bars only)
I tried a range of TOSH TSK gel columns (eg TSK G2000 SW), but with very poor resolution...
Are there websites which could help you find columns from different manufacturers equivalent to specific required specs?
Thank you

[HPLCCONSULT]

Several months ago we had a potential customer contact us with same request and I will reply with the same answer here.

With the basic specs you provided you should have gotten a better result using the Toso TSK G2000SW column. It should cover most proteins in that Mw range. I will assume that you tried several of them in series and not just one column. SEC separations in this range usually require several of the columns in series to obtain the needed total surface area. In my lab, some years ago, we used as many as six columns in series to obtain excellent separations. This is a generalization, but true for many samples which require GPC/SEC type separations.

BTW: I have NO affiliation with Toso or any column manufacturer.

Good luck !

[ScottHorn]

For the range of protein sizes you're talking about, I believe a superdex 75 column might serve your needs better than superdex 200. I may be wrong on this, I use superdex 200 for the most part. I've only used 75 once or twice, but I believe all the proteins you're talking about fall into it's fractionation rage.

[miro2009]

Dear HPLCCONSULT, I tried only 1 TSK G2000SW column with a height of 60 cm, believe me, the profile is horrible where only 2 unresolved peaks appears and the 3rd component is not even separated.. I never tried connecnting several columns in series, is that a practical solution for developing an analytical method?
Dear ScottHorn, as I said Superdex 200 is perfectly fine regarding separation of the 3 components and resolution, I assume Superdex 75 will be ok too, but I'm trying to search for an analytical "stainless steel" column that can tolerate higher pressures therfore increase the flow rate and decrease the run time (currently it is 110 min!!) wit the same separation

[HPLCCONSULT]

Among other things, I have been doing high pressure protein separations for over twenty years and tweaking buffers and using gel columns in series is an ordinary thing to do. Without detailed knowledge of your sample, equipment, mobile phase(s) and columns I can not guarantee that using several TSK 2000's will solve your problem. Just that they often do work. These are typical solutions used by modern chromatographers doing high pressure SEC/GPC work on samples in that range. Your low pressure column is a different story and probably has a tremendous volume of beads to provide a good separation. Changing over to a high pressure column method is not always a simple task. You need to evaluate the mobile phase strength (buffers), column types and quantity of support available to the samples for separation (often rather poor/low). *Take a look in the literature to see what others are using to separate proteins in your range.

[Furry_dice]

Firts thing. This is my first post so please excuse any mistakes in protocol I might make, if I do something wrong please let me know.


I have similar problems. The Superdex columns will produce the separation but I am not willing to wait that long! In addition I find with proteins there is a definite limit to the number of injections I can do before having to replace or at least extensively flush the columns.

The TSK2000 work well but it depends on which column you use. If you have the SWXL the separation (in my opinion) is better but some proteins can stick. The PWXL is not as good for the separation so if you tried this that might be the reason.

As a last resort I have seen data at a conference for a new type column that will be released soon but has not, that I know of, been advertised. This is silica based but with an improved coating and from the data I have seen has an excellent resolution for proteins in the region you are looking at. I am not sure if this is an official product yet but if want to get some info please email mchen@wyatt.com who has used one and may be able to help you get hold of one.

I hope that was helpful

[hplcuser]

Try the Zenix (3um) http://www.sepax-tech.com/SEC.php 100,150, or 300A pore sizes from Sepax Technologies.
Or SRT (5um) 100,150,300,500,1000, or 2000A. I would probably suggest the 150 or 300A pore sizes in either product in my opinion.

[kurian]

i've separated a few years ago 3 proteins with the same Kda.
The most efficient is TSK2000SWxl or TSK3000SWxl.
For inhibited the hydrophobicity you can add a 30% MeOH in mobile phase.

Therefore, you can also use TSK BioAssist G2SWxl/G3SWxl/G4SWxl with the same separation as TSK2000/3000/4000SWxl, but without MeOH.

My Runtime is only 12 min with 1 ml/min and a mobile phase in PO4 50 mM pH 7.00

Good luck