Chromatography Forum

Does any body have experience to separate Snake Venom on HPLC? I plan to use Size Exclusion Column. Is
XBridge BEH300 C4 Reversed-Phase HPLC Columns OK? thanks
Jady

You have mentioned a 300A Reversed Phase C4 column. Now it depends if your venom has large proteins and if they can be eluted from a RP phase. Maybe SEC column can separate the venom better as a first step. What snake venom do you want to analyse?

Crotoxin and Cobra Toxin. I also think Size exclusion column will be good.

Will anybody refer a brand SEC column? thanks

Is TSK's column good? such as SuperSW2000, G2000SWxl Bioasist G2SWxl

Ion exchange also works well for proteins. Are you doing preparative chromatography? If so reverse phase and HILIC is out as it will denature your proteins. I recall my coworkers were doing work with phospholipase from snake venom.

Snake venoms typically are proteins in the size range 15-20 KDa. That means they don't differ much by size, so SEC won't give you much resolution. Also, people usually want to retain the enzymatic activities of the proteins, which rules out denaturing modes like RPC. That leaves HIC (hydrophobic interaction chromatography, NOT HILIC [hydrophilic interaction chromatography]) and ion-exchange. Francis Markland's group at USC has used both modes to purify various snake venom proteins to homogeneity. Here are some references:

1) A.D. Retzios and F.S. Markland, Prot. Exp. Purif. 1 (1990) 33.
2) A.D. Retzios and F.S. Markland, Jr., Biochemistry 31 (1992) 4547.
3) M. Trikha, W.E. Rote, P.J. Manley, B.R. Lucchesi, and F.S. Markland, Thrombosis Res. 73 (1994) 39.
4) S.L. Loayza, M. Trikha, F.S. Markland, P. Riquelme, and J. Kuo, J. Chromatogr. B, 662 (1994) 227.

They used our company's HIC columns and ion-exchange columns from SynChrom. The latter are pretty much gone from the market, but we can supply you with good alternatives for those as well. Contact us offlist if you want to follow up.

Andy Alpert

PolyLC Inc.
(410) 992-5400
info@polylc.com

I agree with Andy. SEC will only work if you have minimum 500 Dalton difference in MW. If you have a TSKgel SuperSW2000 available in your lab, just try it, but remember to modify your SEC instrument, mainly you need a micro flow cell. Otherwise ask the manufacturer for a test column free of charge. I would also aks them for a HIC column to test. Affinity sometimes is also used. Do you want to upscale to semipreparative or even preparative work? Than HIC would be best to use.