Chromatography Forum

Hi !

I'm attempting to isolate C-type natriuretic peptide from chicken brains, which have a pretty high concentration at .5-1 pmol/g tissue weight.

I've found a patent which details the exact isolation method, however it requires the use of HPLC. While I have frequently used column and ion exchange chromatography in the past, I do not possess an HPLC machine and definitely cannot afford one (seeing as they go for 10,000+$ on LabX!).

Could anyone help me with a suitable alternative that is cheap (looking for under 100-200$). I have access to any column/ion exchange columns I need, not sure about gas. The purity doesn't have to be super high, but I would prefer it to be over 75%.

Here's the patent with the isolation method, hopefully there is a different way! The procedure is about 3/4 down the page.

http://www.freepatentsonline.com/5338830.html

And if you don't want to look through it all, it basically entails extracting the tissues with acetic acid et al., running it through an HPLC column, and then running an ion-exchange column to yield 3 fractions, the third of witch is the crude final product.

Do you think I could skip the HPLC step altogether? I am unsure if the HPLC is used to simply purify it further; remember, I don't need 99% purity, simply over 75% will do.

Thanks everyone!

The golden years for HPLC technological advancements peaked some time ago. In other words, you can buy an older model Perkin Elmer pump/detector on Ebay. The shipping will likely be more than the actual sale price, and your system won't be all that different (performance-wise) compared to a later model. Don't expect climate-controlled autosample storage, in-line degassing, or automation of any sort, but, yes, an older pump will get the job done.

You may well end up spending more on fittings, solvent frits, plumbing

It seems that they use HPLC simply as a method of desalting peptides and then ion-exchange for fractionation. In this case you should be able to use Solid Phase Extraction columns instead of HPLC. Gradient ion exchange on SP-Sephadex is a low- to medium-pressure technique - you can do this with almost any kind of equipment: HPLC, FPLC, perystaltic pump, even gravity flow, although quality of result may vary.