I am currently developing a research protocol involving antimicrobial peptides. I have searched various journal articles, and they describe that to purify these peptides I need to have sep-pak cartridges followed by preparative HPLC, followed by semi-preparative HPLC.
In the laboratory I will be working only analytical HPLC is available. So, is it possible to substitute preparative and semi-preparative HPLC with analytical HPLC in any way?
Thanks.
You can easily resolve individual amino acids and peptides by HPLC (do a key word search on the web and you will find plenty of free articles and papers on this), but if you need to collect the peptides for additional analysis or work, you will want to scale up the separation to collect enough material (semi-preparative or preparative HPLC).
How much material do you wish to purify? If you have a working analytical method which easily resolves them, just calculate the scale up ratio to figure out what sized column and flow rate you will need.
Yes, it is commonly done. Depending on the purity, you may have closely eluting peptides (differ by a single amino acid), so you may not be able to get a high load. A 10 mm ID column may only be able to purify perhaps 5 mg in a single run.
Different C18 columns also have different selectivity and resolving power for different compounds, so you may need to try a few different makes of columns to find one that works well for your peptides.
According to my current estimate,considering the number of specimens I am taking, I will have to purify around 50mg peptide mixture. Problem is that I don't have access to larger columns like 10mm or 25mm ID columns.
I don't mind doing many runs, but it doesn't seem like a feasible solution.
May be there are methods that circumvents HPLC??
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