RP-HPLC! Help me!

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dmm520
dmm520's picture
RP-HPLC! Help me!

Does anyone have a good RP-HPLC protocol?  Specifically for caseins would be wonderful but any protein method that is clear and uses TFA:acetonitrile as solvent would work for me..
Also: what are the advantages to a C18 column versus a C8 or C4 for protein purification?
Thanks :) !

Omai
Omai's picture
Here are a couple of

Here are a couple of protocols that might help.
cshprotocols.cshlp.org/cgi/content/abstract/protocols%3b2006/28/pdb.prot4547
cshprotocols.cshlp.org/cgi/content/abstract/2006/28/pdb.prot4546
Here is an answer for you other question:
 "The stationary phase is generally made up of  hydrophobic alkyl chains ( -CH2-CH2-CH2-CH3 ) that interact with the analyte.  There are three common chain lengths, C4, C8, and C18.  C4 is generally used for proteins and C18 is generally used to capture peptides or small molecules.  The idea here is that the larger protein molecule will likely have more hydrophobic moieties to interact with the column and thus a shorter chain length is more appropriate.  Peptides are smaller and need the more hydrophobic longer chain lengths to be captured, so C8 and C18 are used for peptides or small molecules.  Here is an interesting note: Observations have been made that C8 columns are actually better for capturing smaller hydrophilic peptides, the theory here is that the longer C18 chains lay down during the early aqueous period of the gradient and the more hydrophilic peptides are not captured.  We use C8 routinely for all peptide work and this particular alkyl chain length works equally well if not better than C18 for all peptides."
It is a direct quote from this website (a detailed tutorial on RP-HPLC)
www.ionsource.com/tutorial/chromatography/rphplc.htm#The%20Column 
Good luck,
Omai

dmm520
dmm520's picture
Thank you so so much!

Thank you so so much!