Hi everyone, I’d asking about the elution step of protien in sep pak c18 , this is the first time that I’ve made the elution with acetonitrile , the question is when the elution process done the protien and the acetonitrile it will be mixed together right ??? could any one explain to me please
I don't think you would use C18 for proteins because they would be denatured by the acetonitrile and would precipitate. You could use it for peptides, however. If you elute with acetonitrile, the eluted material will be mixed with the elution solvent, of course. It is easy to remove the solvent by evaporation under vacuum or lyophilization because acetonitrile is very volatile.
C18 resins are used for desalting and wash buffers from samples. Hydrophobic peptides will be bind to the resin and then eluted in the organic solvent. I use C18 columns quite often with digested samples and after elution samples are dried as Adam B Shapiro suggested. Hope it helps
Adam B Shapiro Judit Aguilar udit Aguilar thanks for ur answers I appreciate it , is it possible to concentrate the sample by Centrifugal Filter after purification by sep pak c18 ? Does this help to remove the solvent ??
It is possible, but only if the filter unit is resistant to the solvent. Check the solvent resistance chart for the units you have.
Adam B Shapiro Judit Aguilar thank you for your help
I'd like to ask you for my peptide sample it is a transparent sample so when I've made the purification by Sep-Pak I can not see when my sample is clearly purified
so my question here is if I add dye to my sample is it ok? or it may disrupt my sample?
The dye might interfere with whatever you were planning to do with the peptide. If you want to analyze the sample, I suggest you use HPLC or UPLC with a reverse-phase column and 214 nm UV detection. If the peptide is pure, you should see a single peak. If you use LC-MS, you can also determine the mass and sequence. This analysis can be performed at a service lab for a fee.
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