Hi there, I'm trying to separate these compounds (urea, creatine and creatinine) from a mixture of peptides. I'm wondering whether these compounds will bind to the column, too? I'm hoping they won't so that they won't affect the binding capacity. Also, does anyone know a good way to determine whether a compound will bind to a C18 column without having to do an experiment with a standard--i.e. is there some sort of number that can be used, some polarity threshold?
Dear David:
Urea is a non polarizable and polar molecule and consequently not retained onto C18 columns under the experimental conditions used in RPLC methodologies.
HILIC columns are much more suitable than Reverse-Phase High Performance Liquid Chromatography columns for its analysis.
Otherwise, creatinine is more polarizable and can be retained onto C18 columns.
In general, compounds highly polarizable (with conjugated double bonds or polarizable atoms such as I, P...) are strongly retained by C18 columns. Examples of highly polarizable compounds are coronene or phthalates. Otherwise, non polarizable or only slightly polarizable compounds such as urea are not retained.
See this paper on RPLC theory:
file:///C:/Users/auri/Downloads/Andrade-Eiroa%20_HPLC_polarization%20(3).pdf
I hope this will help you.
Best,
Auréa.
@Aurea Andrade Eiroa,
Thanks for the reponse--that link doesn't work though. Can you post another?
You are welcome, David.
Concerning the paper, please find it as attached file.
Best,
Hi Dave!
None of those compounds will be retained by C18 which is perfect if you want to get rid of them from your sample. Good luck!
Andrés, creatinine can be retained and analysed by C18 RPLC columns.
See this paper:
http://www.clinchem.org/content/31/1/109.full.pdf
Best,
http://www.clinchem.org/content/31/1/109.full.pdf
Aurea, totally true but they are using isocratic conditions with a mobile phase composed by more than 99% aqueous buffer and these compounds elute in a few minutes. For preparative purposes, you can easily apply David's mixture to a C18 cartridge and wash away (or collect) urea, creative and creatinine with a few volumes of 97% H20 and 0.1% TFA with minimal peptide lose.
Andrés, yes. I agree with you, polarizability of creatinine is low, on account of this, retention is not strong even under the experimental conditions of the job, with high percentage of water.
Best,
Hello David,
if you want a liitle retention of urea, you could try a amino column. I measure urea from eye chamber water with detection DAD and MSMS. The column is a Fortis Amino 150x3mm 5µ particle, Water and acetonitrile isocratic run 5min 97% acetonitrile. RT of urea is about 2 min. What happen with creatin & creatinine - I dont know, but in my opinion it could be worth to try it. I wanted to try also HILIC, but there was not enough time.
Dirk
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