I am looking to get into CE (mainly because I don't have the budget for an HPLC). One of the things I am looking at doing is separating peptides by size. I have read about CGE mode but it seems more for larger peptides and proteins. I am looking at getting more a profile of amino acids vs dipeptides vs tripeptides very very small peptides. What is the best way to go about this CZE?
CZE separates based on charge-to-size ratio. That means you are hoping your peptides will all have the same charge in solution. This will depend on the pH of the separation background electrolyte you will use.
All I know is that there are many ways you can approach the separation of peptides (additives, pH gradients, capillary coatings etc...). Decide if you want to exploit EOF for your separations or not. Make sure you have a reliable capillary cleaning method for in-between runs.
You will likely want to design a series of experiments in a type of factorial or simplex design to determine which parameters (pH, ionic strength, voltage, BGE composition, etc...) will produce the separation characteristics you are looking for (fast analysis time, baseline resolution, appropriate separation current, best signal-to-noise). Doing this is pretty easy for CZE.
Since your question is very preliminary, I can only recommend preliminary things and not specific things! There are lots of methods in the literature to help guide you along more specific lines!
All the best!
Matthew
That would be the tricky part. For larger peptides I could add SDS and everything will have the same charge/length however that probably wouldn't work with such small peptides. Also I suspect di and tri peptides are too small for something like capillary gell (how about molecular sieving?)
Another possibility is to derivatize the peptides. For amino acids I use ethyl chloroformate. That esterifies all the carboxylates and carbamates the amines and I believe the thiol on cys. That would just leave Arg and possibly Histidine. However that would render them very nonsoluble in aqueous systems.
Basically I am attempting a CE equivalent of size exclusion chromatography.
CZE with a formic acid based electrolyte gives a good separation of amino acids and small peptides. Separation is based on mass to charge, so in the case of peptides is affected by their mass and the number of basic residues (i.e. Arg, His. Lys). Hence, it isn't easy to determine mass based solely on CZE migration, unless of course you know the number of basic resides in your peptides. CE-MS would answer your question, but i'm guessing you don't have access to a MS.
No the main reason I am doing CE is we don't have a budget for an HPLC system let alone a LC/MS or CE/MS.
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