Which chromatography technique is most suitable?

  • #1
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Homework Statement


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Two proteins of similar molecular weight (<900 Da), same net charge in the solution but with different amino acid composition is to be separated. Which one should be used?

a. Cation exchange b. Anion exchange c. gel filtration d. Reverse phase

2. My attempt:

Since the net charge is the same so there's no point using ion-exchange.

Gel filtration or Size exclusion rules out as the sizes are the same.

So the only thing left to be exploited is the hydrophobicity of the side-chain of amino acids.

I have a feeling that hydrophobic interactions are least in proteins, less no. of hydrophobic groups are to be found in one as compared to polar groups. So using reverse will be best because...

Well I think I'm missing out something. Aren't we suppose to decide the combination of stationary and mobile phases based on the component of analyte we want to elude at the end?
 
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Answers and Replies

  • #2
epenguin
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When I saw this question I had a feeling of being out of date, which I am. I have never heard of reversed-phase chromatography used for protein separation. I understand this to be typically nonpolar eluants which denature proteins.

But... the question says <900 Da. I don't call that a protein, I call that a polypeptide, something like a nonapeptide. So the denaturation issue should not arise. You have to consider how you get rid of the eluent after elution. If it's only organic solvent I suppose you evaporate it.

Your reasonings and also your doubts are logical.

The one I go along with is not gel filtration chromatography.

In theory you reasonably exclude ion exchange chromatography. However in practice this is not so absolute because the nonpolar parts and just the spatial distribution of charges makes the binding to the ion-exchange substrates somewhat unpredictable I think, so ion exchange chromatograqphy is still used though you might predict it couldn't be. You're told that the charges are the same (at all pH's?? - well it's possible especially if the peptides are related) but not what sign they are. Anyway you can play a bit with the pH of the solution, e.g. if it's moderately high the peptides will hopefully be more negatively charged, and you can use a resin such DEAE. Elution is done with a salt gradient or maybe a salt plus pH gradient. If the desorbtion conditions of the two peptides are quite similar you have to use a shallow gradient ofeluant concentration in the appropriate range.

Sorry if this doesn't give you a straight answer a, b, c, or d but the question does not IMO lend itself to tht very much; I hope we get some other contributions.
 
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  • #3
Ygggdrasil
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For peptide purification (which is what you'd be doing for <900 Da samples), reverse phase chromatography is the standard. Separation would typically be performed with a water:methanol or water:acetonitrile solvent system and you would empirically optimize the elution gradients for the particular separation. Solvent removal after fractionation would typically be done by evaporation (e.g. in a speed vac or rotovap) or lyophilization/freeze-drying.

For proteins where you do not want to use organic solvents, you can still separate based on hydrophobicity using hydrophobic interaction chromatography.
 

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