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### Topic: Elution order of proteins in a chromatography system  (Read 15551 times)

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#### xc630

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##### Elution order of proteins in a chromatography system
« on: February 03, 2009, 06:20:00 PM »
Hello, I would appreciate some help on how to attack this problem.

You have a protein mixture composed of four proteins with the following properties:

Protein 1: MW= 2000 pI= 4
Protein 2: MW= 18,000 pI=8
Protein 3: MW= 64000 pI=5
Protein 4: MW= 9000 pI=9.5

Predict the emergence order of the proteins when the mixture is chromatographed in the following systems:
- DEAE-cellulose (pka 11) at pH7, with a linear salt gradient elution
-CM- cellulose (pka 4) at pH7 with a linear salt gradient elution
- gel exclusion column with a fractionation range of 1000-28000

For the gel exclusion I think the proteins would elute in the following order: 3, 2, 4, 1. I think the larger proteins would just get around the beads directly but the small proteins would have to traverse through the beads.  However, I'm really not sure how to approach the problem with the first two systems.    I know pI is the pH at which a protein isn't charged so all the proteins would be charged since the pH is 7 in both cases. But how do the given pka's help me? Also do I need to take into account the MW for those two?  Thanks in advance for any *delete me*

#### Arkcon

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##### Re: Elution order of proteins in a chromatography system
« Reply #1 on: February 03, 2009, 06:50:22 PM »
OK, the moble phase is at pH 7, and the proteins are as well.  But, what happens to each column type at that pH?  You're given those pKa's as well, what can you do with them.  Failing that, you can just goggle for the name of the column media, and see what it's made of and try to get some hints that way.
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

#### aHerraez

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##### Re: Elution order of proteins in a chromatography system
« Reply #2 on: February 04, 2009, 06:25:11 AM »

DEAE-cellulose (pka 11) at pH7, with a linear salt gradient elution

DEAE is a cationic (amino) group. Hence, the column matrix will have a positive charge below its pK, e.g. at pH=7. (So, it acts as an anion exchange column)
Proteins are retained stronger the more negative they are.
So elution is in this order: 4, 2, 3, 1
Protein 4: pI=9.5, charge: positive
Protein 2: pI=8, charge: positive
Protein 3: pI=5, charge: negative
Protein 1: pI= 4, charge: negative

CM is an anionic (carboxy) group, so it has a negative charge above its pK, e.g. at pH=7.
Proteins are retained stronger the more positive they are.
(Solve similarly)

gel exclusion column
The larger proteins will elute first, within the fractionation range. So:

Protein 3: MW= 64,000
Protein 2: MW= 18,000
Protein 4: MW= 9,000
Protein 1: MW= 2,000