column chromatography

[pistolpete007]

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kaplan says non-polar cmpns move very quicky, while polar molecules are tightly to the gel....the more polar the solvent, the faster the sample will migrate

destroyer says the motion of solute and solvent through the solid phase is called elution. the more polar a cmpd is, the slower it will travel and the longer it will take to elute

the part in red seems to contradict can some1 explain?

 

[bumpski20]

are you sure they are talking about they same types of column chromatography? it seems like kaplan is talking about gel filtration while destroyer may be talking about hydrophobic interactions

 

[bumpski20]

nvm i take that back. kaplans doesnt make sense to me because first they say "non-polar cmpns move very quicky" but then "the more polar the solvent, the faster the sample will migrate".

gel filtration- kinda the opposite of sds-page in terms of the speed of migration. larger/polar molecules will migrate faster because the smaller ones can get stuck in the matrix.

hydrophobic interaction- hydrophobic (non polar) molecules will stick to the beeds while the polar molecules will run through. usually you rinse your protein in a high salt concentration to expose the hydrophobic core prior to running them through the beeds. you can elute the non polar molecules (which should be sticking to the beeds) after this by rinsing them in a low salt concentration to flip it back.

 

[cm5796]

Actually both are correct, they're just saying the same thing in a different way. Column chromatography relies entirely on the polarity of a molecule. Typically you set up a column full of silica gel which is very polar and adheres to polar compounds. This gel is known as the solid phase.

To move your compound through the gel, solvents of differing polarities are "run" through the gel. Typically you start with very nonpolar solvents (1:40 ethyl acetate: petroleum ether) and work your way gradually up to more polar solvents (1:10, 1:5, etc.). Depending on how polar your molecule is, a particular solvent system should enable you to isolate your desired material by sending the less polar molecules through the silica gel first, hopefully your compound somewhere in the middle, and the most polar compounds near the end.

I think you may be confusing the fact that Kaplan refers to the polarity of the solvent while Destroyer is talking about the polarity of the molecule itself.

Does that make sense?

 

[pistolpete007]

ok kinda of makes sense so will the polar one be mroe closer to the starting point since it moves slow....and the nonpolar be at the top since it moves fast?

 

[cm5796]

Not exactly. Columns work on gravity. There is a piece of glassware that looks like this:

http://www.wfu.edu/academics/chemistry/courses/CC/column.jpg

You load your sample onto the top and dump the different solvents onto it. Then you release the stopcock and collect the solvent that runs through the column in beakers or test tubes referred to as fractions. Theoretically there is one solvent system with the correct polarity that will let only your molecule move through the gel and everything else will either be already through, or stay put.

For instance, if I had an ester on my compound I would expect it to elute when I use a 1:20 mixture of ethyl acetate and petroleum ether. Therefore, I want to start with a 1:40 mixture and gradually work my way down (1:35, 1:30, 1:25) to 1:20 in order to remove all of the molecules that are less polar than my molecule. There could also be side products that are MORE polar than your molecule, but they should stay stuck in the gel until you get to a higher solvent polarity.

You determine whether or not your product is coming off the column by spotting it using TLC compared to a known product. Once you know you've got your product coming off in the right solvent system, you stick with that system until your product is eluted and you can clean up your column.

If you need more, just let me know.

(By the way, I've worked in an organic chemistry lab for the last year and a half so I'm not just reciting something out of a textbook)

 

[bumpski20]

all of this depends on what type of column chromatography you are doing. ion exchange is based on charge. affinity can be based on antibodies, poly A tails, or what ever knowledge you have about the protein or molecule you are purifying.