[nate]

A little guidance with my procedure on this experiment would be appreciated.  I spent an entire day in lab with nothing to show for it.

I am performing column chromatograpy with alumina as my packing material.  I have placed approx. .5 cm of sand below and above the alumina in the column.  The column has a small roller valve and plastic tubing to allow the fluid to drain.  I am separating Fluorene and Fluorenone in the column using solvents HEX, a soln of 70% HEX and 30% acetone by volume, and acetone.  Here is where one of the problem arises.

As soon as I GENTLY place the HEX on the column with pipette, there is a crack or separation in the alumina.  I should note that I did make sure the alumina was packed tightly.

I tried to collect 4 test tubes of fluid from the column and then use thin layer chromatograpy to identify the fluorene and fluorenone.  I never collected anything apparently.

I will say that it felt like I couldn't keep up with the column and the draining and it did run dry a couple times.  BIG NO NO!  

Do you think that I am allowed to close the valve to change test tubes, switch solvents, etc., as long as I have solvent on top of the column?  What is causing the separation of the alumina?  Is it okay to open the valve when first applying the HEX?

Thanks

[Mitch]

It is perfectly fine to close the valve in order to change the test tube. But, anyone experienced won't need to.

To avoid cracking I like to mix my alumina with hexane before I add it to the column. I mix it really well so that there are no air bubbles. Then flush solvent through it for awhile to make sure it is packed well.

The best advice is to ask someone in your lab to help you, because they shouldn't make you do this by yourself. Don't be afraid to ask for help, no one expects you to be a lab genius.

[Nucleophile]

Mitch: I used to do the same ( with silica) but when pouring the premixed silica and hexane, the silica got stuck on the walls above the hexane, and I had a real hard time removing it so it would fall down, and sink.,.

but otherwise, this method can be great.

I never worked with Alumina, what's it's properties?

[Dude]

A few pointers:

1.  The TLC plate must give evidence of complete separation of your two components (with the selected solvent and adsorbent) or you are wasting your time with column chromatography.

2.  Mitch's advice is good and makes preparation easier, although I didn't bother using a pre-formed slurry when I was in school.

3.  When adding eluent, always shoot it off the side of the top of the column and use a reasonably slow addition to avoid disrupting the packed sand and adsorbent (or else you might get the channeling you described).  If you have and maintain a liquid head, then you don't need to worry as much about cracking.

4.  Before adding the sample, allow the liquid to drain to the top of the sand and then close the valve at the bottom of the column.  Dilute the sample in a minimum of the eluting solvent and use the same approach of shooting it off the wall.  When all of the sample is added, open the valve and allow it to drain until it is at the top of the sand again and then start adding the eluent slowly (shoot off the column wall again).  Contine adding until a good liquid headspace is present and maintain it.  To get a good separation, don't exceed 1 g of sample for every 10 g of adsorbent (you should weigh out how much adsorbent you use, silica or alumina).  If the TLC indicates a very easy separation, then you might be able to exceed that ratio a little bit.  

5.  Set up a whole bunch (ie have 20 ready) of collection bottles in advance and change every 5-10 mL when you get close to detecting the compound.  Use a UV lamp (should be easy for fluorene) to determine when the compound comes out.  All you have to do is spot and shine for detection, don't waste time allowing it to elute until you have evidence of material coming out.

[Slyos]

If i were you i would also avoid the use of acetone as an eluent, as silica can catalyse the condensation of acetone, and therefore contaminate your product.

[nate]

Great feedback!  Thank you all.

Unfortunately we do have to work alone.  This doesn't mean I couldn't just get someone to help for a second.  The only problem I might forsee with the slurry is that we are supposed to put sand on the top of the column.  Is that completely necessary though???  If you feel it is unnecessary, I would use the slurry.  How would you add it to the column?  Just funnel it down and then wash the column with HEX until it is settled on the bottom??

I have gathered through the texts, that the first of the four tubes collected should have roughly HEX in it and little else.  The second should contain fluorene because it is non-polar and then tube 3 should have fluorenone because it is non-polar.  The fourth tube should have little more than acetone in it.  We have to use HEX and acetone as our solvents, because it is all we are given.  

If by headspace, you mean a space where the solvent is backed up over the adsorbent, I don't think that ever occurred.  It all seemed to just run off of the column so fast that I had a hard time keeping up with it.  I guess I need to practice with the flow of the valve to slow it down.

Thank you again for the tips.  I am kind of excited about getting back in the lab to finish the experiment where I was rather depressed from such a non-productive day.

Thanks

[movies]

For the slurry method you first add a layer of sand to the bottom of the column, then add solvent slowly and carefully so that you maintain a flat surface on the sand.  Continue until you have around 1/3 of the narrow part of the column filled with solvent.  Then slurry your solid phase (silica, alumina, florisil...) in your solvent and pour in the slurry.  The solvent already in the column should be enough of a buffer that you don't disturb the bottom sand layer.  Use neat solvent to wash down any solids on the sides and then pack the column under a pressure of compressed air or nitrogen.  Push solvent through until you don't see any cracks (air bubbles) in the column and there is no solvent above the top of the solid phase (but don't let the top go dry).  At this point, you can add sand to the top of the solid phase and you are good to go.

Be careful not to add the sand to the top when there is still a little solvent above the top of the solid phase; you'll be more likely to disturb the flat surface on the top of the solid phase.

Slurry packing is definitely faster than dry packing, but some people think it's not as effective for getting a really packed column with no air bubbles (which can affect separation).

[Borek]

I have no practical experience with column preparation, but - won't some vibrations help the slurry to settle down? If vibrated properly the slurry will behave as a liquid and all bubbles will just go up.

Similar technique is (was?) used in construction for concrete slurry to get flat and to fill all the gaps.

[movies]

Yeah, in principle that works.  It's hard to sonicate an entire column though!  I sometimes tap the sides of my column with a piece of rubber hose.  I'm not sure that it has a huge effect though.  I think it is more important to stir the slurry thoroughly prior to loading it into the column.  As Mitch mentioned, a lot of stirring in the beaker will get rid of most of the air bubbles.

[laotree]

Easy. Using vacuum to suck the column after packing with sorbent (silica gel, Al2O3). Adding solution before charge your sample, adding some pressure by N2 or air to make sure there is no crack. Actone is not a good solvent, try ethylacetate.

[nate]

Well, I performed my lab and I used most if not all of the tips that I was given here.  Megathanks!!!

I did "develop" a problem though .  After perfoming the column chromatography, we were to spot a Thin Layer Chromatograpy plate with the compounds in the test tubes.  Mostly to find that we had successfully separated the two compounds fluorene and fluorenone.  After development with Methylene chloride, I found that I didn't get a good separation.  This therefore, affected my melting point of the dried solid that I collected after evaporation.

So, the column chromatograpy went well, procedure wise.  Thanks to all who helped.

[movies]

Did you use methylene chloride as the eluent for your column?  That particular solvent is somewhat infamous for not behaving well on columns even when the TLC looks okay.  Sometimes it is the only solvent that works though!

[nate]

I used the methylene chloride for the development chamber in the TLC.  The eluent for the column was first nonpolar HEX to separate off the nonpolar fluorene and then the eluent was changed to a mixture or 70%HEX-30%acetone.  More polar to separate off the more polar fluorenone.  I didn't have much say in the eluent.  Frankly, I feel like I have studied the lab and the results that I should have gotten so much that I really did learn alot from the lab even though my results were crummy.  I just hope that I can present that well enough in the lab report.  I will just be glad to be done with this experiment.  I was so looking forward to it though.  Such is the life of an organic chemist I assume.

Thanks

[mike]

Why not use the same eluent for the column and the tlc?

[Mitch]

You usually want your column eluent and TLC solvent to be the same.