January 25, 2021, 03:14:34 AM
Forum Rules: Read This Before Posting


Topic: Column chromatography  (Read 1313 times)

0 Members and 1 Guest are viewing this topic.

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Column chromatography
« on: December 30, 2020, 02:52:15 PM »
Hi there,
in my 4 years of studying med I've never come across something so complicated to understand..
How does one go about using (gradient: 0–40% ethyl acetate in hexanes to isolate sugars)

Do you first run the column starting from 0% ethyl acetate in hexanes and work your way up?


Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 4480
  • Mole Snacks: +273/-19
Re: Column chromatography
« Reply #1 on: December 30, 2020, 03:29:01 PM »
Yes, that is what I would infer.  Can you think of a reason for using a gradient?

The manner of increasing the percentage of ethyl acetate might be different depending on the particulars.  Are we talking about high performance liquid chromatography?

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #2 on: December 30, 2020, 03:55:53 PM »
Yes, it will be a high performance liquid chromatography. Also, does running the column with 0% ethyl acetate in hexanes at first mean the whole column into the flask or partially?


Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 4480
  • Mole Snacks: +273/-19
Re: Column chromatography
« Reply #3 on: December 30, 2020, 04:17:36 PM »
It means that solvent from a reservoir of hexanes is pumped through the column at first.  Then there is a mixer which gradually adds more and more ethyl acetate (from a separate reservoir) to the solvent that enters the column.  Have you ever used high performance liquid chromatography previously?

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #4 on: December 30, 2020, 04:48:13 PM »
Thanks for the replies Babcock that was actually a good explanation.

No unfortunately, first time.


Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #5 on: December 30, 2020, 05:16:25 PM »
Sorry.. where it says run through the column first with Hexanes, does it mean making the hexane run through the column into the flask before adding the Ethyl Acetate?

Also, is the ethyl acetate which is added later on diluted with Water or Hexane since it says Ethyl Acetate in Hexanes?

Thanks,

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 4480
  • Mole Snacks: +273/-19
Re: Column chromatography
« Reply #6 on: December 30, 2020, 05:38:14 PM »
It is understood from context that when one reaches 40% ethyl acetate, that the other 60% is hexanes.  Suppose that line A is hexanes and line B is ethyl acetate.  I would let 100% of A through the column at first to equilibrate the column in the starting solvent (this solvent is usually collected in a waste bottle).  Then I would apply the sample to the column.  Then I would start the gradient (the mixing is controlled electronically).  If this is preparative HPLC, I would collect each peak.  Collecting the peaks might not be necessary in analytical HPLC.

Are the sugars that you are working with chemically modified in some way, such as through acetylation?

If you have never worked with HPLC previously, there are a number of issues to be concerned with, such as the quality of the solvents, removing the particulate matter from the sample, and so forth.  I would advise finding a mentor, for lack of a better word.

Offline wildfyr

  • Global Moderator
  • Sr. Member
  • ***
  • Posts: 1582
  • Mole Snacks: +169/-10
Re: Column chromatography
« Reply #7 on: December 30, 2020, 05:39:54 PM »
Hold on here, it would be pretty unusual to run HPLC with just Ethyl acetate and hexanes, but no water. It would, however be very usual to run a benchtop column this way.

Are you using a large piece of glassware, or an instrument connected to a computer?

Offline Arkcon

  • Retired Staff
  • Sr. Member
  • *
  • Posts: 7365
  • Mole Snacks: +533/-147
Re: Column chromatography
« Reply #8 on: December 31, 2020, 01:06:42 AM »
Sorry.. where it says run through the column first with Hexanes, does it mean making the hexane run through the column into the flask before adding the Ethyl Acetate?

Also, is the ethyl acetate which is added later on diluted with Water or Hexane since it says Ethyl Acetate in Hexanes?

Thanks,

Briefly, if you are doing HPLC, then no.  Column chromatography, with a wide-bore glass tube of separation media, can use a pair of flasks, and a mixer, and transfer the gradually increasing mixture of solvents to a column. 

But an HPLC uses bottles and, increasingly these days, a "black box" containing proportioning valves, mixers, a stainless steel tube of media, and a detector to give you an answer.

https://www.pharmatutor.org/pharma-analysis/difference-between-conventional-chromatography-and-hplc


Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #9 on: December 31, 2020, 08:43:23 AM »
It is understood from context that when one reaches 40% ethyl acetate, that the other 60% is hexanes.  Suppose that line A is hexanes and line B is ethyl acetate.  I would let 100% of A through the column at first to equilibrate the column in the starting solvent (this solvent is usually collected in a waste bottle).  Then I would apply the sample to the column.  Then I would start the gradient (the mixing is controlled electronically).  If this is preparative HPLC, I would collect each peak.  Collecting the peaks might not be necessary in analytical HPLC.

Are the sugars that you are working with chemically modified in some way, such as through acetylation?

If you have never worked with HPLC previously, there are a number of issues to be concerned with, such as the quality of the solvents, removing the particulate matter from the sample, and so forth.  I would advise finding a mentor, for lack of a better word.

That's exactly what I thought it should be just like you explained in the lines of A and B.

No, none of the sugars have been modified in anyway and will analyse them maybe later on..

A mentor would definitely help a lot these days

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #10 on: December 31, 2020, 08:47:40 AM »
Hold on here, it would be pretty unusual to run HPLC with just Ethyl acetate and hexanes, but no water. It would, however be very usual to run a benchtop column this way.

Are you using a large piece of glassware, or an instrument connected to a computer?

A glassware is what I'm currently using, nothing connected to a computer.

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 4480
  • Mole Snacks: +273/-19
Re: Column chromatography
« Reply #11 on: December 31, 2020, 09:53:40 AM »
Are you following a protocol that was published in the scientific literature?  One reason that I am asking is that the solvent system you are describing does not seem polar enough to elute polyhydroxylated compounds.

Offline wildfyr

  • Global Moderator
  • Sr. Member
  • ***
  • Posts: 1582
  • Mole Snacks: +169/-10
Re: Column chromatography
« Reply #12 on: December 31, 2020, 10:06:43 AM »
Ok everyone, lets stop and read. OP is doing what I think is plain, bulk silica gel column chromatography. Not HPLC.


OP, are you using reverse phase silica gel? That would be somewhat common for sugars, but kinda expensive. As someone mentioned before, also for sugars, Ethyl acetate/hexanes is usually not polar enough to elute such molecules. That is a solvent system more suited to greasier fully synthetic molecules, like say, benzophenone. Often acetonitrile, methanol, dichloromethane, even small amounts of water or acetic acid are part of the elution system for sugars. Are you following a procedure or trying to develop one yourself? Even if you are trying to create one, looking in the sugar synthesis literature for solvent systems other people use would probably be very useful for crafting you own.

Are you testing eluents on TLC paper with a stain? Otherwise you are working blindly on the full scale each attempt.


To get back to your original question, this is how a gradient silica gel column is run, say from 0% ethyl acetate/100% hexanes to a 50:50 mix.

I would disperse my silica gel in hexanes, then run most of the solvent out of it. Then I add a few hundred mL more hexanes to  and run it down to <1 cm above the silica gel pack the column. Some people repeat this. I'd add some sand as a physical buffer for splashing and then drip on my crude product onto the sand. I run that product into the silica gel then carefully add a couple hundred mL more hexanes on top of that and run down to the silica gel. Then I make 10:90 EtOAc/hexanes and add that on top, and run most of it down. Then 20:80, add that on top etc, etc, until im adding 50:50 mixture and finish my column at that ratio.
« Last Edit: December 31, 2020, 10:19:11 AM by wildfyr »

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #13 on: December 31, 2020, 10:19:15 AM »
Pardon me when I said sugars, not long ago (around 3 weeks ago) I had been given an assignment of isolating sugars, due to working on it for so long I ended up typing "sugars" instead of "compounds" (occupational hazard I guess). 

Offline Jason09

  • Regular Member
  • ***
  • Posts: 12
  • Mole Snacks: +0/-0
Re: Column chromatography
« Reply #14 on: December 31, 2020, 10:47:17 AM »
Thanks wildfyr truly appreciated.

But don't you think a couple more 100ml's of hexanes once the crude has been dripped on to the sand will be too much solvent for the column to handle? or would you just remove some of the solvent if it reaches the top of the column?
« Last Edit: December 31, 2020, 10:59:23 AM by Jason09 »

Sponsored Links