January 25, 2021, 02:17:38 AM
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Topic: Column chromatography  (Read 1312 times)

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Offline wildfyr

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Re: Column chromatography
« Reply #15 on: December 31, 2020, 03:18:37 PM »
I said a couple hundred mL, but this is very dependant on the diameter of your column. I often did cokumns that had 1-2 liters total volume. Let's call it filling about 10-20 cm above the level of the silica gel.

Offline Jason09

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Re: Column chromatography
« Reply #16 on: December 31, 2020, 06:52:32 PM »
Never knew columns can be that big unless made for industrial use. 

But what do you think would be the right size of Silica mesh to be used in this type of separation?


Thanks,

Offline wildfyr

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Offline wildfyr

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Re: Column chromatography
« Reply #18 on: December 31, 2020, 11:56:03 PM »
By the way, I assume you are using some air pressure and doing a flash column?

Honestly... There isn't anyone around to show you how to do this? Any grad student in an organic chemistry lab does many columns per week and you could watch them run a few. Learning online via text is a garbage way to. There are YouTube videos if all else fails.

Offline AWK

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Re: Column chromatography
« Reply #19 on: January 01, 2021, 08:02:33 AM »
Let's start from the beginning. You present a very broad problem that you do not understand. No unmodified sugar can be chomatographed in the chromatography system you are discussing. Unmodified sugars in HPLC can can't even be chromatographed in pure water.
If you use the appropriate HPLC equipment, you only program it appropriately (also the concentration gradient), the computer does the rest for you.
For you, the most important thing is to understand the basics of chromatography, be able to choose the solid phase of the column packing and be able to choose the developing system. So you have to read a few chapters of the manual on the basics of chromatography, the instruction manual of the apparatus, a few publications on the separation of sugars by HPLC, and then have a long conversation with your mentor. He will show you what else you should read to be well prepared to perform the analysis (or analyzes).
By asking imprecise questions on the Forum, you waste your time and the time of people trying to figure out how to help you.

fixed typo
« Last Edit: January 02, 2021, 04:51:46 PM by wildfyr »
AWK

Offline Jason09

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Re: Column chromatography
« Reply #20 on: January 02, 2021, 03:20:24 PM »
By the way, I assume you are using some air pressure and doing a flash column?

Honestly... There isn't anyone around to show you how to do this? Any grad student in an organic chemistry lab does many columns per week and you could watch them run a few. Learning online via text is a garbage way to. There are YouTube videos if all else fails.

No, I'm currently using a normal phase chromatography not flash.

Offline wildfyr

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Re: Column chromatography
« Reply #21 on: January 02, 2021, 04:53:21 PM »
Flash refers to whether you use air pressure or gravity to run a full size preparatory scale column. Normal phase vs reverse phase refers to whether the silica gel is bare silanols or if it has been functionalized with a greasy alkyl or aryl silane.

Most often (but not universally) an HPLC is reverse phase, and a bench top scale preparatory column is normal phase.

Offline Jason09

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Re: Column chromatography
« Reply #22 on: January 05, 2021, 12:54:10 PM »
I have always thought that flash meant when you use air pressure during chromatography, by the way I am looking forwards to packing my column with Alumina, will it be a wise approach to use silicon dioxide (sand) to not let solids escape from the column and at the top for buffering when using alumina?

Offline MOTOBALL

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Re: Column chromatography
« Reply #23 on: January 05, 2021, 03:51:33 PM »
What ??
Alumina???
You are on the way to snatching defeat from the jaws of victory!!!

At this point, I believe that you only have a gravity column to work with NOT HPLC---is this correct??

But seriously, you need to name,
1. your sample matrix (aqueous; water/methanol; acetonitrile...??)
2. the individual compounds that you wish to separate/isolate,
3. name the likely ester or ether groups present
4. name the starting material (SM)
5. name ALL chemical/enzymatic steps performed on the SM and ALL reagents
6. decide on the scale of this endeavour (low mg to low g of matrix)
7. name the detector systems available to you
8. give column dimensions (mm)
9. do you have any gradient forming apparatus for an open-column (i.e. gravity) set-up?

If you give us 1-9, we could suggest a thin-layer chromatography (TLC) system (using silica gel plates) to help you scout for a suitable LC system.

There is more than enough expertise on the forum to guide you through this, but very difficult to hit a moving target.
Please help us here to help you.

Regards,
Motoball


Offline Jason09

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Re: Column chromatography
« Reply #24 on: January 15, 2021, 05:55:20 AM »
Hi Motoball

Due to having a shortage of info based on the extraction I'm trying to carry out I'm unable to provide you with answers for some of your questions but hope this helps.

The starting material will be: White Ginger (from Southern Asia)
The compound I need to isolate is: DPPH (2,2-diphenyl-1-picrylhydrazyl) due to its ability to boost the immune system.
The sample matrix: Ethyl Acetate in Hexanes
And the scale of this endeavour is low g of matrix

Hope this helps Motoball, please let me know what you thing.


Thanks

Offline wildfyr

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Re: Column chromatography
« Reply #25 on: January 17, 2021, 10:00:55 PM »
2,2-diphenyl-1-picrylhydrazyl

What a fantastically wacky molecule. Before you do anything you need to come up with some way to run the chromatography system on a TLC plate of either silica or alumina and identify the spot that is the target molecule. If you can't do that, then everything else is moot.

FYI you are about the most difficult actually earnest poster I've ever met on the forums. I can't tell if its a language barrier or a total lack of education on chromatography, but we have zigzagged all over the place. Half the answers here are for HPLC. It would be FAR more efficient if you could find literally anyone in real life to show you how to run a column. Almost every chemist in the world was shown by a more experienced lab mate the first few times they did this sort of thing, because its so easy to suck at it.

Offline MOTOBALL

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Re: Column chromatography
« Reply #26 on: January 18, 2021, 12:51:18 PM »
Jason,

BEFORE you set foot in the lab,

1. Order DPPH from Sigma Chemical Co. for use as a standard
2. Order tlc plates (silica gel) approx 5 cm x 15 cm glass plates OR equivalent
3. Mason jars with screw-on lids, in which to perform the tlc (say 2 or 3).
4. Large filter paper to line inside of jars.

Get hold of “ Thin Layer Chromatography “ by Egon Stahl for everything you ever wanted to know abou t tlc.
I’ll add more after lunch.

Regards,
Motoball

Offline MOTOBALL

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Re: Column chromatography
« Reply #27 on: January 18, 2021, 03:35:42 PM »
Jason,

You have struck it lucky, because DPPH is a free radical (unpaired, single electron) and has a lambda max at 517 no, i.e. is a purple solid and in solution.
Easy to detect on a tlc plate or column visually, UNLESS obscured by crud in the ginger extract.
I would suggest that you aim for an Rf value between 0.2 and 0.5 on the tlc plate; this will allow for other components of the extract to be separated from the DPPH.
Once you have isolated the DPPH, in several fractions, combine them, reduce the volume to concentrate and then re-analyze by tlc to check purity of DPPH; for this you will need a spray of 5% conc. H2SO4 in Ethanol and a hot plate or oven.
Other organic s will be charred to give brown/black spots.

Check the directory of your Chem Dept to see who, if anybody, deals with natural products.
Do some quick reading about DPPH and tlc/ column chromatography before you shoot over to their lab.
Everything is at your Google fingertips!

Good Luck,

Motoball
 

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