Chemical Forums
Chemistry Forums for Students => Analytical Chemistry Forum => Topic started by: Jason09 on December 30, 2020, 02:52:15 PM
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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?
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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?
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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?
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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?
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Thanks for the replies Babcock that was actually a good explanation.
No unfortunately, first time.
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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,
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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.
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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?
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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
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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
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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.
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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.
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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.
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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).
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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?
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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.
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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,
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https://www.google.com/search?q=mesh+silica+gel+chromatography&oq=mesh+silica+gel+chromatography+&aqs=chrome..69i57.5165j0j7&sourceid=chrome-mobile&ie=UTF-8
Have you tried this yet?
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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.
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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
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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.
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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.
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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?
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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
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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
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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.
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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
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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