Author Topic: Column Chromatography Tutorial  (Read 2663 times)

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bigblue

  • Guest
Column Chromatography Tutorial
« on: May 24, 2004, 01:49:00 AM »
For the bee's reading pleasure. 

http://www.chem.ubc.ca/courseware/121/tutorials/exp3A/columnchrom/



And Hello to all.

bigblue

  • Guest
Doesn't seem too difficult.
« Reply #1 on: May 24, 2004, 10:40:00 PM »
Doesn't seem too difficult.  Looks like a handy bee could whip up it's own column (if that was some bee's intent) rather easily.  Seems a little involved but nothing too difficult.  Any thoughts on this?  Swim is going to do some reading.  In search of the universal cure....

and hello back to myself :(

Aurelius

  • Guest
column
« Reply #2 on: May 24, 2004, 10:56:00 PM »
it's a good idea to have several sizes of columns (vary the width and the length) for different size samples


wareami

  • Guest
Nice Find!
« Reply #3 on: May 24, 2004, 11:22:00 PM »
I was meaning to reply at the time this was posted.
This info is right UP the Bees ally concerning front-side extractions.
Osmium has been pushing bees in this direction and this link takes some of the scare-E-ness out of the terminology.
Thanx bigblue!
Don't hurt yerself looking for a Uni-Cure cause the pHarmcritters ran off with the HenHouse this time.
Painstaking processes can be employed but it's like a crapshoot!
Hey? Where is that wiley old Crapshoot anyway?

Good Lookin out!


Chimitant

  • Guest
Very informative!
« Reply #4 on: May 24, 2004, 11:45:00 PM »
I really like this link. Written in a very simple style and not to detailed. Excellent reading for newbees!


amalgum

  • Guest
Why are you people scared by chromatography?
« Reply #5 on: May 25, 2004, 11:57:00 AM »
Why are you people scared by chromatography?  I KNOW some of you have steam distilled before, and that is harder to do than run a chromatogram.

The real main reason why the chromatography method for pseudo hasn't been developed yet, is becuase it's a trial and error process that takes a lot of time and tedious procedures to do.  There is no and cannot be a "universal" chromatography cure.   You don't just run the shit through a column and thats that. All pills are different, have different compisistions and gaks. Plus sometimes ingreidients for one type of brand can be different than the same brand in other stores/states/countries.  People have to learn chromatography themselves, and learn how to get it working for them themselves because of this.  Here's why:

Let's say your gonna use silica gel in your column as the absorbant, mainly because you have a bunch of silica gel tlc sheets (the same absorbant in column has to be used as whats on the tlc plates).  First, you'd have to extract the pseudo as best as you can anyway using conventional methods because in raw pill mass there is just way to many chemicals in there to be separated.  You'd have to do several long tedious trial and errors and column runs before you narrowed it down to just the pseudo.  This would take way to much time.  If you extract the pseudo as best as possible first, you not only do you have a better chance at separation, but it should take as many runs either.

Once you have done that, dissolve in some methanol in conc. suitable for tlc spotting (read up on tlc), and use it to spot several plates.  Then you have to develope the plates, one by one in different mixtures of eluents (again read up on tlc). Actually, once you get good at tlc, you will be able to do more than one at once, as you'll already have an idea at how polar the eluent needs to be to get good separation.
Once the first plate is developed, spot it.  Look at results, in tlc it's never perfect at first, in fact the results will be unusable.  See, in tlc, you have to see how the spots that you do see on that first run have risen up the plate, as the polarity of the substance you are trying to get an analysis on plus the polarity of the eluent will cause some compounds to move faster than others.  If the polarity is to far to one side (again which side depends on shit your purifying), all the spots will run farther up and closer together.  This will tell you if the eluent should be more polar, or more non-polar.  So, based on that you create a new eluent mixture, and develope the second plate.  Keep doing this until the spots are well separated, but not well enough so that the top fastest rising spots start rising off the plate.  When you develope a tlc plate, the solvent is only allowed to rise to a certain distance from where it is originally spotted.  Pick a spot, and measure distance in cm from starting place to center of spot, and divide by the distance from the starting spot to the top where the solvent stopped.  You should get a decimal number, which is called an Rf value for that particular compound.  Calculate all Rf values.

Then, you have to mix up some kind of testing solution, like a marquis reagent or something that'll change a unique color only for pseudo.  Get as many test tubes as you have spots on the plate.  Since each spot on the plate conatins such small amounts of that compound, you may have to develope several plates with the eleunt you found to work.  Now you'll scrape off the abosrbant where each spot is into each test tube (I said you may need several tlc plates so you can scrape the same spot from each plate into a test tube, to build up conc. of the compound).  Now, add your testing reagent to each test tube.  Whichever spot was the pseudo, will cuase the desired reaction in the test tube.  So remember to begin with which spot is in which tube, so the tube that has pseusdo in that is matched with the Rf value. You now have a good idea on how to separate using the column.

In the document at rhodium's, it says the desired component has to have an Rf value of like .5 or something (I think, don't quote me, you'll have to read the article yourself).  So now you must take some more of the crude compound, and spot several more tlc plates.  Based on the Rf of the pseudo spot on the plates used to indentify the spots, adjust the eluent polarity to make it run higher if it's Rf is lower than .5, or vice versa.  When you get it to hit that certain Rf value, NOW it is ready for the column.  Since you adjusted it to have a the Rf value the document at rhoduims already designed the proposed seperation column to work with, just follow the directions it gives you for that column.  This would be size of column, weight of absorbant to use (or it's height in column), the eluents to use, as well as how much should be collected in each fraction.
 Now, after all that tedious and very boring work, evaporate each fraction, cause there is more tedious work.  Now you have to add some solvent to each fraction collected suitable for spotting tlc plates, and spot a plate for each fraction.  Now develope all these plates at once in the same eluent that caused the pseudo to have a certain known Rf value.  When the plate or plates that give spots at that Rf value are noticed, then those are the fractions you'll save, and throw out the rest.  Then, if there ends up being more than one fraction containing the pseudo, then you'll have to pool them, evaporate, and start all over to separate it even further (which is almost garunteed).

Most bees do not have those fancy automatic pipetting machines and tlc developers. For those who don't know what that is, ever see those DNA labs where they have a humungous block with asses of tiny test tubes in it, and they have this overhead robotic pipetting block that you'll see dip into on block of tubes, move to the next and dip in there, going back and forth between the blocks, these are automatic tlc machines so the sceintist can do things like chromotagraphy in an afternoon.  This would make the thought of separating pseudo out of pills more practical.

We have to face it, right now, chromatography is just way to impractical and for pseudo extraction.

Osmium

  • Guest
You do not have to extract the pfed and clean...
« Reply #6 on: May 25, 2004, 01:03:00 PM »
You do not have to extract the pfed and clean it up like people are doing it now. You only need to extract as much of the pfed as possible. Contaminants will not matter since they will be removed during chromatography. The extract may contain pfed and all the gakks, chromatography will nevertheless clean it up.

You also do not need to achieve good separation between the individual pill components. All we want is the pfed, all the other crap doesn't need to be separated from each other. It won't matter if the other components will end up with an Rf of either 0 or 1, as long as the pfed is in the Rf=0.1-0.9 range.

Also, people should NOT do columns (well, maybe once or twice, for learning purposes). There is a much easier method on Rhodium's, which uses a buchner funnel and a filtration flask. This method is MUCH more economical since it uses way less SiO2 (or other absorbent) and solvents, and it's also much faster to perform.

> We have to face it, right now, chromatography is just way to
> impractical and for pseudo extraction.

Wrong!


amalgum

  • Guest
You do not have to extract the pfed and clean...
« Reply #7 on: May 25, 2004, 03:48:00 PM »


You do not have to extract the pfed and clean it up like people are doing it now. You only need to extract as much of the pfed as possible. Contaminants will not matter since they will be removed during chromatography. The extract may contain pfed and all the gakks, chromatography will nevertheless clean it up




No shit!  I was saying the exact same thing, I meant to only do a rough extraction, or else why would you even need to chromatograph?



You also do not need to achieve good separation between the individual pill components. All we want is the pfed, all the other crap doesn't need to be separated from each other. It won't matter if the other components will end up with an Rf of either 0 or 1, as long as the pfed is in the Rf=0.1-0.9 range.




I KNOW GODDAMNITT!  I said you have to adjust the tlc plates at first until you get good separation (on the plates), then use a reagent to identify each spot to find out which is pseudo, then adjust until your pseudos spot has Rf of .5.  I got that from here:

https://www.thevespiary.org/rhodium/Rhodium/chemistry/equipment/flashpad.html


THEN run it through the column.



Also, people should NOT do columns (well, maybe once or twice, for learning purposes). There is a much easier method on Rhodium's, which uses a buchner funnel and a filtration flask. This method is MUCH more economical since it uses way less SiO2 (or other absorbent) and solvents, and it's also much faster to perform.




Again follow the link above and you'll see the flashpad thing is what I was referring to all along. I've read that whole section at Rhodiums at least 100 times (along with probably the rest of the damn page, I've been going to Rhodiums for several years now).

I know my writing may be a little hard to understand or decipher sometimes, and I know it was a long post, but you should actually read them next time instead of skimming and then attacking my credibility.

If you think it's so pratical, what ideas have you got?  Seriously, I am interested, cause this is a good direction to lean toward for pill extraction.  Is there an easier way?


amalgum

  • Guest
sorry Os
« Reply #8 on: May 25, 2004, 03:55:00 PM »
For that initial burst of anger.  And I do realize that when I said:


First, you'd have to extract the pseudo as best as you can anyway using conventional methods because in raw pill mass there is just way to many chemicals in there to be separated.




It may have been a little misleading.  I wrote that in mind however, thinking of the methods used in the past on the pills SWIM used to use, which will yeild gakked crap on the ones with the new gaks upon extraction.  I simply meant that sometimes some simple procedures people DO still use now, should be ran still, and the separation is to be done using this.


Osmium

  • Guest
> https://www.rhodium.ws/chemistry/equipment...
« Reply #9 on: May 25, 2004, 07:02:00 PM »
>

https://www.thevespiary.org/rhodium/Rhodium/chemistry/equipment/flashpad.html



Yes, that's the one.

> adjust until your pseudos spot has Rf of .5.

The Rf doesn't matter. 0.5 or bigger is best, but any Rf will do as long as the other components are separated.


Rhodium

  • Guest
Dry-Column Flash Chromatography
« Reply #10 on: June 04, 2004, 05:32:00 AM »
Dry-Column Flash Chromatography
Alan J. Shusterman, Patrick G. McDougal, and Arthur Glasfeld

J. Chem. Educ. 74, 1222-1223 (1997)

(https://www.thevespiary.org/rhodium/Rhodium/chemistry/equipment/dry-column.flash.chromatography.html)

Abstract
Dry-column flash chromatography is a safe, powerful, yet easily learned preparative chromatography technique. It has proven useful in research, and an adaptation of the technique for use in large teaching laboratories (general chemistry, organic chemistry) is described here. The student version is similar to vacuum filtration, uses the same compact, readily available glassware, and inexpensive and safe solvents (ethyl acetate and hexane) and adsorbent (Merck grade 60 silica gel). The technique is sufficiently simple and powerful that a beginning student can successfully resolve diastereomers on sample scales ranging from 100 mg to >1 g.


Rhodium

  • Guest
Dry Column Vacuum Chromatography
« Reply #11 on: June 23, 2004, 05:04:00 AM »
Dry Column Vacuum Chromatography
D.S. Pedersen & C. Rosenbohm

Synthesis 2431-2434 (2001)

(https://www.thevespiary.org/rhodium/Rhodium/chemistry/equipment/dry-column.vacuum.chromatography.html)

Abstract
Chromatographic purification is an integrated part of organic synthesis. The Dry Column Vacuum Chromatography presented here, has excellent resolving power, is easily applied to large scale chromatography (up to 100g) and is fast. Furthermore, the technique is economical and environmentally friendly due to significant reductions in solvent and the amount of silica used. Therefore, it is an excellent alternative to the commonly used Flash Column Chromatography for purification in organic synthesis.



biotechdude

  • Guest
on your marks...
« Reply #13 on: June 25, 2004, 08:34:00 AM »
Dry Column Vacuum Chromatography

Looks good Rhodium.  Now the annoying part of working out which fractions to collect.  Any hints on TLC visualisers?  Or column solvents?

Rhodium

  • Guest
Suggestions
« Reply #14 on: June 30, 2004, 12:36:00 AM »
TLC visualizer: UV light or Iodine vapor
Elution Solvents: Same as a successful TLC (the mixture with which the amine Rf becomes 0.2-0.5).