Low tech flash chromatography

[Beaker]

This is a slide show post. My care provider routinely gifts me samples in these small containers to try. This particular one was very strong in pinene. I have wanted to try a switch from Ethyl Acetate to acetone as the polar solvent in Chromatography so I gave it a try. The ratio as a gradient started at 20% acetone/80% hexane and proceeded slowly up to 100% acetone. I learned a bit about initial solvent used in packing and from now on intend to include 1-2% of the polar solvent in the hexane used to pack the column. It takes a bit to explain the subtle nature of why but that is the point of these labs is to observe the fine details and adjust.

The raw crude testing 60% nominal THC

The crude shown dissolved in acetone now sitting in a PTFE evap dish. These dishes are perfect for pouring.

The acetone and compound was poured through a lab filter rated “slow” which is very fine mesh.

I estimated the amount of Celite 545 here and needed actually about 50 more really after it dried because it was sticky. A fine powder is wanted for dry loading the compound like this. So I added more Celite then redisolved it all and remixed in hexane and dried again. Then ground to a fine dry powder.

Here is the silica gel column being packed. A cotton ball in the bottom plugs the column for the gel bed. A sand layer is added on top of the cotton ball and up until the column is a constant diameter. Then the silica gel 60 is added dry. Then a diaphragm vacuum pump is connected to the outlet and turned on pulling the gel down.

While the pump is on a hand massage unit with vibration is held against the lab stand causing brisk vibration in the gel during vacuum. This packs it in a highly consistent manner (Brownian motion). Finish packing with a spatula then a sand layer on top to protect the gel from turbulence. This shot shows the hexane descending under gravity through the newly packed column and is only an inch or so past the top so far. The line of hexane is descending evenly and straight and level. It did so all the way until the hexane eluted indicating that the column had been properly packed.

Shown here is the cake decorating airbrush pump with a tape modified end to provide the air pressure for the “flash” part of the process. Gravity feed could take hours. Flash is tedious enough lolz even with air pressure but it works fine. Note the color seperations in the column. The Celite is seen on top of the sand layer and has been washed clean of compound.

The browns on top represent highly polar compounds that even with 100% acetone just barely moves. These would be called waxes by most. It takes 100% methanol to flush these last parts off the column. The methanol causes the silica gel to swell and “cracks” the column run like this so a methanol flush ends the useful life of the column.

I will add that compared to DCVC this style of seperation is tedious lolz! It is hard to switch modes of thinking from the instant sort of results of a DCVC run to the more time consuming flash method I employed. However for tiny samples flash is a fun way to test ideas without using gobs of resource.

Last thing as a question. If I vibrate the tube long enough with sand in it the largest particles and pebbles bubble to the top. They are easily removed by spatula that way. So here is the question for The Fellowship of the Dab…

I use 35-75 micron size silica gel. Does this mean that if I vibrate the column but not under vacuum and do so long enough as loose loaded dry gel that the larger 75 micron particles would tend to migrate and pack on top leaving a much tighter 35 micron packing on the bottom once the vacuum is applied?

[alienrocketscience]

what is the make/model of the diaphragm pump your using

[Beaker]

From my order history.
https://www.amazon.com/gp/product/B01BH5W26G/ref=oh_aui_search_detailpage?ie=UTF8&psc=1

[skankhunt420]

I have had a read of your DCVC thread and I notice that the DCVC column you used (vac filter funnel), looks considerably smaller than the column you have shown here for the flash chromatography (FC). Are these columns so different in size due to the amount of product you are running or is it possible to use a much smaller column for DCVC compared to FC?

Are there any notable differences in quality or efficiency of seperation for DCVC vs FC? Based off what I have read from your posts, DCVC seems faster and more space efficient.

Are you aware of any isocratic chromatography methods for resolving crude? I would love to find a stationary phase that could use butane as a solvent to seperate crude as it leaves the extractor through a flash column

[Beaker]

For DCVC the length of the column becomes almost irrelevant beyond about 3.5 inches of total length. What dictates then the choice of column for DCVC is the diameter. This is because you are pulling the column dry of solvent between each gradient and so is a totally different dynamic than swamping a column with solvent and pushing it through. My explanation is not going to be high tech but maybe it works.

With flash chromatography the compound of interest is likely always on the move as are all of the other compounds in the solvent. As the solvent (mobile phase) passes down through a column the compound is continuously sticking to the stationary phase (the gel) and then unsticking in a continuous process all the way until it elutes from the bottom. The mobile phase is better at “unsticking” some compounds than others so some spend very little time stuck on gel while others spend more time. So a longer column gives each compound now traveling downward at different speeds the distance they need to seperate in the column.

The “flash” part is simply the notion that this can be sped up by pushing or pulling the mobile phase through the gel instead of gravity. With the right technique this style can produce high resolution seperations of small samples.

DCVC is a different thought process. The compound still sticks to the gel and is washed off as it moves through the gel but the column is not swamped with the mobile phase at all. The mobile phase is divided up into tiny discrete measurable units and applied one at a time then vacuumed through the gel. If the solvent is not strong enough to move the compound then it stays stuck or is super slow but as the solvent front passes and is pulled out the compound remains stuck in position. So it is not a continuous process of sticking onto gel then washing off. That part of the process is only given seconds to work really in my column because each fraction of just 20 ml is pulled through in about 30 seconds leaving whatever was too sticky behind in a dry column.

Then the solvent gradient is applied. Gradient just means with each fraction I make the solvent stronger in the sense that it is more capable of unsticking stuff from gel. With standard silica gel 60 this means I make it more and more polar with each fraction. Each fraction is very small and the power to move compound keeps increasing a bit at a time until all have moved through. The exploit here is the precise tuning of the mobile phase with each fraction. Flash certainly runs as a gradient if desired but it is not so nearly as easy to precisely control that gradient in such a targeted manner and with such ease. The difference between the two styles are subtle but as a practical matter the DCVC can process orders of magnitude more compound in just a fraction of the time of flash chromatography.

I have indeed tried a multitude of isocratic columns meaning of course no solvent gradient with DCVC. I have yet to find an isocratic mobile phase that can resolve (seperate) as nicely as a gradient does which frankly is the reason to run DCVC for me - precise and flexible gradient control.

I have also run a bunch of just gravity seperations of tiny samples in the small column. It’s fun and is how I started with the chromatography. I learned pretty fast about “slow” hehe with gravity fed chromatography but it absolutely reveals on a small scale what you are faced with when scaling up. It shows what is in the med visually.

[skankhunt420]

Nice explanation of key differences, as well as a quality introduction to chromatography theory.

Running the DCVC with a solvent gradient definitely seems like the way to go due to the control over the polarity of the gel and the resulting selectivity of what you elute from the column. I’m looking into getting some glassware now thanks for the help

[haloscorp]

Yes, using vibration alone should be enough to make the larger particles migrate to the top.

[skankhunt420]

@Beaker
Have you tried separating THCA via chromatography methods? Considering it is slightly more polar than THC perhaps it will have less affinity to the non-polar stationary phase and hence be able to be resolved through a column with a low polarity mobile phase such as butane? Just a thought…

[Beaker]

I identify THCa by evaporating the fraction seperately. All my evaporation is at room temp and ambient so overnight generally unless I am in a hurry. The fraction with the THCa will be shot through with bubbles and none of the rest of the fractions will.

On crude the THCa overlaps others of course but always elutes at the front of the solvent gradient ahead and overlapping the other cannabinoids.

So on crude generally as I run DCVC the THCa nearly always elutes at about the third fraction of 20 ml fractions just after the brighter yellow or orange carotenoids. They absolutely move through the column faster. If it was my intention to crystallize THCa I would do a DCVC first just to narrow it down but I really do not run big enough samples as a patient to fiddle with crystallization.

As far as solvent this means it is eluting at a gradient that is more toward the non polar solvent than the polar. The polar of course washes it through very fast but it is the non polar end of the mobile phase itself over silica gel 60 that gets the THCa to elute in my process.

[DrJosh]

how long will diahragm last?

[Bruce_leeroy]

For all eternity