[Mr.rose]

Hey guys,

I have a solution of the following two chemicals:

-Testosterone propionate (4-Androsten-17ß-ol-3-one Propionate)
-Estradiol Benzoate (17β-Estradiol 3-benzoate)

I'm Going to Run a Gravity Column chromatography with silica gel as my stationary phase to separate the two chemicals from one solution. Firstly I'm having trouble with picking an eluent. I don't know how i can obtain my Rf value when i already have my two components in solution. Do i just run a sample of it on a TLC plate and see how far the compounds separate? I'm not sure how easy this is going to be since both molecules are pretty similar, I mean Testosterone Proprionate is C22H32O3 with a MM of 362.5 and Estrodiol Benzoate is C25H28O3 with a MM of 376.5.

Secondly, these compounds are colorless in solution, which in this case they are both dissolved in Methanol (could i use this an an eluent since they are already dissolved in it?). My understanding is that they will not visualize under UV light because they need to have a florescent added? Since my TLC plates are homemade with silica gel and plaster of paris as a binder, i don't know how accurate everything will be. All i want is to be able to tell my fractions apart at the end of the column chromo experiment. But with these molecules so similar, I don't know how i can tackle this.

Any help will be kindly appreciated.

[Mr.rose]

Any one?

[sjb]

Typically, yes you'd run a TLC or two to try and see where the two compounds elute, and then collect many fractions in small vials to try and get separation

It's possible that the oestradiol will fluoresce by itself, but you may need to stain your plates to see where the testosterone is - perhaps something like permanaganate or similar?

[bolo]

Or you can use a 10% solution of H₂SO₄ and carbonization on a heater. It is typical procedure to visualisate steroids.

[Mr.rose]

Hey guys, thanks for your replies.

I cannot figure out a good eluent, would Methanol (99%) work as a good eluent? What are some common eluents that work well with many chemicals?

I can stain the sample with potassium permanaganate, would this be a good option if it does not originally show up under the black light?

What type of procedure are we looking at for the carbonization with H2SO4?

[sjb]

Methanol might be quite a bit too polar, ideally you'd be looking at Rfs of ~ 0.3 - 0.7 to make sensible suggestions from TLC. Perhaps something like ether, or DCM; though it is a bit empirical.

[Mr.rose]

The chemicals are already dissolved in Methanol, that is why i am asking. I would have to form a re-crystallization method to remove them from solution, or would i be able to evaporate on a hot plate?

Hmm, either and DCM are both extremely pricey in my part of the world, are there any other common eluents that may work? Do you have a link to a site which contains them, then i can experiment?

Also I'm having trouble finding a suitable silica gel, which one of these would work?

1. http://www.shopdynamicadsorbents.com/index.php?main_page=product_info&cPath=88_98&products_id=1096
2. http://www.shopdynamicadsorbents.com/index.php?main_page=product_info&cPath=88_96&products_id=1073
3. http://www.shopdynamicadsorbents.com/index.php?main_page=product_info&cPath=88_89&products_id=1006

Thankyou.

[sjb]

The silica itself should be OK, I think (have not made my own plates). Once you have the plates, you should be able to spot them, then perhaps heat before eluting to ensure a dry spot to drive off the methanol.

[Mr.rose]

With a bit of reading, i came across that methanol and CH2CL2 don't have that much of a difference in polarity.

Predicted dipole moment of methanol
1.882 Debye

Predicted dipole moment of CH2Cl2
1.807 Debye

So im assuming that use of either as an eluent could be considered?

Any very commonly available chemicals that can be used as an eluent, Im looking for a selection so i can calculate a good Rf.

[Doc Oc]

Just looking at those two structures I think you might be able to separate them with a combination of hexane and ethyl acetate.  The free hydroxyl of the estradiol should make it move much more slowly than the testosterone.  I agree that the estradiol should show up under UV, and it's possible the testosterone will as well due to the a,b-unsaturated ketone.  If not, KMnO4 (permanganate) should certainly get them both to show up on a silica plate.

I only use DCM/MeOH combinations when I'm trying to separate things like peptides that have very sticky hydrogen bonding components that make them slow-moving in silica.  I wouldn't recommend this as a go-to solvent combination.

[OC pro]

I (as a steroid chemist) strongly recommend hexane / ethyl acetate as eluent. Try a mixture consisting of 90%hexane, 10% ethyl acetate. Instead of hexane one normally uses now petroleum ether since hexane is highly neurotoxic.
Staining solutions: anisaldehyde solution (especially for steroids) or Ce(SO4)2/phosphomolybdate.
The androstenone will also be UV-active since it contains an enone structure. These steroids are normally UV-active. Therefore, staining solution is not really necessary.

[Mr.rose]

Thankyou for your help,

One problem is that i do not have enough hexane to preform a proper procedure, I have 100mL and just checked with my supplier, and i won't be able to receive any until the 6th of next month. I have an abundance of ethyl acetate (well 500mL or so) and an abundance of methanol. I was thinking a running the methanol first, then running the ethyl acetate afterward, hopefully obtaining a good separation.

Thank you for the staining solutions, i have seen those on my suppliers list, i will give them a go.

After a bit of consideration, i was thinking, i could create a very alkaline solution using a supersaturated solution of NaOH with at a 10:1 molar ratio to the quantity of estrodiol benzonoate, which should turn the estrodiol into a salt and leave the androstenone untouched for a period of time. Then the androstenone can be recrystallized out, and i can obtain proper values for these compounds individually, as on a TLC plate i will not be able to tell them apart.

[Doc Oc]

If you have pentane or heptane, those are perfectly good substitutes for hexane (the petroleum ether is a mixture of several hydrocarbons and it's cheap so that works well).

DO NOT use the NaOH solution on the estradiol, it will hydrolyze the benzyl ester that is protecting the alcohol as well as the propionate of the testosterone.  This will not improve separation and you will ruin your products.

Also, don't do MeOH then EtOAc, you're running the column backwards (ie; most polar solvent first, then less polar).  Even in pure ethyl acetate the two compounds will likely run together.

[Mr.rose]

Yes sorry you are right, i meant, run EtOAc before MeOH.

Ok I will run hexane and ethyl Acetate. So i get a picture of how much i would need, now many mL of eluent will it take before you estimate that both molecules will completely separate. There is 20,000mg of androstenone and 2,000mg of estrodial benzonoate in solution.

I will run the ethyl Acetate first then the hexane. But i do not know how long to run each for?

Yeh i realized that it would hydrolyze the proprionate ester as well, but was thinking that it would take at least 15-30min before that started, whilst a majority, if not all of the estrodial ester. I'll stick with the chromatography.

So how can i calculate separation time, as my biggest fear is that i wont be able to tell them apart because they are so similar.

[OC pro]

They can be easily separated...trust me.
Some advices: never use pure methanol in combination with silica gel TLC plates. It will simply dissolve the silica gel. Maxium is ~10% methanol. More doesn´t make sense.
Back to your problem: have you run a proper TLC with the androstenone/estradiol solution yet? This is a must before you run the column.