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  1. #1
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    Column Chromatography

    olumn Chromatography: The Smart Man's Synovex Conversion.
    by: Mr.Rose

    Disclaimer: The contents of this publication reflect the opinion of the author only. This publication is entirely fictional and for informational purposes only. Under no circumstances should one attempt to replicate the provided procedures and methods. The use of the compounds in the manor described in this publication is against federal law in many countries and should never be attempted by any person.

    As many of you might know I have been working on a number of procedures to separate the Estrodiol Benzoate molecules from the Testosterone Propionate molecules in the Synovex-H Implants. After many failed experiments I have come up with two methods. One, being a method known as Fractional Distillation, is relatively simple and cheap to perform, however, this procedure is still not ready to be published and will not be covered in this article. The second, known as Column Chromatography, is relatively difficult, however, once understood is easy to grasp and achieve. The only cons to this method is the high start up cost once one factors in the laboratory equipment required, however, no other method will give a 100% separation and yield rate.

    In the following publication I will be referring to two techniques that go hand in hand; Gravity Column Chromatography and Thin Layer Chromatography. Don't feel disheartened as these methods are much more simple then they appear.

    Column Chromatography is a common method used by analytical and organic chemists to purify chemical compounds from mixtures of compounds. A common method used by 'backyard' chemists is gravity column chromatography. This method involves the use of gravity to help push the eluent, or mobile phase, through the glass column. Gravity fed columns are much slower then flash fed columns, which often use pumps to force the eluent through the solution, greatly reducing the running time of the column.

    Column Chromatography has three main components, the column, the stationary phase and the eluent, otherwise known as the mobile phase.

    The Column: is a glass apparatus similar to a burette that is used to hold the stationary phase and the mobile phase.

    The Stationary phase: Is a micro porous substance otherwise known as the absorbent, that is used to fill the column. The purpose of the absorbent is to form temporary weak polar bonds and in some cases hydrogen bonding between the molecules that are to be separated. It is the difference in the polar bonding strengths between molecules that determine how fast the molecules move down the column. Two of the most common absorbent's in gravity fed systems are silica and alumina at mesh sizes around 60-240 at 60A for gravity fed systems. Mesh size just refers to the size of the particles. To keep things simple, we will say that 1 mesh = 1um.

    The Mobile Phase: otherwise known as the eluent is a single or mixture of organic solvents that is mixed with the compounds needed to be separated and then poured into the column containing the stationary phase. As the eluent moves down the column it drags the compounds with it and the molecules will begin to separate and exit at the end of the column with the eluent at different rates.

    Ok, let me simplify this for you. Think of the column as a hallway. Now in that hallway I have put many, many poles of different thicknesses; these poles are the stationary phase. Now at the beginning of the hallway I have a bag filled with air and two different size doge balls. In this case, let's say the air is the mobile phase (it carries the balls) and the balls are the compounds, the testosterone propionate and the estrodiol benzoate. When I empty this bag down the hallway the balls will start to interact with the poles and the smaller balls will end up on the other side of the hallway first. Hence we separated the balls. Offcourse in chemistry it is a lot more complicated, but this simple explanation will do.
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    Thin layer Chromatography

    Thin layer Chromatography uses the same principles as Column Chromatography, however, the sample, instead of moving down a Column packed with silica gel, it moves up a TLC plate, which is a glass plate with silica gel bound to one side, using a phenomenon known as capillary action. The same separation occurs as in the Column setup, however this method is used to obtain data values and cannot be used to obtain pure samples. This method is important because we need it to determine Retention factor (Rf) values of the compounds and to determine which compound is which. The following link will provide some good information on obtaining Rf values.

    As you can see in the link, the compounds will separate. We need this separation as it will tell us which samples at the end is our testosterone proprionate and which is our estrodiol benzoate. I will explain how to obtain these values in the procedure below.

    The Procedure

    Now that I hope we got the nitty gritty out of the way, let's begin the procedure.

    Let's look at what he have to deal with. We have two compounds that are EXTREEMLY similar, all but for one little thing, the estrodiol benzoate has a free hydroxyl end. The free hydroxyl of the estradiol should make it move much more slowly than the testosterone . Now let's get started.

    Firstly we have to calculate the total volume of absorbent needed. In this case we will use silica since it is cheaper, alumia however is more stable. Silica gel has a density of around 0.7g/cm3 wet. So to separate 22g of material; Usually if you have good separation (>0.2 Rf), you want something like 25g silica/g material to be separated. That's about 550g. Now we have an approximate value for the amount of silica gel needed.

    Next we need to calculate Rf values. This however I cannot do for you guys as there are so many variables that will change this value. The solvents I have experimented with should give you a Rf value greater than 0.2. But we still need to run a TLC or we will be doing this experiment blindly, and won't know which is which at the end.

    Firstly, let's discuss our materials needed.

    This is what we need:

    - Glass Chromatography column with a Teflon or PTFE stopcock, 2in Inner diameter, at least 18in long.
    - A 500mL - 1000mL reservoir (If your column didn't already come with one)
    - 1kg Silica Gel, 60-2240mesh, 60A.
    - Silica Gel TLC plates (you need at least 20+ 4cmx8cm plates, either glass backed or flexi)
    - Retort Stand with grasping hooks.
    - 20x 500mL Glass beakers, or 40x250mL Glass beakers. More is better.
    - Cotton balls
    - Pure (very clean) sand
    - Capillary tubes
    - 10L n-Hexane, or 10L n-Heptane, or 10L n-Pentane or 10L Petroleum ether, whichever is cheaper for you, or easier to obtain.
    - 2L Ethyl Acetate
    -1L methanol
    - Black light (UV lgiht)

    Everything above can be bought of eBay, or laboratory suppliers relativity cheaply. It should not cost you more than a few hundred dollars at most.

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    Step 1: Extracting the hormones.

    1. Dissolve 100 doses ( so total 20g Test P and 2g Estrodiol) into 500mL of methanol in a glass beaker.
    2. Filter out the binders, but DO NOT throw them away.
    3. Using a hotplate or a NON open flame heat source, heat the filtered methanol to about 60-80*C. Keep an eye on this as the methanol evaporates away.
    4. Scrap off the powder and weight. If you did not get 22g (+/- 0.5g say for small losses) then add 200mL methanol to the binders I told you NOT to throw away and then mix it around and refilter. Again evaporate the methanol and add that powder to your total.
    5. It should be 22g (allow a little losses, but it shouldn't be more than 1g, unless you spilt something), of not then repeat until it is 22g. Then now you can discard your binders and gunk.
    6. Now you have pure test P and Estrodiol B powders.

    Step 2: Obtaining TLC values

    Now we need to see how our samples separate.

    1. Dissolve that powder into 100mL of a mixture of 90%hexane, 10% ethyl acetate. If it doesn't all dissolve, then add more of the mixture.
    2. Put on some latex gloves and pick up a TLC plate, if you got a sheet, then cut it up to obtain a small 4x8cm strip. Do not touch the face of the TLC plate. Now draw a line with a pencil about 1cm from the bottom of the plate. This will be our starting line.
    3. Using the capillary tubes place two TINY drops about 1.5cm apart just above that line. make sure they are in line with each other.
    4. In a glass beaker, place a small amount of the 9:1 solvent mixture you made, just enough to reach the pencil line of the TLC plate.
    5. place the plate carefully into the beaker so the solvent line is in line with the pencil line.
    6. Place a glass plate or a plastic plate on top of the beaker and leave it for a good hour or two.
    7. Now you will see that the solvent would of moved up about 3/4 the way up the TLC plate. With a pencil, roughly draw a line where the solvent has reached. We need this because the solvent will evaporate quickly.
    8. Now in a dark room, turn the black light on and observe the TLC plate. You will see two glowing spots vertically up from both drops you places. One of those dots is the testosterone , and the other is the estrodiol. Get a pencil and circle those dots.
    9. Now which one is which? Well since I did the maths, I will tell you to save you time, the testosterone should be the higher dot as it does not have the hydroxyl tail, hence it moves faster and further. Now keep this TLC plate as your reference card because when you obtain your fractions at the end you will need to do a TLC for each fraction to determine what it is.
    10. Now repeat, yes repeat this whole thing again with a solvent mixture of 80%:20%, 70%:30%, etc until you find the best separation. The reason I tell you to do this is because if you choose to use heptane instead of hexane you will get different results, or if you solvent is a different purity etc. We want the mixture that gives us the best separation, the further apart the dots are, the better the results.

    Step 3: Running the Column

    1. Set up the apparatus as shown below, but you don't need to put the reservoir in until after step 8.
    2. Place a cotton bud into the column and with a long stick press it into the bottom.
    3. Place about 1-2cm of sand above the cotton bud.
    4. With a funnel place 550g of silica in. This should go 3/4 or 4/5 up the column, if you can't fit it, then you got a small column!
    5. Place another 1-2cm layer of sand above the silica gel.
    6. Create a 10L mixture of hexane:ethyl acetate. mixture you found to give you the most separation between the dots.
    7. Using a glass rod, SLOWLEY add the solvent mixture into the column until it is about 5cm above the silica gel.
    8. Now place the Reservoir on and add more solvent until the reservoir is full.
    9. Place a 500mL beaker under the stopcock and open it. Run the column until the reservoir empties and you have about 1-2cm of solvent still above the sand line. Now what this did is removed any bubbles trapped in the column. NOW NEVER EVER let the solvent drop below the sand line. otherwise everything will be ruined and you will introduce bubbles into the system.
    10. Now the fun begins. Now we can make 40x 250mL collection (know as fractions) or 20x 500mL collections. Don't look so worried, you can pick up bulk beakers for dirt cheap, they don't even have to be beakers, they can be CLEAN glass jars. Ok so now SLOWLEY place the entire volume of the solvent and test p/estrodiol mixture we made before into the column. and then add more solvent mixture until the reservoir is full.
    11. Now start running the column at about 5 drops per second into a beaker, (250mL requires more beakers but will give you a better result.)
    12. Periodically change the beakers as the previous one becomes full. Label the beakers as you go.
    13. Once you have gone through all your beakers and you have run through the 10L of solvent (remember NEVER let the solvent fall below the sand line) I want you to run a TLC on EVERY fraction collected.
    14. Now these TLC's will tell you which beaker has what compound in it. You will use your original TLC plates as a reference to see which dot lines up with which. Remember to use the same solvent mixture you did for the first TLC plates or you will screw up big time. label which beaker had which dot.
    15. Now one of the beakers may contain both dots, and will have both estrogen and testosterone . I doubt you will get this, but if you do you can run that one through the column again, or just use it because there will only be a very very small amount of estrogen in there.
    16. Evaporate these beakers at 80-90*C. Combine the powders of the beakers with the same dot pla***ent you found using your TLC and weight it. You should have 20g +/- of Testosterone propionate with absolutely NO estrogen.
    17. Enjoy, and be proud that you just completed a University grade chemistry experiment. You can now throw away the silica and solvent and clean the column and beakers for next time.

    What you choose to do with the estrogen is up to you, personally I would give it to my enemy as test prop to give them gyno . No I'm just kidding, I just needed some relief after writing that mammoth post.

    Look, I know this seems so complicated for something like Synovex-H, but I wrote this for those people who have access to labs, or have the money to buy the equipment for a home lab. For everyone else I am working on another method that is cheaper, but does not yield 100%.
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