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Author Topic: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)  (Read 1616 times)

aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #120 on: April 30, 2011, 01:28:01 PM »
it is under a blacklight, and the purple spot (center) is the ergot alks; the yellow perimeter is just the solvent front
« Last Edit: April 30, 2011, 01:29:35 PM by aniracetam »
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Vesp

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #121 on: April 30, 2011, 08:40:13 PM »
What does this look like in normal light? To me this image only looks like how wet paper would look under a black light. If normal light revealed purple on white, that would be impressive.

Do you have any quantitative instead of qualitative results?
 
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aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #122 on: April 30, 2011, 10:08:48 PM »
nope

it was a culture aliquot that had been sitting for a few days. the spot was dry, and checked it again under the UV
light this morning. it's definitely an indolic complex.
already adsorbed the culture on bentonite, next step is elution/extraction, conversion to a salt.
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aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #123 on: May 01, 2011, 12:41:10 AM »
here
« Last Edit: May 03, 2011, 12:18:29 AM by aniracetam »
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Vesp

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #124 on: May 01, 2011, 07:01:52 PM »
All right, the second picture is more helpful.

Good luck!
Looking very forward to the final write up, hopefully from start to finish.
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aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #125 on: May 02, 2011, 04:19:49 AM »


raw alks from the filtrant (most are still in bentonite)

gonna test on MALDI, 2,5-DHB as the matrix
« Last Edit: May 03, 2011, 12:12:49 AM by aniracetam »
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Tsathoggua

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #126 on: May 02, 2011, 06:30:19 PM »
Sick, sick sonofabitch!
Nomen mihi Legio est, quia multi sumus

I'm hyperbolic, hypergolic, viral, chiral. So motherfucking twisted my laevo is on the right side.

aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #127 on: May 02, 2011, 09:38:05 PM »
'ey y0...


reflectron, positive mode. 2,5-DHB matrix. m/z threshold cutoff at 200.

base peak looks like elymoclavine +4  (+ 0.021 m/z deviation)

..

this is just a raw alk sample from the filtrant, no chrom.
 the real goods are in a brick of bentonite in the vac des. ;)
« Last Edit: May 03, 2011, 04:05:54 PM by aniracetam »
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xxxxx

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #128 on: May 02, 2011, 10:47:18 PM »
Wow aniracetam. This is really amazing work! This is the first time I have ever seen someone making such practical progress towards ergot alkaloids! I hope this works out for you and I know many people here would love a full writeup when you finish. BTW it might be a good idea to upload those images here rather than hotlinking them, they can give away members information and they can also get lost easier leading to broken images.

Anyways keep up the fantastic work aniracetam!

akcom

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #129 on: May 03, 2011, 02:00:17 AM »
Wow, I don't know whats more impressive: your results or your lab.  If you don't mind my asking, are you doing this in a professional setting or are you just well funded?

aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #130 on: May 03, 2011, 02:42:21 AM »
I just know how to shop for equipment.
the mass spec is something I work with, it'll be a little while before I have my own.
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akcom

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #131 on: May 03, 2011, 06:41:51 AM »
That makes me feel better haha, what is that big hunk of junk next to your rotovap?

Vesp

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #132 on: May 03, 2011, 08:13:55 AM »
Where did your graph picture that was in this post go?
http://127.0.0.1/talk/index.php/topic,1383.msg21357.html#msg21357
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Tsathoggua

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #133 on: May 03, 2011, 08:26:51 AM »
Aniracetam, have you got a writeup for the bentonite separation protocol? I have heard nothing but good things, but its one of the things I lack in my library.

I'll say it again...sick, sick fucking fuck 8) Any idea yet what your strain is producing per liter, and what culture medium are you using?
Nomen mihi Legio est, quia multi sumus

I'm hyperbolic, hypergolic, viral, chiral. So motherfucking twisted my laevo is on the right side.

aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #134 on: May 03, 2011, 02:48:00 PM »
akcom, it is a UV-Vis detector with a capillary flowcell, syringe pump, and an ultrasonic power supply under them.

vesp, I took it down to edit it...i'm actually doing another run now, to improve resolution.

tsathoggua, it was a patent I looked at, for the process of separating raw ergot alkaloids. basically loosely followed two patents. the first was the tonolo et al. patent, which uses 5% mannitol, 1% sucrose, 2% succinic acid, 0.5% KH2PO4, 0.03% MgSO4; added 0.5% tween 80 for good measure.

latest batch follows the same formula, but I added 1 g l-tryp.

for this prior batch, I added 50 g/L bentonite, stirred for an hour, and vac filtered. put the cake in the oven at 80 C for about an hour and a half, then transferred to a vac des.
« Last Edit: May 03, 2011, 02:54:49 PM by aniracetam »
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overunity33

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #135 on: May 03, 2011, 04:07:58 PM »
makes me happy to see this, things have come a long way since the lsa bioreactor post...

Look forward to getting more experimental details on the bentonite elution and further purification... youre a hero bro, this is a gamechanger..

aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #136 on: May 03, 2011, 04:09:45 PM »
it appears degradation of the the sample has occurred; relative abundance for 258 has dropped considerably, and 273 has increased, from the run yesterday and the run today.
did several sample runs.

thanks, overunity. been 15 years since I daydreamed about this; now I'm able and willing.

this a killer resource...The Handbook of Secondary Fungal Metabolites (Vol. 1 - 3),  52 MB.
hxxp://megaupload.com/?d=7PMP6KJM
« Last Edit: May 03, 2011, 04:28:01 PM by aniracetam »
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overunity33

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #137 on: May 03, 2011, 04:20:45 PM »
Do you think that the degradation products will still hydrolize to LSA? Can you speculate as to what change occurred? i cant interpret spectra just yet..

Those daydreams man... its funny how fast capable people end up staring their daydreams in the face..

aniracetam

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #138 on: May 04, 2011, 06:03:18 PM »
ok...after further examination, it seems 273 is background signal for matrix. 258 is part of the sample.
I loaded a couple spots on the plate with matrix only, shown in red, and matrix with sample, shown in dark blue.
the masses listed are for the blue peaks.



 258 is likely chanoclavine (m.w. 256.343)  +2.
pubchem CID 5281381. it has two proton acceptors

again, this is just from the filtered broth (did a base/acid extraction on it), i haven't eluted from the bentonite.
« Last Edit: May 04, 2011, 06:10:14 PM by aniracetam »
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Tsathoggua

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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #139 on: May 05, 2011, 07:42:37 AM »
You baked the bentonite in the oven at 80 C ?

that sounds like it would wreak havoc on the alkaloids absorbed.
Nomen mihi Legio est, quia multi sumus

I'm hyperbolic, hypergolic, viral, chiral. So motherfucking twisted my laevo is on the right side.
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