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OfflineBIG_Steve_01
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Help needed with HPLC
    #28367968 - 06/20/23 11:43 PM (7 months, 3 days ago)

Hey guys! I've recently got my hands on a waters 2695 separation module, a 2487 absorbance detector, and a 2996 photodiode array detector. I want to use these to analyze the psilocybin contents of my grow, but the problem is that I have zero experience using HPLC, and my chemistry knowledge is extremely limited. I was wondering if anyone knows where I should start to learn how to set these up and use them. I'm located in Vancouver, BC, Canada; if anyone is close by and can teach me how to use these I will be more than happy to compensate your time. Any information will help! Thanks guys!


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OfflineTri-Polar
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Re: Help needed with HPLC [Re: BIG_Steve_01]
    #28367999 - 06/21/23 12:31 AM (7 months, 3 days ago)

Start with ChatGPT and supplement with youtube, google, and reddit until someone can maybe help you 1 on 1, I was able to get this basic outline with a single question, you can then ask it to expand further on specific topics if need be, has helped me workshop a variety of things recently

sorry if not super helpful


--------------------
Intro to Shroomery:raver2:The Sexy TEKs

IF I HAVE SEEN FURTHER IT IS BY STANDING UPON THE SHOULDERS OF GIANTS, BITCH.

hey fuck you dont look at me


Edited by Tri-Polar (06/21/23 12:32 AM)


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OfflineBIG_Steve_01
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Re: Help needed with HPLC [Re: Tri-Polar]
    #28368090 - 06/21/23 01:43 AM (7 months, 3 days ago)

Thanks! I forgot how powerful AIs are these days lol, still getting used to it


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Offlinetholos
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Re: Help needed with HPLC [Re: BIG_Steve_01]
    #28369379 - 06/21/23 08:33 PM (7 months, 3 days ago)

How much experience do you have with analytical chemistry? Are you familiar with Chromatography at all?

The best starting place is to find a paper that has their method parameters listed out for you, I will include an example for caffeine

https://www.agilent.com/cs/library/applications/5991-2851EN.pdf

You need to find a "version" of this, but for Tryptamine Alkaloids instead of Caffeine

It's going to be the exact same process as getting any other method online


Just to reiterate the information that you will find in the Agilent Caffeine Method

1) What solvents are you going to use for your Mobile Phase? Are you going to acidify/buffer them?

2) What type of stationary phase (column) are you going to use? C18? Reverse or Normal Phase Chromatography?

3) What wavelength are you going to use to detect your analytes?

4) Where are your going to obtain your CRMs (Certified Reference Materials) aka standards for your alkaloids

This process can broadly be split up into two parts:

1) Getting your instrument online/calibrated/your method dialed in

2) Getting your extraction parameters dialed in and making sure that you are recovering as much analyte as possible


Edited by tholos (06/21/23 08:43 PM)


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Offlinetholos
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Re: Help needed with HPLC [Re: Tri-Polar]
    #28369384 - 06/21/23 08:36 PM (7 months, 3 days ago)

Quote:

Tri-Polar said:
Start with ChatGPT and supplement with youtube, google, and reddit until someone can maybe help you 1 on 1, I was able to get this basic outline with a single question, you can then ask it to expand further on specific topics if need be, has helped me workshop a variety of things recently

sorry if not super helpful





This is actually a great starting point. Although I fear there is too many technical terms/jargon to be palatable to someone without any previous chromatography experience


Edited by tholos (06/22/23 05:31 AM)


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OfflineTri-Polar
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Re: Help needed with HPLC [Re: tholos]
    #28369414 - 06/21/23 08:49 PM (7 months, 3 days ago)

Thats the beauty of chatgpt though imo, unlike static info you can ask chatgpt clarifying questions and to simplify terms and all sorts! I'm constantly impressed and disappointed by it :biggrin: definitely has its limitations especially when trying to research the thermal stability of PsiK :mad2:


--------------------
Intro to Shroomery:raver2:The Sexy TEKs

IF I HAVE SEEN FURTHER IT IS BY STANDING UPON THE SHOULDERS OF GIANTS, BITCH.

hey fuck you dont look at me


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Invisiblepablotx
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Re: Help needed with HPLC [Re: tholos]
    #28370203 - 06/22/23 01:33 PM (7 months, 2 days ago)

Yeah, where do you get your reference standards? And diluting the standards appropriately ain't easy - requires some nice glassware and maybe a Mettler balance that can read 0.1 mg resolution?

Maybe you could make an extraction in MeOH, then send 1/2 off to another lab to see what they get for the various tryptamines? Not a perfect solution, but better than nothing.


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OfflineBIG_Steve_01
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Re: Help needed with HPLC [Re: tholos]
    #28370552 - 06/22/23 06:53 PM (7 months, 2 days ago)

I got absolutely no experience with analytical chemistry or chromatography. My friend has had these HPLC in his storage for years and recently he moved and left the HPLC at my place. I thought maybe I can make use of it. I'll try to find a paper that layout the procedures, but there are a lot of terms that I have no idea what it means. I'm basically a 5-year-old that happens to have an HPLC.


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OfflineBIG_Steve_01
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Re: Help needed with HPLC [Re: pablotx]
    #28370555 - 06/22/23 06:57 PM (7 months, 2 days ago)

I'm thinking of getting the standard from Toronto research chemicals, haven't gone through with it yet. I got an analytical balance reads 0.1mg and a couple of mechanical pipettes that I use for PCR work. I should have most of the equipment needed to do it, I just don't have the knowledge.


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Offlinetholos
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Re: Help needed with HPLC [Re: BIG_Steve_01]
    #28370573 - 06/22/23 07:22 PM (7 months, 2 days ago)

Quote:

BIG_Steve_01 said:
I'm thinking of getting the standard from Toronto research chemicals, haven't gone through with it yet. I got an analytical balance reads 0.1mg and a couple of mechanical pipettes that I use for PCR work. I should have most of the equipment needed to do it, I just don't have the knowledge.





I would verify/tune your auto pipettes if you're going to be using them for dilution work. Unless you spend big $ on a nice autopipetter, my experience in the lab setting is that air tight syringes are much more accurate for a much more cost effective price point


Read the link I sent you of the Caffeine method -- if you use this exact outline but replace caffeine with tryptamine alkaloid then you are in the exact right analytical framework


Edited by tholos (06/22/23 07:29 PM)


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Invisiblepablotx
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Registered: 05/06/23
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Re: Help needed with HPLC [Re: tholos]
    #28371452 - 06/23/23 03:07 PM (7 months, 1 day ago)
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A good set of calibrated micropipettes used in PCR should be ok. My standard serial dilutions with them usually end up with an r^2 of 0.999 for the linear regression.

Attached is another ref using reverse phase chromatography for psilocybin and psilocyn.


Edited by pablotx (06/23/23 03:08 PM)


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Offlinetholos
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Re: Help needed with HPLC [Re: pablotx]
    #28371478 - 06/23/23 03:38 PM (7 months, 1 day ago)

Quote:

pablotx said:
A good set of calibrated micropipettes used in PCR should be ok. My standard serial dilutions with them usually end up with an r^2 of 0.999 for the linear regression.

Attached is another ref using reverse phase chromatography for psilocybin and psilocyn.





As long as they are properly calibrated, agreed. The key is that they are properly calibrated.

R^2 value can be a little misleading. Yes, it is the one of the criteria for a good calibration curve, however, if your observed volume from your pipette is 20% off from your expected volume, and the pipette is consistent from plunge to plunge, you can produce a curve with a high r^2 value that is inherently 20% off and your R^2 value alone cannot tell you that. You need a proficiency test or a secondary CRM to cross reference to be able to tell that


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Invisiblepablotx
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Registered: 05/06/23
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Loc: Hot & humid
Re: Help needed with HPLC [Re: tholos]
    #28372460 - 06/24/23 09:26 AM (7 months, 13 hours ago)

With a balance capable of 0.1 mg readings, at least you can calibrate your pipettes! Doesn't mean you do it, but it helps. I lucked into mine horse trading, and balance came freshly calibrated!


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