Sciencemadness Discussion Board

Accurate Transfer of Volatile Liquid Standards

Pseudosapien - 31-5-2019 at 05:21

Hi, first post on this forum! Haven't been on one of these before so I'm just gonna wing it and hope for the best.

Okay, long story short I work in a lab which has been working on cannabinoid analysis (in the licensed Canadian market) and have had a lot of issues surrounding the standards/response factors varying over time.

They are only available in pure methanol or ACN, are very expensive, and only come in 1mL quantities. I currently micropipette 150uL of these into a vial and then dilute, though the vapour pressure and of the solvent causes it to immediately push droplets back out of the pipette tip, meaning the volume changes slightly based on the speed at which you transfer, and I think this (among other things, like slight, slow evaporation of retained stocks in storage) is causing variation. Relative response factors (assuming concentrations consistent with the CoA, assuming no change) between cannabinoids vary up to 30% between different preparations on different days, which is unacceptable.

So I need to both conserve the liquid I use and find a way of transferring that can reduce the effect of volatility. I proposed weighing the ampule, breaking/pouring the entire contents into a volumetric, rinsing/transferring rinses to same flask, drying and weighing again, then correcting for the density of methanol, though my manager seems to believe back calculating volume via density would be unreliable. A reason was given but I don't remember it because honestly I don't think it made sense.

My question is, would my proposal be reliable? Is there a better way to quantitatively transfer small volumes of volatile liquids? Even just a reference to further reading would be great, Google isn't so great for these sorts of narrow technical things.

Ubya - 31-5-2019 at 06:30

Have you tried cooling? I mean keeping the standards in a fridge, or better in a freezer, and maybe even using a cooled pipette

Fulmen - 31-5-2019 at 07:12

I assume it's specified as weight/volume only? IIRC (it's been 30years) we used regular GC-syringes for this sort of work. I believe it was the lack of air that made the difference.

DavidJR - 31-5-2019 at 09:57

For pipetting volatile liquids, it's useful to draw up some liquid and expel it a few times before you actually draw the desired amount to use. The goal is to saturate the air in the pipette with vapour. It helps a lot, especially for stuff like DCM.
Quote: Originally posted by Pseudosapien  

... though my manager seems to believe back calculating volume via density would be unreliable. A reason was given but I don't remember it because honestly I don't think it made sense.

Assuming that these are very dilute solutions, and that you take into account the actual temperature, I think this could well be a workable method.
Quote: Originally posted by Pseudosapien  

... (among other things, like slight, slow evaporation of retained stocks in storage) is causing variation.

There's a reason these certified reference materials are supplied in flame sealed ampoules not screw cap vials. It's strongly preferable to use the entire vial in one go. One approach is to (accurately) make a large dilution (eg 1:100), and store aliquots of this separately. This more dilute solution can be measured easily and, although you still need to take care to minimize evaporation of solvent, any evaporation has a proportionally smaller effect.

Of course, this assumes that you can tolerate using a much more dilute solution.

[Edited on 31-5-2019 by DavidJR]

[Edited on 31-5-2019 by DavidJR]

Metacelsus - 31-5-2019 at 22:38

Maybe you could use a positive-displacement pipette instead of a standard micropipette. This should solve your vapor pressure issues.

https://en.wikipedia.org/wiki/Positive_displacement_pipette#...

Dcormieb - 3-6-2019 at 17:25

(FYI I'm the OP, I'm just a dum dum and lost access to that account, workin' on it.)

Standards are stored at -20*C due to cannabinoids being inherently unstable in solution, though I consider doing this cold would not be effective enough and would cause a lot of hassle with condensation. I'd try other things first.

The positive displacement pipette sounds perfect actually. While I'm ending up going a different route, I've petitioned to buy a few of these because wow those would be useful to me.

The total mass of all dissolved cannabinoids in the stock is 2.75mg in 1mL of solution. I have since clarified with my manager what the concern was, and it was to do with the partial molar volume effect on mixing/ how much this would throw off density or concentration on the manufacturer's end/ are their provided numbers reliable? I didn't remember this because intuition said at such low concentrations it would obviously be negligible and I didn't understand the question :cool:. I ended up writing a pseudo proof for this arguing 2.5mg solute of any kind out of 786mg solvent would produce a variation of <0.5% in density even when exaggerating any possible partial molar volume effects, and our chief scientific officer reviewed and signed off on it so that part's good to go.

Additionally, I have since figured out 2 more things:
1) The manufacturer prepared the mixture gravimetrically, at a known temperature when ampule was sealed.
2) Knowing both these things as well as the mass I transferred means I have very precise knowledge of the mg amounts contained in the ampule.

I would also prefer to use the whole ampoule in one go, though for various reasons including difficulty establishing stability in aqueous solution, the stupid requirement that the standard must be diluted in the same (aqueous) diluent as the sample, and the eye-watering price of $1500 for a single mL of standard, I was forced to re-pipette small amounts of 11 individual standards every time I did the test and retain the stock. I actually told management earlier today that I refuse to follow some requirements that nobody's been able to adequately explain or justify, and I basically am the entire cannabis analysis department so tough luck. TL;DR we're doing things my way now.

Current plan is to transfer whole ampule contents by weight, dilute to a large volume in pure ACN to match the stock's solvent and therefore knowing it's ~as stable, which I'd probably portion off into smaller containers I can seal extremely well and then pop them all in -20*C.

Thanks everyone!:D

Sulaiman - 3-6-2019 at 18:56

I'm in no way expert, but if you change your procedures then you will also need to change THE (written, formal) procedures,
otherwise results may be considered invalid ?

Dcormieb - 4-6-2019 at 04:05

Quote: Originally posted by Sulaiman  
I'm in no way expert, but if you change your procedures then you will also need to change THE (written, formal) procedures,
otherwise results may be considered invalid ?

Yes, though this is R&D work and isn't subject to the same restrictions, as we don't issue "official results" and don't assert their accuracy. Once I'm finished/have finalized a method then we'll do validation and it will become the official version that must be stuck to by the letter.

Overall this project is messy. Cannabis is a brand new industry (well, new *legal* industry anyways) and I get a new dosage form or some other one-off technical request pretty much every day, such so that ~50% of the time I'm just making things up as I go.