[scar]

Hi!  Wow, what a vast forum!  i hope this is the right area.  I am looking for a solvent we can use with high performance thin layer chromatography (HPTLC) and a glycerin-based mixture.  Currently we use chloroform for other mixtures, but it will not dissolve the glycerin.  I tried using larger sample sizes (up to 8x normal) and some cotton to filter the the pickup of the solution for placement on the plate, but results were still very faint and unreadable.  I'm wondering if a 50/50 v/v mix of the glycerin with some water will help before mixing with chloroform?  I will try that soon, but was wondering if there is a better solvent we can order from our chemical supplier?  Also was reading about 2-D chromatography, is that something we might like to try?  Thank you

[Arkcon]

This reference may help you, I can't view it myself, so I'm not sure, but I hope you have luck with it.:  http://www.sciencedirect.com/science/article/pii/S0016236108001695

[scar]

Thanks but i'm not sure... all i can see is the abstract and it sounds like they are trying to detect glycerol in biodiesel.  And we are trying to detect different substances in the glycerol.  So i'm not sure if the method they are using would still apply to our method.  Would like to be sure of that before i spend money on the article or invest time in trying to find it at a library....  btw i like your icon Arkcon

[MOTOBALL]

Scar,

If you provide information along the following lines, you will get a much better response from the forum.

1. SOLVENT----to load the sample onto the plate or to develop the plate ?

2. HPTLC----normal or reverse phase (or other) ?

3. SAMPLE---what classes of compounds are present ?

Motoball

[scar]

Thanks, as i'm just an amateur trying to bring a currently-outsourced job in-house to our pharmacy, i may not describe things correctly and appreciate all of your patience.

Currently the solvent we use is chloroform, and we use it for both loading the sample on to the plate and for the next step where it moves up the plate.  For developing we use a powder called Fast Blue B Salt (CAS 14263-94-6) mixed with some water and either spray onto the plate or dip the plate into this developer for a second.

Not sure what you mean by normal or reverse phase but the method is pretty straightforward so hopefully that will answer your question: take the sample, mix with chloroform, put 2-4ul of that onto a silica plate, put the plate into jar with more chloroform and wait for it to move up the plate, then develop with the dye and interpret results.  The compounds analyzed are cannabinoids.

[MOTOBALL]

Scar,

You are doing TLC on silica gel---that is NORMAL phase (only capitalized for instructional purposes, since you are a beginner)
You are using CHCl3 for both LOADING the sample and DEVELOPING the plate, i.e. performing the chromatography to separate the components of the mixture.
You are using Fast Blue B salt for DETECTION.

Re-reading your first post, it seems that the issue is very low levels of cannabinoids in the glycerin which makes them hard to detect.  You also have a second issue, which is the presence of glycerin in the spot that is applied to the TLC plate.  The normal procedure with TLC is to air-dry the spot to remove the solvent that the compounds are dissolved in; this will not work with glycerin (not volatile; high B.pt.), and the residual glycerin will distort the chromatographic separation. Try the following....

1. Take glycerin extract (100 uL) in a test-tube or small vial; dilute with H2O (400 uL).

2. Add CHCl3 (500 uL) shake well; remove lower CHCl3 layer with Pasteur pipette and save.

3.  Add a second aliquot (500 uL) of CHCl3 to remaining aqueous glycerin solution; shake well; remove lower layer (CHCl3) and combine with the CHCl3 from step 2.

[4.  Dry the CHCl3 extract by shaking with a small amount of anhydrous sodium sulphate and stand for 5 mins.]

5. Remove the dried CHCl3 solution (Pasteur pipette) to a second vial; blow down to near dryness with stream of air, or allow the CHCl3 to evaporate naturally, to around 20 uL

6.  Use the 20 uL CHCl3 extract to perform TLC.

Step 4 can be omitted, if you do not have an anhydrous drying agent.

Good Luck,

Motoball

[scar]

Thanks i will give it a shot.  Based on how the glycerin tincture is made, there shouldn't be low levels, but i will look into this more since we make it. 

Some questions though.  Is using more glycerin extract going to skew the measurement of the results (we have transparent sheets with various spot sizes on them to measure the %), or will it just make the spots darker and easier to measure?

Also you say to remove the 'lower layer' of CHCl3, so is that 500 uL?  Should i remove this layer immediately after shaking, before the glycerin separates to the top, or should i wait a few seconds for the glycerin to coagulate at the top?

[DrCMS]

Before we all get more helpful to scar lets consider what they are doing - a non chemist testing THC in glycerin for sale.

[billnotgatez]

@scar
Please read forum rules if you have not already

I am going to lock this thread for at least a while - at least until we sort this out
We are getting close or maybe past the forum rule limits

@scar
If you have concerns you can private message me using forum messaging
or
use the Report to moderator link
And we will respond

[Borek]

Thread reopened.

Please note we can close it when we decide it crosses the line.

[MOTOBALL]

Also you say to remove the 'lower layer' of CHCl3, so is that 500 uL?  Should i remove this layer immediately after shaking, before the glycerin separates to the top, or should i wait a few seconds for the glycerin to coagulate at the top?

SAFETY

Working with volatile solvents should be done in fume hood.  Also, I have read that dichloromethane (CH2Cl2) is less toxic than chloroform, while having very similar extractive properties.

Each layer will be about 500 uL; you want the lower layer (CHCl3). After vigorous shaking to thoroughly mix the two layers, allow to stand for 1-2 mins for layers to separate.

Is using more glycerin extract going to skew the measurement of the results (we have transparent sheets with various spot sizes on them to measure the %), or will it just make the spots darker and easier to measure?

For detectability, more is always better as long as the loading does not degrade the chromatographic separation or saturate the detector.
Suggest that initially you run three spots, with loadings of 1, 5 and 10 uL, respectively to see (i) if the chromatography is affected by heavier loadings, and (ii) how the size/color of spot varies.

Motoball

[scar]

Hi i got good results with your method!    I attached a scan of the plate.

But i think evaporating down to 20 uL might be a little too much, because the first lane appears saturated to me.  the first lane is 4 uL, and the second lane is 2 uL.  it seems like the two biggest dots in the first lane are saturated; they both appear to be the same size but probably shouldn't be since they are different sizes in the second lane.   Then again, the dots are still not as large as our measuring sheets go up to....  How to precisely measure the dots/percentage is still pretty vague to me.  The measuring sheets we have are supposedly calibrated to 100 mg plant matter in 1 mL of CHCl₃ so i wonder if they are accurate when using the above method with 100 uL glycerin and 1 mL of CHCl₃ evaporated down to 20 uL, or would it be more precise to weigh out 100 mg of glycerin instead, or evaporate down to a different volume?

Evaporating down to 20 uL was difficult to measure using the microcentrifuge tubes we have since the lowest measurement is 0.1 mL.  Is there a more accurate way to measure down to that small of an amount?  but i did eye it pretty close! ...maybe to 22-24 uL.  I used 8 uL on the plate and then took out about 14-16 more uL using a minicap.  So maybe i can try evaporating down to 50 uL?

[MOTOBALL]

The methodology that I suggested has got you into the ballpark, but fine-tuning the details is up to you.

Regarding concentration to an exact small volume, you can instead blow down or air-dry the extract to dryness; then add the required volume, say 20-50 uL using a syringe.

There are two potential issues with going to dryness,

(1) some compounds may be irreversibly adsorbed to the glass/plastic surface of the vial,

(2) vial plastics may contain additives (plasticisers, anti-oxidants etc) that will be extracted into the CHCl3.

So, you will need to run some experiments with known standards of your cannabinoids to determine if you get good recovery, and blanks to determine if any interfering chemicals are eluted  from plastic equipment.

I would be interested to see how a pure CHCl3 tincture (no glycerin) compares to the TLC plate shown above.

[scar]

As i mentioned, the calibrated rulers i've got call for 100 mg dried plant sample with 1.0 mL CHCl₃.  The rulers are to measure the results (sizes of the dots).  Now we've established that glycerin tincture needs to be mixed with some water first to help break it up and get the chloroform to soak it up better, but how to properly do this so the calibrated rulers can be used?

It seems to me that if i added 100 mg H₂O to 100 mg of glycerin and then performed the test as usual, i would double the final results.  Is that right so far, or should i somehow be working with volumes?  In lieu of doubling, could I evaporate the test solution down to 50% before running the test?  Or do i not need to be doubling anything at all?

If it's right then it follows that if i use 400 mg H₂O then i would multiply the final results by 5 correct?  Or evaporate down to 20% instead?

[scar]

if i use 400 mg H₂O and evaporate down to 20% and the results are still too low to be readable, i need to evaporate more.

if i evaporate down to 10%, then i would need to divide my results by 2?
and if i go down to 5%, divide results by 4?