Glycerin related compounds

Discussions about GC and other "gas phase" separation techniques.

8 posts Page 1 of 1
Hello,
I'm working on the USP related compounds for glycerin for the first time. I was trying to convert the method over to columns/conditions we already have in my lab. I am having problems with peak shape, broad 2 minute wide glycerin and diethylene glycol peaks. Resolution is at most 4, but spec is 7.
I'm also occasionally seeing odd peaks.. almost like glycerin is splitting in the injector. I saw this with limit of ethylene glycol and diethylene glycol, adding a pressure pulse and speeding up the injection resolved those odd peaks.

My Conditions:
Restek 624 30m, 0.3-mm, 1.8um.
Carrier: nitrogen,
Column flow is set at 0.8mL/min with a pressure pulse of 16psi (to avoid backflash). I calibrated the septum purge for the 16psi.
Liner- restek cyclosplitter, pn 23312. (220C), Split 10
Samples are dissolved in water, 0.5uL injection volume. Washes are water.
Oven: 100C to 220C at 3.8C/min, hold 8 minutes.
Detector: FID at 240C

Original USP Method:
30m x 0.53mm x 3um
Carrier gas: Helium
Linear Velocity 38 cm/s
Liner: cup or spiral structure
Un specified sample preps, but I believe it's water. 0.5uL inj. volume
Oven 100-220C at 7.5C/min, hold 4 mins

I've tried adding a temperature hold, slow the gradient, increase split, and played with different pressure pulse settings (best results so far). One question I have, for the liner, restek says this part is for an agilent, but I have a varian. The basic dimensions are the same, but could the split location be an issue?

The column is fairly old. I can order a new, identical column, since we use it for other tests. Or, I could order the one specifically for this test. Alternatively, I could try switch out my carrier gas. The last two options are not ideal.. so, I'm wondering if one would be more promising than the other. Also, could my gas settings on my detector or gas quality be impacting the poor peak shape and tailing? Generally, I think of this as a column chemistry issue, but I'm no GC expert. Thanks!
Can I ask why you have changed from the USP method? Is your in-house method one you have been using for a while, or are you just transferring it in-house now? A flow of 0.8ml/min N2 on a 0.32mm ID column does not seem like it would give you a high enough linear velocity for optimum resolution, but maybe I'm wrong.

Going from a 0.53mm x 3um to a 0.32mm x 1.8um column will definitely reduce the sample capacity of your column - is it possible you are seeing overload with the Glycerin peak?

The USP method is a pain in the arse (as with most GC-based related subs USP methods) but it might be no harm going back to the original USP settings and seeing how things look, if you have the correct column in stock and access to Helium.
I have considerable expertise in the analysis of glycerin and similar materials, typically derivatized those for GC analysis.

When our glycerin manufacturing plant had to begin assaying for such components to demonstrate lack of adulteration as a requirement, we just followed the USP method as written (even though our glycerin was made directly from fats and oils, so ethylene glycol and diethylene glycol were not possible). So even though a pain, was simpler to follow the USP procedure to the letter, even though that procedure kept changing - it seems yearly. Yes, quite silly that adulteration concerns would require testing at such low levels, even though it would not be "profitable" to intentionally adulterate at such low levels. I think GC after derivatization would be a more robust test scheme to look for adulteration in glycerin.


AaronAIT wrote:
Can I ask why you have changed from the USP method?


I, too, am curious. Seems like a lot of wasted labor costs to try to make do with a used GC column of the wrong dimensions. I would dedicate the new column to this USP assay as well. I fully understand that your pointy-haired boss is trying to keep supplies budget as low as possible, but you've spend more company dollars trying to "make it work" than buying a new column.
Image


AaronAIT wrote:
The USP method is a pain in the arse (as with most GC-based related subs USP methods).


I couldn't agree more.

Denise - I'm also curious: is your company making glycerin or just getting running the required USP no-adulteration test on incoming raw material? If the latter, is this glycerine used in a topical product or for internal consumption or injection?
Wow, thanks for all the advice! Quick update, I did discover that my GC's heated locations were not responding to their settings and my injector was not heating up! It happened recently with a memory loss. I fixed that and my peaks are sharper but still not resolved to 7.

We are testing the glycerin as a new raw material. It's for a suppository.. so not a syrup but also not a topical. We will be using it for a topical later on too. It begs the question, does this test make sense if the consumer isn't digesting it? We will be submitting this to the FDA; so, I want to make sure we sail through with no issues from them. But, yeah, this is very low risk.

I think I'm breaking down to purchase the intended column and the helium. Unfortunately.. I'm the boss and the analyst here. We are a very small shop. And, yeah, my labor is definitely being wasted!

Just to confirm with you guys, the diluent on this method should be water, correct? I don't see what it is in the method. But, I did see historical comments from you that you dissolved in water... I thought maybe methanol would improve things.
Water would be my last choice...
Your friendly Restek/Phenomenex/Agilent tech support ppl may be able to suggest something better, but if you do continue w/ water, try injecting samples very slowly and cold trapping it to prevent the flashing. That's how we tested aqueous solutions for injection for extractables back in my GC days.
Almost any organic solvent into which your compounds of interest are soluble would be preferable.
Thanks,
DR
Image
DeniseHeinz wrote:
We are testing the glycerin as a new raw material. It's for a suppository.. so not a syrup but also not a topical. It begs the question, does this test make sense if the consumer isn't digesting it?


Your finished product will be used internally, so my guess is that it will need to be tested.


DeniseHeinz wrote:
I think I'm breaking down to purchase the intended column and the helium.

Just to confirm with you guys, the diluent on this method should be water, correct? I don't see what it is in the method. But, I did see historical comments from you that you dissolved in water... I thought maybe methanol would improve things.


Denise - I really think you would be better off purchasing the detailed column, inlet split liner, and helium, that's what we did at R&D and at our manufacturing facility. The column and inlet split liner were used only for this assay. We used an inverted cup type (Restek #20990 mini-Lam split liner) or a spiral type (Restek #20706 cyclosplitter) in our Agilent GCs.

I'm retired now, don't have access to the glycerin monograph. I remember that in the early 2000s, when this testing was first added to the USP monograph, the test was modified a few times, and methanol was used in at least one revision. But last time I looked at the monograph, the solvent was water.

From https://www.uspnf.com/official-text/acc ... -monograph "Compliance may be determined also by the use of alternative methods as permitted under the USP General Notices. Such alternative methods shall be validated by the user to demonstrate the methods ability to meet the specificity and detection limits as outlined in Category II—Limit tests in USP General Chapter <1225> Validation of Compendial procedures."
So, I had a look at the current Glycerin monograph...
Good news:

•B. Limit of Diethylene Glycol and Ethylene Glycol
Standard solution:2.0 mg/mL of USP Glycerin RS, 0.050 mg/mL of USP Ethylene Glycol RS, 0.050 mg/mL of USP Diethylene Glycol RS, and 0.10 mg/mL of 2,2,2-trichloroethanol (internal standard) in methanol
Sample solution:50 mg/mL of Glycerin and 0.10 mg/mL of 2,2,2-trichloroethanol (internal standard) in methanol

Carrier gas:Helium
Injection size:1.0 µL
Flow rate:4.5 mL/min
Injection type:Split ratio, about 10:1
System suitability
Sample:Standard solution
[Note—The relative retention times for ethylene glycol, 2,2,2-trichloroethanol, diethylene glycol, and glycerin are about 0.3, 0.6, 0.8 and 1.0, respectively.]
Suitability requirements
Resolution:NLT 1.5 between diethylene glycol and glycerin
Analysis
Sample:Sample solution
Acceptance criteria:If a peak at the retention times for the diethylene glycol or ethylene glycol is present in the Sample solution, the peak response ratio relative to 2,2,2-trichloroethanol is NMT the peak response ratio for diethylene glycol or ethylene glycol relative to 2,2,2-trichloroethanol in the Standard solution; NMT 0.10% each for diethylene glycol and ethylene glycol is found.
•C.Examine the chromatograms obtained in Identification test B. The retention time of the glycerin peak of the Sample solution corresponds to that obtained in the Standard solution.

Column:0.53-mm × 30-m fused-silica analytical column coated with 3.0-µm G43 stationary phase, and a deactivated split liner with glass wool
Temperature
Injector:220°
Detector:250°
Hold 100° for 4',
50°/min to 120°, hold 10'
50°/min to 220°, hold 6'.
-and-
Organic Impurities:
•Procedure 1: Related Compounds
System suitability solution:0.5 mg/mL each of USP Diethylene Glycol RS and USP Glycerin RS
Sample solution:50 mg/mL of Glycerin
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode:GC
Detector:Flame ionization
Column:0.53-mm × 30-m fused-silica analytical column coated with 3.0-µm G43 stationary phase, and an inlet liner having an inverted cup or spiral structure
Temperature
Injector:220°
Detector:250°

Start at 100°, 7.5°/min to 220°, hold 4'
Carrier gas:Helium
Injection size:0.5 µL
Linear velocity:38 cm/s
Injection type:Split ratio, about 10:1
System suitability
Sample:System suitability solution
Suitability requirements
Resolution:NLT 7.0 between diethylene glycol and glycerin
Analysis
Sample:Sample solution (above)

Hope these help.
Thanks,
DR
Image
Thanks, DR, for posting the current glycerin monograph; methanol as solvent is much more robust than using water. I wonder when the USP went back to methanol as solvent?

Just changing this test procedure so often in the last dozen years or so just reinforces my "confidence" in the USP.
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