Chromatography Forum

Hello everyone

One more question on residual solvent analysis, the solvent of interest is benzene.

I am using the method of Ph Eur 20424 (system A). Here below are some details: (other parameters are set as per 20424)

Diluent: DMF
Carrier gas: Nitrogen
Column: DB-624, 30 m, 0.32 mm, 1.8 μm
Headspace oven: 80℃, loop: 90℃, transferline: 105℃
Equilibration time: 45 min

In the chromatogram obtained with reference solution a and reference solution a1, there is not any peak corresponding to class 1 solvents, including benzene. Seems like a sensitivity problem.

Wondering if anyone can point me in the direction.

Hi terry

Spike some DMF with benzene at 10 and 100 times the concentration in the reference standards - do you see peaks. If not go to 100 times just to be sure. If still no peaks sensitivity (detectability) might not be the only problem.

Are there any other substances that you can detect, what are they and at what concentrations ?

Do you see any peaks at all on the chromatogram ? If not, put some cigarette lighter gas into a vial and inject from that. If there is no peak either the detector is off, or there is a terrible problem with the gas flows.

Peter

Hi Peter

Spike some DMF with benzene at 10 and 100 times the concentration in the reference standards - do you see peaks.

At 10 times of the concentration in the reference standards, I can see all the class 1 peaks.

Are there any other substances that you can detect, what are they and at what concentrations? Do you see any peaks at all on the chromatogram?

On the chromatograms of the reference solution a and reference solution a1, there are peaks due to impurities in DMF and DMF itself, and they resemble what in the reference solution d.

Other in-house test method for residual solvents run properly on this instrument, Agilent 7890A & G1888A.

I doubt the compendial method itself is the problem.

Hi Terry

How big are the peaks at ten times reference concentration ? - if they were ten times smaller could you still see them ?

I do not know the method that you are using - do you make up the reference solutions yourself, or buy them ready made ?

Peter

Hello Peter

At ten times reference concentration, the peak of interest has a signal-to-noise ratio of about 10. This means at ten times smaller, it would disappear in baseline noise.

I bought the offical stock solution, and diluted it to target reference concentration. I have also prepared an in-house stock solution, and made dilutions to the same concentration. Chromatograms of the offical reference solution and in-house reference solution look quite alike: no corresponding peak.

I would use helium as carrier gas, and optimize headspace vial pressure to get maximum response. I will let you know the result.

Regards

Terry

Hi Terry

What (if any) is the split ratio and the loop volume ? Check that your vials are not leaking - put a little bit of a volatile solvent into a vial, crimp the cap as usual, then immerse the whole vial in hot water. If you see a stream of bubbles you have leaky seals and you need to tighten your crimper.

Peter

Hi Peter

The split ratio is 5:1, loop volume is 1 mL. These two parameters can not be modified.

I would check the vial to see if it leaks.

Terry

Your loop and transfer line temperatures are lower than I would run. I usually run these at least 120C to avoid condensation in the lines. How long is your loop fill time?

Your loop and transfer line temperatures are lower than I would run. I usually run these at least 120C to avoid condensation in the lines. How long is your loop fill time?

Would even go further (155°C) as DMF has a boiling point of about 155°C.

I understand Terry, that you are switching to Helium as carrier gas so let see whats happen, just make sure that the total flow is not less than about 10ml/min otherwise your instrument might not be too happy .

The loop fill time under these condition would typically be in the area of 0,15-0,40min.

I agree that you should look in to pressurisation time, at 80°C in the HS oven the vial pressure itself could be enough. Start with putting on the vial pressure but pressurisation time to zero seconds/min then maybe increase just to 0,1min/6s. Pronglonged pressurisation may increase risk of leaks and cause dilution.

Even so I would consider increasing sample amount in vial and standard concentrations. If performed as per EP: 0,2g sample in 20ml=>5ml in vial=0,05g x 2ppm benzene(limit)=0,1ppm Benzene in Vial.

If possible, dissolving 0,2g sample directly in vial and adjusting standards accordingly you would increase concentration of benzene and the other solvents 4 times.

Thanks a ton for sharing

Peter

Vial leaking check: No bubble

Ron

The loop fill time is 0.20 min

Krickos

I am using the offical EP 20424 method, to avoid the tedious validation of in-house method, which the inspectors may pick on. I am trying to adjust the compendial method without changing it.

In the EP (5.0) monogrph of 2.2.46. CHROMATOGRAPHIC SEPARATION TECHNIQUES, under the item of ADJUSTMENT OF CHROMATOGRAPHIC CONDITIONS, there are words like these:

Gas chromatography
Stationary phase:
— column length: ± 70 per cent,
— column internal diameter: ± 50 per cent,
— particle size: maximal reduction of 50 per cent, no increase permitted,
— film thickness : −50 per cent to + 100 per cent.
Flow rate: ± 50 per cent.
Temperature: ± 10 per cent.
Injection volume: may be decreased, provided detection and repeatability are satisfactory.

Pressurization time and sample preparation procedure are not mentioned, can I adjust/change them?

Terry

Hi Terry

hmm the replies have been truncated after server move it seems,

Still seem to recall one or two questions:

Yes, you may adjust vial pressure time (EP states the following settings may be used).
The need (and effect of) vial pressure depends on HS instrument type, vial size and HS oven temperature. The combi PAL HS can not pressure the vial (heated syringe injection) to mention one type. And as mentioned before pressurization may increase leaks and dilute the gas sample, as you have 80°C in HS oven start with pressure on but set time to zero increase pressurization time stepwise to see effect.

Hello Krickos

A few lines of words in my last post has gone.

The instrument of I am using is a typical pressure-loop system, Agilent G1888.

I have checked the impact of vial pressure on peak responses. Vial pressure is set as 10, 11, 12, 13, 14, 15, in psi, respectively, no response difference is observed.

I would change the pressurization time to see if it makes a difference.

Hello Terry

What are you able to change and still stay within the official method ?

Peter

Hi, Peter

This is a very good question.

I think I am not supposed to change the parameters which are specified in the official method, such as the equilibration temperature, equilibration time, sample preparation.

I would you the method if you are interested.

I have 'adjusted' the method to maximum extent which is permitted by the Pharmacopeia, but still the acceptance criteria can not be met.

My final decision is to come up with an in-house method, which is to be validated.

Terry

Hi Terry

I think that your decision is the right one - you needed ten times more response and you cannot get that by minor adjustments. Good luck with the method development, let us know what you come up with.

Peter