Chromatography Forum

Hello everyone, i'm writing here because after months of tries i still can't get last 4 PAHs on my chromatogram.
I'm talking about dibenzo(a,i)(a,l,)(a.e)(a,h)pyrene, and i have some issues with indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene and benzo(g,h,i)perylene too.
I have never seen the first 4 i mentioned, the only thing i see is a small unresolved peak (it's more like a hill actually) at the end of the chromatogram, and about the last 3 i can see them but they are very discriminated and bad shaped compared to the others.

I work on a Varian Saturn 3900/2100 in MS/MS, i have a varian factor four 5ms column 30m, 0.25 both ID and film.
Conditions are:
Injector: 300 °C with pulsed splitless @30psi for 0.5min, then split open at 50 ml/min.
Oven: 100 °C x 3min, @6°C/min to 320 x 20 min.
Xferline= 300 °C
Trap= 230°C
Manifold= 50°C (the real T is ~70°C)

I inject a STD mix 22 @2ppm (diluted by me) from an ultracientific solution.

The story is: i couldn't see anything past 202m/z, then i have read a topic in this forum where someone had his trap cleaned and his column cut and solved the problem so i did it as well and it actually worked, but i still can't get those.
The trap is clean, the column is cut, the PAHs are in the solution (std mix22 from ultra) the only thing i didn't check is the injector. Could it be the liner? If so which liner should i use? Currently i'm using a splitless with single taper.

The instrument works well, i think my problem is that i'm not getting the heavis in my system, can anybody help me? i'm desperate with this.

Davide.

Although it's certainly application specific, the "default" operating temperature for this particular ion trap is 150C. If you try to run the system at this trap temperature, keeping all other parameters the same, does this change anything for you? You will probably need to retune the trap before running a standard, once it has equilibrated at 150C.

When you say you couldn't previously see anything above 202 m/z, what exactly do you mean? On a siloxane bleed peak, are you unable to see the characteristic 207, 281, 355 m/z? Check your RF adjustment in MS Workstation (in manual control, adjustments). Make sure it seems reasonable all the way to 650 m/z. The software should tell you the RF response is within limits.

What is the scanning range of your analytical method? Ensure your rf dump value in the MS segments section is at least 50 m/z greatest than the upper scanning range.

As far as liners are concerned, the Restek SkyBlue seem like a great choice. Highly inert.

Good luck!!

Another thought is to increase your injection temperature to 325C. You might try this prior to lowering the trap temperature to 150C since it's a quick parameter adjustment. Let us know what happens.

Sorry for the late reply, i wasn't at work yesterday.
When i say i couldn't see anything past m/z202 i mean i couldn't see any PAH in my chromatogram with a molecular mass greater than 202 (which are fluoranthene and pyrene) but after i got the trap cleaned and the column cut i had no problems seeing any 228, 252, 276....i still miss the last 302 m/z.
I see any characteristic bleed peak and my instruments reads any mass fine to 650.

I'm gonna try trap T at 150°C and Tinj 325°C today, since the instrument will be PAH dedicated i don't care about retuning etc.

Thanks a lot, i'll post any result as soon as possible.

Davide.

Lowering the trap temperature didn't work, now the PAHs with 252 and 278 m/z have lower intensity and have a bad shaped peak, the 302s still don't exist.
I will now try with the trap at 200°C and with TInj at 325°C.

Isn't method development fun?? Sounds like the higher trap temperature is the way to go for this particular analysis.

Another thing we noticed about your analytical parameters... Your column is temperature ramped to a final operating temperature of 320C. However, your transfer line is only to set to 300C. At the very least, it should at least be set to the highest temperature value used in your ramp (30C above your maximum ramp is often a good rule of thumb). The Saturn can be programmed to a maximum temp of 350C on the transfer line. You should increase it to 320C. Guessing your column max programmable temp is 330C? You should definitely consider a high temperature column for this specific application. We're also wondering if the column is tailing up considerably at the higher temps? Hope your analyte(s) aren't getting lost in the presumably higher noise. Is your analytical column plumbed directly into your ion trap or have you installed a guard column into the MS that your analytical column is connected to?

Another suggestion is to increase the splitless time from 0.5 to 0.75 minutes. The pressure pulse should definitely help you, but the splitless time might benefit from an increase.

Good luck!

Another thing we noticed about your analytical parameters... Your column is temperature ramped to a final operating temperature of 320C. However, your transfer line is only to set to 300C. At the very least, it should at least be set to the highest temperature value used in your ramp (30C above your maximum ramp is often a good rule of thumb). The Saturn can be programmed to a maximum temp of 350C on the transfer line. You should increase it to 320C. Guessing your column max programmable temp is 330C? You should definitely consider a high temperature column for this specific application. We're also wondering if the column is tailing up considerably at the higher temps? Hope your analyte(s) aren't getting lost in the presumably higher noise. Is your analytical column plumbed directly into your ion trap or have you installed a guard column into the MS that your analytical column is connected to?

Another suggestion is to increase the splitless time from 0.5 to 0.75 minutes. The pressure pulse should definitely help you, but the splitless time might benefit from an increase.

Good luck!

Thanks for the reply.
So, yesterday evening i finally saw the dibenzo pyrenes for the first time. It happened after i raised every temperature of the system, i had Tinj= 325°C, Trap= 230°C, Column last T= 325°C, Xferline= 300°C.
Their shape is very bad but at least i can see them and now i can work on it. I'll definitely try xferline T= 325°C now, and i'll raise the splitless time as well.

The noise is very low because i work on MS/MS.
The column goes directly in the trap.
Column max T is 350 programmed and 330 isothermal, and yes, they are tailing and eluting together.

I'm working on it all the day, i'll post results this evening.

Davide.

I increased splitless time and xferline T to 325°C, now i see a clean peak on dibenzo(a,h)pyrene (the heaviest).
Now i can finally start calibration. Thanks a lot for the help, much appreciated.

Davide

So glad you were able to get it to work! The Varian Saturn ion trap is a great mass spectrometer. The MS/MS feature is a really great way to screen for target analytes.

Good luck with the calibrations.