Chromatography Forum

I posted a few months back about Endrin breakdown issues and I am still fighting with it. Quick background again... I'm running EPA method 505 pesticides. Endrin breakdown must be below 20%, injected as Endrin only standard (No DDT), and injected at a midpoint level of our calibration (which is 1ppb) and with the same parameters that we run calibration & samples with. Of course my trouble is that no matter what I do I can't get Endrin breakdown below 20%; and I've tried pretty much everything. Like I said I've been battling this for several months now so here are all the things I've gone through. And I'm running on a Thermo trace 1310 GC

-Started with basic inlet maintenance, Changed septa, liner & gold seal (many times over the few months)
-Cleaned the inlet several times with several solvents, most notable cleaning was using Agilent's method with a brass gun barrel brush.
-Clipped a good 24 inches off the column
-Replaced the carrier and makeup charcoal gas filters (this is part of the 1310 inlet design).
-Tried different oven ramp settings including starting the run at higher & lower temperatures
-Tried different inlet temperatures ranging from 170C to 250C
-Used different type and brand liners
-Ran in splitless mode (our normal run mode)
-Ran in split mode
-Used different split ratios
-Ran the standard at different concentrations in both split and splitless mode (Would not be able to do this on a normal run but wanted to try it anyway)
-Tried different manufacturer standard (Ultra, AccuStandard & SPEX)
-Ran Endrin & DDT standard (the one that we use for Method 525)
-Ran Endrin only on method 525 breakdown (no breakdown when run on method 525 MS/GC)
-Put new triple filters on both carrier and makeup gasses (the Aglient Big Universal Trap that filters moisture, hydrocarbons & oxygen)
-Triple checked anything & everything for leaks.
-Changed the injection needle several times
-Made the endrin standard dilution in different solvents (Hexane & methanol... we normally dilute it in Hexane for the run)
-Triple checked that Endrin, and both aldehyde & ketone were properly identified
-Made sure there was no contamination or carryover by injecting a hexane blank (that always came out clean)
-Used different sources of gasses (main line gasses and from cylinders)

I know there are a few other minor things that I did but they are escaping me right now.

** A few things to note: All things listed above are listed in no particular order but were performed very methodically, I didn't go changing and/or cleaning things all willy-nilly. When cleaning the inlet I know for most GC's to not forget about cleaning/changing the split valve/line. Unfortunately I cannot do this because of the modular design of the inlet. I checked with Thermo's techs and they said it could no be accessed. The same thing goes for changing out the inlet, it's built into the module and cannot be taken out. Poor design, I know. Changing the septa & liner were done many times throughout. I did a few hot swaps but I mostly changed it like you are supposed to by purging any oxygen in the inlet before heating it back up and running. The same lot of hexane was used to run hexane wash blanks & to make the Endrin dilution. The hexane always showed it was clean, no hexane contamination. All Endrin standards used were also run for breakdown on method 525 GC/MS. That always show no breakdown meaning that the standard was not breaking down in the vial. Everything that was done made little to no change in Endrin breakdown. Average breakdown was 35%-45%.

** Last thing to note, and this is oddball. For 4 days recently (5/10/16 to 5/13/16 a Tuesday to Friday) endrin breakdown was at 13%. For the pervious week and a half all that was done was change the septa & liner first thing in the morning then run a few injections of the endrin standard. During that time endrin breakdown was at the normal 35%-45%. During the 4 days that breakdown was at 13% the only thing that was done was change the septa & liner first thing in the morning then doing some runs throughout the day. Endrin breakdown was "good enough" leaving that Friday afternoon. came in Monday morning, changed the septa & liner and breakdown was back to 35%-45% and has not gone back down since. What happened in those 4 days for it to work I have no clue, I did nothing different or out of the ordinary.



I know this has been a lengthy post and if you have read this far thank you very much.

I'm completely out of ideas. My best guess right now is that there is an active site somewhere in the inlet module that I cannot access to clean. So given all that I have done so far and nothing has worked do any of you have suggestions or ideas of what may be going on or what else I can try?

Thank you in advance for any help.

What is the endrin breakdown using 525? If it is acceptable, what differs from the 525 and 505 set ups?

If I remember correctly 505 is an ECD method, and I hate to ask this but, are you sure you identify EA and EK peaks correctly?

With all that you've done, I'm beginning to think that the GC is not the issue. I'm wondering if the glassware might be the problem, and in those 4 days when it worked well you were using "good" glassware.

What is the endrin breakdown using 525? If it is acceptable, what differs from the 525 and 505 set ups?

If I remember correctly 505 is an ECD method, and I hate to ask this but, are you sure you identify EA and EK peaks correctly?

505 is GC/ECD and 525 is GC/MS. Running the standard on 525 there is no breakdown. Outside of one being ECD & the other MS the other big difference in setup is the instruments themselves. 505 is being run on a Thermo Trace 1310 and 525 is run on an Agilent 7890.

And yes, EA and EK have been correctly identified. I ran separate EA & EK standards to make sure they were the correct peaks.

With all that you've done, I'm beginning to think that the GC is not the issue. I'm wondering if the glassware might be the problem, and in those 4 days when it worked well you were using "good" glassware.

I don't think it's the glassware. I had used the same Endrin dilution on both the days it worked and did not work. The only thing that changed from day to day was the sample vial cap all of which came from the same bag of caps.

Just curious, why is it that you change septa/liner every day before running?

Is it possible that there are active sites in your liner of choice? Possibly a liner that has been run on for a longer period has been "de-activated" therefore giving you lower breakdown %'s.

Normally a new liner, septa should have minimal breakdown. I know I have heard that metal/steel injectors can cause Endrin breakdown and a Silco-coating is helpful.

Just my 2cents. I used to run 8081B via GC/ECD so I'm a bit unfamilar when it comes to GC/MS.

I've been looking into my breakdown problems a little more an I think I may have figured something out.... nothing that will fix it but I think I've found a contributing factor. I think one of the issues may be with the baseline and how the software (Chromeleon) interprets peak area. The baseline is given in kHz and peak area is read in kHz/min.

I had a few days last week when Endrin breakdown passed but then my calibration looked terrible. That's when I noticed my baseline had slowly risen. It was up at 12kHz when ideally it should be around 1.5kHz - 3kHz. I also looked back at the other days a few weeks ago when breakdown was passing and the baseline was trending around 5kHz-6kHz (which is still acceptable). Going forward I adjusted pulse width settings (which adjusts the baseline) so that the baseline was reading 1.5kHz.

**Side note: Where the baseline was trending also dramatically affected my calibration. I ran calibration points with the baseline at 12kHz and it was all over the place. Adjusting the baseline to 1.5kHz I re-ran the same vials and this time the calibration was almost better than perfect.

After that adjustment Endrin breakdown went back to failing (around 30%). Here's where peak area comes into play. The higher the baseline is trending the larger the peak area reads. Examples:
Baseline at 1.5khz Endrin peak area 0.0224kHz/min - Breakdown failed
Baseline at 5.0khz Endrin peak area 0.2020kHz/min - Breakdown passed
Baseline at 12.0khz Endrin peak area 0.5922kHz/min - Breakdown passed
The baseline shift also effects the ketone and aldehyde peaks the same way just not in such drastic proportions. I think between this and with calculating larger numbers you have a little more wiggle room for deviation, could be contributing factors to my problem. If anything I believe this explains why I was able to pass Endrin breakdown those few times. Unfortunately it still doesn't get me any closer to figuring out why and where Endrin is breaking down.

After all I've done thus far and with these new developments I'm still at a loss. Could it be possible that the instrumentation and/or software is not reading and interpreting the data correctly and giving me bad numbers?