Method development for 525.2

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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Right now I'm developing a 525.2 method for some PAHs, plasticizers and a couple herbicides. I'd like to get a better handle on the process of method development, from a theoretical and practical perspective. I'm going to ramble for a bit, the point of which is just to lay out my thought process so that you all can (hopefully?) let me know if I've missed anything.

I've been doing GCMS - largely wastewater analysis for VOC content - for the better part of a decade, but this is the first time I have been involved in this part of the job. Up to this point it's all been routine, with some maintenance (GC and MS side) and troubleshooting from time to time.

My understanding, from reading up on my target analytes, is that the plasticizers and some PAHs are surprisingly sensitive to heat and light. So, to optimize my recoveries I'll want to avoid any excess of those things in the process. Also, avoid contact at any step of extraction with anything that might contain the target analytes (which, for the plasticizers, might be more an exercise in Whack-a-mole than for the rest). Run some checks on new lots of solvents, to make sure there's not contamination, and just in general document everything because of course. On to the GCMS side.

The general process of optimizing the GCMS hardware is one of making sure that the column is properly selected (length, composition, and size), the injection port is optimized (liner composition, liner geometry, septum composition), and the MS hardware is up to spec (source cleaned, electron multiplier in good working order, etc). Maintaining that is a big day-to-day thing, but so is finding the ideal starting point for method development. However, that all is also fairly straightforward.

On the software (I'm using that term rather loosely) side, we have things like the GC temperature program and MS parameters. That's where things get a bit tricky for me, as I've never been involved in temperature programming at all.

Right now the method operates on a total run time of about 38 min with a 45 C starting point, a fast ramp to 160 C, a slow ramp to 300 C, and a 10 minute hold at the end. That 10 minute hold at the end SEEMS excessive, since the last peak elutes at 28 min and my baseline creeps up past 27 min or so. Also, starting things at 45 C SEEMS low, since the lowest BP solvent (EtAc) boils at 75ish C. The first peak of interest comes out at 5.5ish minutes, which is 1.5 min past the solvent delay.

Is that thought process where I ought to be?

Operating on the notion that it is, I adjusted the temperature program so that it started at 65 C and shortened that hold at the end to be 2.5 min long. After I did, I saw the average responses for basically everything drop by about a factor of ~2 (from ~450K to ~225K for the largest non-target, but consistent throughout).

Ought I to reconsider the program changes, based solely on that response change? The change moved one of the leading non-target peaks into the solvent delay period, the column bleed at the end was largely eliminated, and it significantly shortened the run time. But, a short(er) run time not beneficial if my sensitivity is insufficient to actually do what I need it to do. So, is a higher response categorically better? Also, why did making those changes reduce the response to the degree that it did?

Also, what else should I be keeping in mind to make this method work as well as it can? Since we're not up and running "for realsies" samples right now, I want to get it to as good as place as possible, which is why I'm focused on optimizing things.

This is my first time going through method development. So, am I asking the right questions? Is my head in the right place, more or less?
Your questions are very good for this stage of the work.

As far as the lower response with changing initial temperature. 10c below the boiling point will give the solvent effect when injecting ethyl acetate, but since ethyl acetate is mid polarity and the 5 phase column normally used for this is non-polar, you can be forming droplets at the head of the column which can affect peak shapes and sensitivity. I find it best to put a 1-2m length of intermediate polarity deactivated guard column in front of the analytical column to help better focus the ethyl acetate solvent front. You also have to give it time for the analytes to transfer from the inlet to the column, and if the temperature is higher then they are moving along faster and will not form the same sharp peaks unless you are increasing the inlet temperature or doing a pressure pulse to push them onto the column more quickly.

Inlet liner choice is also part of the development. Adding glass wool with give larger responses for the late eluting PAH analytes but can also cause some tailing and will definitely result in more breakdown of the DDT and Endrin in the initial checks. Double tapered liners lose a little on PAH response but give the best protection against breakdown of DDT and Endrin. I split the difference and use the double taper cyclo liners so I have more surface area but less active sites to deal with.

Column placement is critical also to avoid tailing. I normally set the tip of the column so that is it about 1mm above the bottom of the inlet( in an Agilent split/splitless port, other models will differ and you need to find the optimum for each type).

I also like using 0.18 columns so that even with lower oven temps you can still have higher injection port pressures to keep the solvent slug as small as possible for better peak shapes.

Splitless times are also something to optimize as shorter can help the early eluting analytes while longer can help the later eluting ones.

I would work on getting the inlet setting adjusted first so that you have the sharpest peaks, then adjust temperature ramps to optimize separation. Most column manufacturers will have a list of near optimum parameters for each column and method combination and they are typically a good starting point.
The past is there to guide us into the future, not to dwell in.
"10c below the boiling point will give the solvent effect when injecting ethyl acetate, but since ethyl acetate is mid polarity and the 5 phase column normally used for this is non-polar, you can be forming droplets at the head of the column which can affect peak shapes and sensitivity."


Oh! I forgot to mention that there is an initial 1 min hold at the start temperature (45 in original, 65 in altered), to allow the sample to vaporize. You're right that we are using an HP-5ms column, so non-polar.

You also have to give it time for the analytes to transfer from the inlet to the column, and if the temperature is higher then they are moving along faster and will not form the same sharp peaks unless you are increasing the inlet temperature or doing a pressure pulse to push them onto the column more quickly.


I will watch out for that, thanks :)

Inlet liner choice is also part of the development.


Yeah, it seems like it is pretty significant but often neglected. The folks who were working on this prior to me were using four or five different kinds of liners, at various points, and it seems like the decision to go with a particular one is based more on which look the newest and not what geometry works best with the method.

I split the difference and use the double taper cyclo liners so I have more surface area but less active sites to deal with.


I'll keep that in mind, thanks! Right now I'm using a single taper without wool.

I would work on getting the inlet setting adjusted first so that you have the sharpest peaks, then adjust temperature ramps to optimize separation. Most column manufacturers will have a list of near optimum parameters for each column and method combination and they are typically a good starting point.


Makes sense. That way, you are working "early" to "late" in terms of the analytical pathway, and solving each problem in a methodical way rather than fixing a problem only to undo your solution later. I'll keep that in mind, thanks!
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