EPA 515.4 Diazomethane

[Cody]

I have a question for those of you who run EPA 515.4. After injecting the diazomethane solution into your extract, does your extract stay a slight yellow color and if so, for how long? Mine barely stays yellow (maybe 10 seconds) and then the color fades. I'm having trouble replicating analyte responses from one of the earlier times I performed this method and I'm wondering if this is my source of error. Anyone know of anything I can look for that might be preventing the diazomethane from methylating my analytes? To my knowledge, I am following the EPA method exactly as written unless I'm just missing something. Any help is greatly appreciated.

[Don Shelly]

Your extract should remain yellow for 30 minutes after adding the diazomethane. If it fades before then, your diazomethane is too weak.

If you are using carbon black stoppers in your diazomethane generator, the gas will bind to the stoppers and give you a weak solution. Switch to silicone stoppers and see if that helps.

Don

[Cody]

Don-

I'm using a diazomethane generator purchased from Ace Glass. It has plastic connectors that screw into each compartment with O-rings.

-Cody

[Don Shelly]

Are you reacting the extract and diazomethane in borosilicate glass or soda lime glass? Ideally, use deactivated borosilicate glass.

Could there be some small particulates in your extract that are causing the diazomethane to effervesce? Think Mentos in soda.

[Cody]

Don-

It's borosilicate glass.

Also, how much acidified sodium sulfate do you use to dry your extract? And for how long do you let it sit before transferring for the derivatization process?

Also, if you'd be interested, you can email me to discuss some of the issues I'm having. It would be greatly appreciated. I've been having these issues for months and I can't figure anything out. My email is cody.dickinson@me.com.

-Cody

[Cody]

I'm going to post some of the things I tried yesterday to rule out where the error was coming from. Hopefully someone will help me find an error that I'm doing.

1) I put only MtBE in a glass vial (the ones I use to derive in) and injected 250 uL of the diazomethane solution in it. The solution stayed yellow for well over 30 minutes. I tried this to see if there was anything wrong with the container itself. With the solution staying yellow, I assumed that the vial wasn't the issue.

2) I made a solution of the analytes, surr. std, and int. std. in MtBE in another glass vial. I injected 250 uL of the diazomethane solution and the solution stayed yellow for well over 30 minutes. I tried this to see if there was anything possibly wrong with the stock standard solution that I was using. With it staying yellow, I assumed that this wasn't the issue either.

3) I made another solution of the analytes, surr. std., and int. std. in MtBE. I placed this solution in 0.6 grams of the acidified sodium sulfate, left it for an hour while swirling it by hand every 15 minutes. I then transferred this solution to another glass vial and placed 250 uL of the diazomethane solution into it. The solution stayed yellow for more than 30 minutes. I did this to see if there may have been an issue with contaminants or something else in the acidified sodium sulfate that I had been using. Since the solution stayed yellow for more than 30 minutes, I assumed this couldn't be the culprit either.

4) I made a standard equal to my highest level standard and ran it through the EPA 515.4 standard method. I raised the pH by adding 4N NaOH, left it for an hour while shaking every 15 minutes. I added 10:90 MtBE:Hexane solution, shook, and took off the upper organic layer and discarded. I lowered the pH by adding 2mL of sulfuric acid, added 2 g of copper sulfate, 16 g of muffled sodium sulfate and mixed. I then added 4mL of the extraction MtBE solvent with the internal standard, shook for 5 minutes and let it sit for 5 additional minutes. I took off the upper organic layer and added it into a glass vial with the acidified sodium sulfate (however, I used 1.5 grams instead of the 0.6 grams). I let it sit for an hour while swirling it every 15 minutes. I then transferred this to another glass vial, added 250 uL of the diazomethane solution and the extract only stayed yellow for 10-15 seconds. I added another 250 uL of diazomethane solution and the same thing occurred where it only stayed yellow for 10-15 seconds. The reason I used 1.5 grams of acidified sodium sulfate this time instead of the usual 0.6 grams is because I was afraid that maybe I wasn't drying out my extract efficiently and that dissolved water might be reacting with the diazomethane.

To be honest, I'm at a complete loss of what to do next. The very first time I did this method, I passed all of my QC requirements and also my performance evaluation samples. Ever since that first time, I haven't been able to get anywhere close to the response on the instrument and I'm suspecting it has to do with the extract not getting methylated properly due to the solution not staying yellow.

If anyone has any suggestions, it would be greatly appreciated. If you would rather email me, my email is listed in one of the responses above.

[jdjaw]

We currently do this method in our lab and the diazomethane causes the extract to remain yellow for a long period of time. I think the way it looks it would take an entire day to for the color to fade on its own. The first thing I would look at is if your diazomethane is concentrated enough. If there are any cracks or leaks in your closed system then it will not be concentrated enough and therefore not be strong enough to completely derivatize your extract and yield consistent results. When your diazomethane is done generating it should look like a dark yellow. I guess the best way to describe the color is almost like Mountain Dew. I am not familiar with the system you are using to make your diazomethane but the apparatus we use to generate diazomethane is the the one from Wheaton. We allow the diazomethane to generate for 45 minutes before transferring and after 45 minutes there should still be some diazald in the apparatus indicating we had enough to saturate the MTBE.
We generally generate our diazo the same day as the extraction and store it in an amber voa covered in foil inside a refrigerator. At my previous job we didn't save the diazo in a container away from light and you could tell that it would fade throughout the course of the day.
We use 500 uL of diazomethane for our derivatization just to be on the safe side. For the drying step we use 0.5 to 1g of acidified sodium sulfate and simply swirl it a bit then let it dry for about 10 minutes. I highly doubt this drying step has anything to do with your diazomethane issue though. If there are any questions or anything is unclear I'll be happy to give you more information on our extraction but our results are coming out pretty good these days.

[Cody]

jdjaw-

How do you prepare your diazald solution that you use in your diazo generator?

And our generator is a glass setup that was purchased from Ace glassware long before I got here. As far as I can tell, all of the connections are tight and the apparatus is identical to the one shown in the EPA method. I also make the diazomethane solution same day as I make the extraction.

[jdjaw]

At my current workplace we do not use a diazald solution. The setup is pretty simple where you simply put the diazald powder into the inner tube and put in 2 mL of GC grade MeOH. At the top of the tube is a small hole. An outer collection glass tube has 5 mL of MTBE which "captures" the gas that is generated and travels through the small hole in the inner tube into the outer collection tube. Everything is sealed in a closed system and capped off with a black cap with teflon septum. Then we use a syringe and add 6N NaOH dropwise through the septa onto the diazald MeOH mixture very slowly. This starts the reaction and gas gets generated. We also do this all in an ice bath. At my previous work place we did use a diazald solution but I do not remember exactly how we made it since it was a couple of years ago, sorry.

We are actually doing 515.4 today and I am making the diazomethane. I can take a few pictures and upload them so you have a better idea of what is going on as well as the color our solution is at the end.

[Cody]

That would be greatly appreciated.

Unfortunately, I doubt I can use any other method of generating diazomethane. My governing authority over my lab doesn't like it when the "recipe" (in terms of reagents) is changed.

If I decreased the amount of MtBE I used in the collection vial from 10mL (which is what the method states) down to 5 mL, do you think this would help concentrate it down enough to be successful?

-Cody

[jdjaw]

Decreasing the MTBE could help make it more concentrated or using more of the diazald solution and what I'm guessing is KOH might work as well. Either way I'll document what I do today and at the very least you can see the color and compare.

[Cody]

Thank you!

-Cody

[jdjaw]

Here is the process we use to make diazomethane:

This is all the parts to our apparatus



This is the apparatus with diazald in the inner tube



This is the fully assembled apparatus. Inside the inner tube with the diazald is 2mL of GC MeOH and the outside is 5 mL of MTBE.



This is the gas being generated after adding the 12 drops of 6N NaOH



This is the final product with excess diazald in the inside so we know that the solution is saturated.



This is what the extract looks after 30 minutes of derivatization



It is darker than it looks the condensation on the outside from the ice bath makes it look a little lighter. The last picture is actually 20 minutes into the generation but the process is pretty much done we just leave it in for 45 minutes because that is what the directions from Wheaton state. After all this we take it apart and transfer to a 40 mL amber voa covered in aluminum foil and throw it in the fridge to be used in a few hours. One last question is how do you store your diazald solution?

[Bigbear]

I do this method and don't have issues. I made a "generator" with 2 septa voa vials and plastic tubing. Vial one has some MtBe in it, and the second has a mixture of 4ml1:1 MTBE/carbitol and 400mg diazald. I bubble N2 through the apparatus, collecting in 10 ml ICED Mtbe. Icing the collection MTBE helps and sometimes I mix some NaCL with the ice to really cool the MtBE.
My extracts turn slightly yellow (all that's needed) after adding 300ul diazomethane solution.
I store the unused diazomethane in a capped tube @ -80C and have used it for months.

[James_Ball]

I just mentioned this to our Herbicide Analyst and he mentioned that if there is any elemental sulfur in a sample it will cause the color to disappear quickly. Could you have some residual sulfur present in your copper sulfate?