Chromatography Forum

Hi.

I'm currently putting a method together for derivitised phenols from water. It's a relatively large suite of 27 phenols from phenol up to Dinoseb.

The problem I'm having is derivitising the phenols after extraction. I believe I have enough checks in place to be confident that the extraction and analytical phases are working.

Here is a brief outline of the extraction.

100ml of sample is acidified with HCl to pH 2 then passed through a conditioned SPE cartridge.
The cartridge is left to 'dry' with full vacuum on for 15-20 minutes.
The column is then eluted with acetone followed by ACN.
The extract is passed through a column containing magnesium sulphate (anhydrous) before derivitisation with BSTFA/TMS

The final concentration after extraction is 1000ppb of each phenol.

1ml of the final extract is then spiked with 50ul of Int Std (including bromophenol to check effectiveness of derivitisation) followed by 100ul of BSTFA/TMS.

The reason I am confident that the extraction is working is that if I do a X10 dilution on the extract the derivitisation works perfectly, but on the undiluted extract derivisation doesn't work at all, as confirmed by the addition of the bromophenol after extraction.

I believe the problem is residual water inhibiting derivitisation but none of the techniques I have tried so far have helped.

So far I have tried:-
Leaving the column with vacuum for 1 hour
Drying the column in 60 Deg C oven for 30 mins before elution
drying extract in sodium sulphate and magnesium sulphate.

Any bright ideas out there ?

Thanks

Rich

Elute with a very volatile solvent (diethyl ether). Then dry the solvent by magnesium sulfate, and allow the solvent to evaporate completely. I'm guessing you might lose some of the lower phenols, but hopefully not much since the ether has such a low BP.

BTW, a friend of a friend has been running phenols (nonylphenol etc) without derivatization for a long time. He said that his columns don't last long because he loses separation efficiency quickly due to active sites building up in the column. If you have plenty of money and limited time, you might want to try this route.

If water is the problem you are not going to get rid of all of it by sucking lab air through the cartridge (not to mention the large quantities of contaminats that you are loading onto the cartridge). Try blowing dry air or preferably nitrogen through instead of using vacuum.

I'm not sure whether when you dilute 10 x you then take 1 ml of the diluted extract and derivatize, or do you derivatize the whole diluted 10 ml ? If the latter then water is not likely to be the (only) problem, since you have just as much there as before. If the former, try increasing the amount of derivatizing reagent so that there is enough excess to react with the water and have some over for the analytes.

Peter

Sassman,
I'll try your suggestion with the diethyl ether and get back to you. We'll being sticking with the derivitised phenol method for now I think because it offers us another level of security for identification purposes. Its a SIM method so we should be pretty sure but the clients like the extra level.

Peter,
I'll look into the practicalities of blowing nitrogen through the cartridge. I can't see it being an issue for testing but may be a problem when/if the method goes live.
With regard to the dilutions. I take 1 ml of the X10 diluted sample and derivatise that. Thinking about it as I type I should be able to add 10 times the amout of the derivitising agent to the neat extract to acheive the same effect.

Thanks for the suggestions. I'll post feedback as and when I have it.

Rich

I'll post again after testing.

If you are stuck with using vacuum you can try putting a large SPE tube filled with dry silica gel above the sample SPE cartridge, then the room air gets dried (or at least dryer) as it is sucked through.

Peter

All,

Too much chemistry for this nuts and bolts guy, but here are my thoughts. First, if too much water, excess BSTFA should help resolve that problem since it will react with the water and the phenols? However, even if water is at 1% in the elution solvent, that is thousands of times more than your analyte.... Second, why use BSTFA? Ugly, gooey mess..... yech! Why do that to your poor injector? Why not use BF3/meoh or even, perhaps, diazomethane (handled judiciously.) Third, pulling 30 minutes on raw phenol, I would suspect it is evaporating off the SPE tube. Which leads me to ask the question; all phenols with low recovery or just some (like phenol?)
Fourth, no mention of salt in the sample. Might be worth a look.

Just some thoughts.

Best regards.

If you are able to use alternative derivatizations (so many posters are stuck with methods that they can't change) making the sample alkaline and then acetylating with acetic anhydride is reputed to work well on (some ?) phenols.

Peter

From where/what are your phenols derived? A few years ago I helped some aggi group put through a doctoral candidate by working out the synthesis and purification of 14C-nanophenols. We did analyses and purification with both RP and NP without derivatization. The problem centered around nanophenols in soil, suppoedly they got there via detergent derivatives of nanophenol. A researcher of this group did GC of these and got some 10 to 5 times as many peaks as we did with HPLC, needless to say, the soil analyses were also done with GC-MS.
Thus, I would also appreciate a more detailed explanation as to why you prefer derivatization.

Putting a SPE tube of silica gel above the the SPE catridge is an excellent idea. Sound like one borne of experience as well.

Why use BSTFA ? Partly because its an inherited project and the previous work was done on BSTFA and partly because it works very well on all 27 phenols in the standards. As for being an ugly, gooey mess...thats something I haven't come across yet but I haven't been running production levels of samples either.
The recovery when derivitisation works is excellent for all analytes, even with 30min draw through the SPE tube.

Switching to a different derivitising agent is something my manager doesn't want to do since so much time has been spent on BSTFA. Although if no more progress is made on BSTFA then it will have to be considered.

The phenols are derived from environmental water samples, surface and ground waters. Derivisation is a preference of the client and the industry in general.

Rich

When you're spiking, is the solvent the spike is in compatible with your derivitization?

Do you have a decent KF instrument, check the amount of moisture in your ACN and acetone.

Sodium Sulfate: As I recall from the EPA methods for herbicides (very similar procedure since they're organic acids) they have you acidify the sodium sulfate to stop any potential absorption or chemistry from happening.

Sodium Sulfate: Double-check the drying temperature to ensure it's "cooked" enough to drive off any moisture.

Are you doing HPLC-MS or GC-MS? (I suspect HPLC-MS due to the ACN but GC-MS due to wanting to derivitize). If I was running HPLC-MS, I would be very tempted to go back to the clients and see if I couldn't get them to back off on the derivitization. The main reason we're derivitizing historically, was with the older, less inert GC columns, it was the only way to get them through a GC. If you're running an HPLC-MS, you're adding a level of complexity that even when you get it working right, is another point where something can go wrong (I'm a strong advocate of the KISS principal).

Sorry, with "derived" I meant to find out what phenols are you analyzing. If you are doing nanophenols with HPLC I would certainly like to see details on how you separate 27 analytes. If you are doing GC of nanophenols I just wonder how someone can force anybody to ignore some 10 years of development.

"Nanophenols" or "Nonylphenols" (common industrial chemical of current interest due to endocrine disruptor potential)?

Greg

Ups, made that mistake again, the correct word is "nonyl". Anyway, I am restating one of my questions: Is Dinoseb a nonylphenol as well as all others, except of course, phenol?

Nonylphenols are formed during degradation and/or production of polyethoxylated surfactants. They are common contaminants in the environment. Dinoseb, although somewhat chemically similar, is a pesticide and is not related to the nonylphenols.