Chromatography Forum

Hey All,

I am seeing a strange occurrence with a headspace method we are trying to validate.

I am experiencing a response suppression of the six residual solvents being determined (methanol, ethanol, acetone, ethyl acetate, THF and dioxane) when the material under test is present (cholesterol, 50mg/mL). The responses are approximately 20% lower than the standard without cholesterol.

Has anyone seen this before when working with large molecules? Is the steroid backbone doing something or is the method at fault? I doubt p-xylene is acting as the suppressant.

Here is the method.

Diluent: p-Xylene
Test solution: 50mg/mL cholesterol
Standard Conc.: 19µg/mL to 250µg/mL
Static Headspace: 22-mL vials, 4 mL total of solutions, heated for 30min @ 120°C
Transfer Line: 125°C
Inlet: 150°C with 2mm liner
Injection Volume: 1 mL (sample loop)
Column: DB-5
Detector: FID

All the responses of the Standard are linear from 10-250% of the nominal concentration. Once spiked into the headspace vial with the test solution, a suppression occurs.

I then tried to convert the method to a standard addition method to account for this issue. Now we are seeing recoveries all over the place (75 to 175%). It seems as though some variance is occurring here too between the test solution and the spiked test solution.

Basically, I have linearity, precision, specificity, but no accuracy. Recoveries by standard (bracketing) calculations gives me 20% lower values than expected with the cholesterol solutions and the standard addition approach gives me variances at the different concentration levels being evaluated.

Any clue as to my problem?

Thanks in advance,
Burt

You wrote: "I doubt p-xylene is acting as the suppressant. "

Oh ye who don't doubt when YOU SHOULD. Learn to doubt unless proven.

QUESTIONS:

You are adding a solution of cholesterol in toluene? What volume?

The solution of residual solvents is also in toluene? What volume?

If I read your post correctly you are putting 4mL of toluene into a sealed vial and heating it 9 degrees above its boiling point. This is NOT recommended IMHO.

The matrix solvent you choose MUST have an adequately low vapor pressure at the temperature you choose to partition the volatiles or you can saturate the headspace with matrix solvent vapor. Then poor Henry will never get his law to work. It doesn't work under saturated or supersaturated conditions.

Suggstions:

Put your solvents and cholesterol into dimethylacetamide instead of toluene. Don't add more than a total of 100µg of volatile residual solvents in your vial unless you HAVE DONE THE RESEARCH to show you are not reaching a saturation point.

ND

Don't use more than 1mL of total DMAc solution in your vials.

Remember you don't have to dissolve the cholesterol before you heat the vials, just watch and make sure the DMAc dissolves the cholesterol and that the static heating of the dissolved sample lasts for at least 15 minutes.

Good luck.

Rodney George

Hi

Seems like p-xylene (bp 138°C) and not Toluene (bp 110°C), but the effect is nevertheless the same. Ie you get too much diluent vapor in gas phase acting as a suppressor and making the partion coefficiant unstable.
This can happen with a lot of typicall headspace diluents even water at too high tempertures.

Yes then we have the matrix effect ie the samples effect on the partion. The most common one is an increased response of polar samples on polar solvents. Sometime one can use this effect to increase sensitivity (or even out matrix effect to avoid std addition)if water is used as diluent by adding a salt like 0,5g sodiumsulphate to all vials. In this case you have a "fatty" sample that attracts your solvents giving you negatie efect though hard to be certain due to your choice of diluent and headspace temperature.

So as Rodney stated, go back and chose a wiser diluent like DMF, DMSO or DMAA, and it is true that sample do not need to be dissolved at room temperature it is enough if it dissolves in oven.

Oops !

Meant to type "heating it 9 degrees within" not "9 degrees above".

This old man can't seem to always type with his fingers what his mind orders them to type. Hmmm. Well, at least I still have most of my hair.

Moral of the story.

Do your research. Sadly many companies don't want to anymore.

And it usually means either you don't have a product or the product will end up biting you in the A**.

Have a good day !

Rod