Chromatography Forum

I have a few comments from a previous post:

[quote]The Sigma-Aldrich product manager indicated that he often sees large, elevated baselines with inexperienced biodiesel producers. This can be attributed to two sources:
1. Incomplete derivatization of the sample with MSTFA. This can be caused by:
a. using the wrong MSTFA reagent;
b. not waiting for the reaction to complete;
c. waiting too long after preparing the samples;
d. excess water or alcohol in the sample that uses up the MSTFA.

Elevated baseline due to incomplete derivatization can be overcome by baking out the column.

2. Poorly made biodiesel that contains large amounts of unreacted vegetable oil. This problem cannot be overcome and the column and precolumn must be replaced.

He also provided the following recommendations:

Calibration standards and samples should all be prepped at the same time, using the same lot of derivatization reagents.
The MSTFA derivatization reagent should be purchased in 1-ml ampoules, and fresh reagent should be used every time you prepare standards and samples. The only MSTFA customers should use is "Sigma-Aldrich Derivatization Gradeâ€

The purpose of preparing the standards fresh every time you run samples is that you use the same derivatization reagent since the reagent is not very stable. If it is half bad and gives incomplete derivatization, then at least your standards will have the same level of incomplete derivatization that your standards do. I don't think it is critical to run the samples immediately following derivatization since the derivatized product is quite stable. You do however have to avoid evaporation of the solvent that the sample is dissolved in and/or precipitation of any components of your sample.

Yeah I agree. However it isn't always economical or practical when the GC is down for a day to have to re-silyate all the samples and throw the rest out.

I agree with analytical chemists have about quality, but I think sometimes people go overboard on stuff.

You do however have to avoid evaporation of the solvent that the sample is dissolved in and/or precipitation of any components of your sample.

The reality is to avoid evaporation of the samples, you have to have a controlled temperature and humidity chamber or environment, which most people don't. The solvents are going to evaporate at least a little bit no matter how quickly you cap it.

As long as your samples and standards were all derivatized at the same time with the same batch of derivatization reagent, they should be stable for at least a few days if not a few weeks. So in this case you don’t have to throw everything out and start over again. As I mentioned, the biggest concern is derivatizing samples with a different batch of derivatization reagent (or the same batch that has been sitting around for a while) since these reagents are often not very stable. Everytime you prepare your samples you have to derivatize them, so why not just derivatize new standards at the same time?

Yeah I pretty much derivatize all my samples at the same time. I also have been using the same derivatization reagent as the method specifies.

As noted in earlier threads, the 100 runs per column cited is much lower than typical users get with most columns and Biodiesel, but it depends on the initial lipid quality, and the derivatisation protocols followed.

If you are reporting results to a formal method, and that method lists maximum permitted time/temperature between derivatisation and analysis, then you are required to adhere to that, unless your client agrees otherwise.

The problem is that some lipd derivatives are more unstable than others, especially from highly unsaturated lipids, consequently the method may be based on a worst case situation.

Some FAMES can autocatalytically polymerise, which then report low in unsaturates, and start gum forrmation - which may not be miscible with your solvent, and which will not elute.

It's not really about how stable your samples are, but what conditions were evaluated during the method development and validation.

Bruce Hamilton