Chromatography Forum

We determine aflatoxins in precolumn derivitized samples by HPLC Fluorescent technique. It was working well for many months until last two weeks when at broad peak started appearing at the retention time Aflatoxin B1 making it difficult to determine the peak area of AflatoxinB1 (at 8.1 minutes)

we checked the chemicals used in the derivitization (acetonitrile, hexene and trifruoroacetic acid). Only TFA even gives the said peak which over the AFB1 peak.

We used another bottle of TFA but it did not solved the problem.

Can any friend help me to know this problem and how to solve it

Best Regards

Zargham

Did I understand this correctly, you injected TFA and got a broad peak in fluorescence detection? I would expect a tiny wiggle if anything, as TFA does not fluoresce. Is this a gradient method?

HW Mueller
You got the point correctly. The broad peak occurs when TFA is injected alongwith ACN with or without sample

We use isocratic system and mobile phase is Methanol+ACN+Water (22.5+22.5+55)

Zargham

Does the peak appear on all injections, and if you inject a lower concentration of TFA ( 50% ), does the peak decrease by the same amount?.

I would suspect rubbish in the injector, guard or column is being mobilised by the TFA, perhaps appearing in later injections. The most likely source is your samples, but don't exclude mobile phase or reagents. You can test for those by changing the equilibrium time between injections and, separately, varying injected quantity.

If deposited rubbish is the cause, you could change the mobile phase to flush it out, change the guard, clean the filter, or perform multiple injections of TFA, and see if the peak area decreases. If you have another column, try that.

Simply put, you need to ascertain what causes the peak, and then make changes to sample preparation or the chromatography to separate it from your analytes.

Please keep having fun,

Bruce Hamilton

Perhaps a clarification on what Bruce said: He is telling you that TFA does not fluoresce.
It´s also possible that you are injecting gunk from the TFA itself.
It might be interesting to note that there are some methods in which strong acids convert non-fluorescing compunds to fluorescing ones (this is digging into remote corners of the brain, . . . someone did this with ouabain?).

HW Mueller and Bruce Hamilton

I will start working as per your suggestions from Thursday and let you know the results. Many thanks for advises

Hi M Zargham Khan,

I’m guessing now, but maybe you’ve observed the following situation:

You install a brand new column, equilibrate it and inject your analyte. The first several injections result in increasing peak heights (and areas) until you reach a level at which the peaks get constant.
If you’ve experienced the above described situation, then the answer to your question is as follows:

Some of the analyte, or another part of the sample, sticks (irreversibly under the utilized conditions) to the stationary phase support, until the latter is saturated with the compound and thus all subsequently injected amounts of analyte elute completely/reproducibly.
At the describe conditions, the chance for active silanols is very high indeed. So, my hypothesis is that the retained analyte is sticking by interaction with these silanols. And when you inject the TFA, the picture changes – TFA interacts with the silanols thus releasing the retained analyte from its “captivityâ€