materials and methods

[stecrom84]

Good morning at all, I created this section of the forum to discuss and find solutions to some methods in hplc

start with a problem:
I can not see sorbic acid on the chromatogram; I explain you

I'm trying to separate sorbic acid from transmuconic acid:
I dissolve the standard of sorbic acid in ethanol and then diluted it in water to inject it onto the hplc; The mobile phase consists of a 1% acetic acid solution (phase A) and methanol (Phase B), the column being a Perkin Elmer "COL - C18 3um 100 x 4.6mm". The standard of transmuconic acid dissolved in ethanol and diluted in water and injected is seen at a retention time of about 5 minutes at 264 nm. Under the same conditions, the peak of sorbic acid does not appear; sorbic acid is very similar to trasmuconic acid , how come you do not see it ??

Can anyone help me understand this arcane?

[Gerhard Kratz]

No chromophore group. Try an RI detector.

[Rndirk]

We measure sorbic acid in food by HPLC-DAD daily. You don't need an RI.

We don't know if there's something wrong with the chromatography. What concentration, injection volume and what is the mobile phase composition (and gradient program)? I think 1% acetic acid is quite high.

Is the detector a DAD or a UV? If DAD, measure at wavelengths between 230-270nm. If UV, try 254nm for sorbic acid (maximum).

[stecrom84]

I dissolved 1 mg of sorbic acid in 10 ml of ethanol to obtain a concentration of 100,000 mcg / l, then I diluted 1: 100 by pipetting 100 μl of initial solution with 9.900 ml of water hplc gradient to obtain a solution of 1000 mcg / lt and I injected 25 μl of this solution in hplc, now i try to make a reading at 254nm in uv
I know a portion of sorbic acid ingested with the diet is converted to muconic acid (it seems to me 0.12%) but the rest of it? is sorbic acid in the urine? And if it is confirmed I should find the molecule of sorbic acid near or overlapping with muconic acid I explain?

[stecrom84]

the method is isocratic: Phase A (1% acetic acid solution) and Phase B (Methanol)
is the classic method for detecting muconic acid and we were told that with this method we could also see the overlay of sorbic acid