[lamaksha]

Hi,
I am doing a project involving extracting different lactones from the supernatant of bacteria (which produce it as the chemical messengers for quorum sensing) using HPLC.

As a standard, I ran a sample containing 100nM C8 lactone (octanoyl homoserine lactone) on a C18 Reversed Phase Column (flow rate 1ml per minute, gradient from 50% methanol to 90% methanol), for 50 minutes, and fractions collected every 2 minutes. The protocol was given to me by my supervisor, and the added C8 lactone was supposed to elute in the 10th fraction. While I do get C8 in the required fraction, I also get C8 in a much later fraction is almost equal amounts. Does anyone have any reasons as to why this is happening?
(I tried with another lactone, C10HSL, and once again, the lactone elutes in the 'proper' fraction, and in another later fraction as well.)

Thanks for your help, and sorry in advance if I sounded oblique here.

[macman104]

Different configurations that result in different polarities of the compound?  Just a random guess.

How do you confirm you have the lactone, is it possible it is opening resulting in a much more polar acid? (This seems much less likely, but again, just a random guess).

[enahs]

Unless you are getting one long peak and collecting it in the fraction(s) immediately following, you are getting two different products.

If you have a few minutes of separation, they are different compounds.


Unless of course your column turns out to be a racemic column. Is your C8 lactone racemic?