Chromatography Forum

we here at work want to know why we have to wait 10 hours after derivatising our samples and stds. We work on a RHPLC and we are working with aminoacids (20 of them) we use the Accutag derivatising reagents and have a 66min runs for each samples. Could any one tell me why and how important this may be.
Tracy

Just off the top of my head, possibly because the derivatization reaction is slow and you need to allow that long for it to go to completion.

If you are running a validated method, then you have no choice about the conditions; it should be run exactly as written (even if some aspects seem counterintuitive). If it's not a validated method, then you presumably have some leeway and it might be worth investing some time to try systematically decreasing the wait (perhaps cutting in half each time) and plotting the response for standards and then "known" samples to establish how short you can make it.

It is probably the case that 19 of the aa reactions are done in a minute or two. It's that 20th aa that takes a couple of hours. Include the changes in reaction rates that are a function of relative concentrations of the other aas present and you arrive at, probably 2.5 hours. Then someone discovers that the %RSD goes down 0.1% for a rack of validation samples that was left out over the course of a couple of meetings and lunch.

Voila, 6 hours.

Uwe would be in a better position to answer this query, but i would say check your method description again. The Waters AccQ.Tag derivitisation method is not 10hours, but 10min in a oven at 55C. To a 10ul aliquot of your sample add 70ul borate buffer and mix, then add 20ul of the AccQ.Fluor reagent, mix immediatley, then heat at 55C for 10min.

We inject straight after this proces and have no problems with repeatability in standard or sample injections with respect to peak area and internal standard normalisation.

Greg

I will check with our experts tomorrow, but to the best of my knowledge, the reaction is over in seconds, and the reagent is completely consumed after about a minute or so. It makes no sense to me to let the stuff sit around for 10 hours.

It's maybe your samples takes 10 hours to "hydrolise". If your samples are in Carrier and not in Amino acids.
The derdivatization take a few minute, and for de derivatization of Cysteine in Cystine, head 10 min in 55°c listed here.