Chromatography Forum

Hello everybody!!!

i am a beginner in the LC world and here is my problem...

i measurend twice the same compound leucine enkephalin (LE) which is my ISTD in my study... once (4 weeks ago) LE was coming out after 5.44 mins in my 23 min gradient - today LE is coming out at 5.28 mins

i am using Mobile phase A Water:Acetonitrile (95:5 v/v) with 0.1% AA and Mobile phase B 100% Methanol with 0.1% AA

the only thing that changes between the two measurements is that i had to do again my solevnt A and B

is there any trick to make my LE coming out again near 5.44 min

thanks a lot for you help

Alain

That is a rather modest shift in retention time. Most likely it is due to variations in the making of the mobile phases. If you want very reproducible mobile phases, make them by weight instead of volume. Also, if you don't use a column oven, the retention can vary. Your column should be quite stable with this mobile phase, but just to be safe, store it in unbuffered MeCN when you are not using it.

This is a very small change. It could be due to the accuracy of mobile phase makeup. It could be due to column use/age. Did you use the column continuously for this time? Apparently not... If not, how did you store it (in the care and use manual it prescribes that the column should be stored in 100% acetonitrile for an extended period of time)?

hey thanks so much for these quick replies

i stored the column in 100% ACN... the problem for me is that i am using markerlynx from waters to detect and collect m/z peaks inside my LC chromatograms... and the LE is used as an ISTD - the ITSD is used for retention time correction and for this reason would be really nice (important) that hs retention time doesnt vary that much!!

so doi have to redo my solvent or is there an other option

best,

Alain

How many times did you measure that at the two different times? (How do the values vary within one session?).

hey the first retention time 5.44 has been measurement 964 times (i am doing a metabolomic study) with an UPLC-MS instrument

and the second (6 months after) has been measurement without the matrix already 10 times

and as u probably know uplc gives very reproducible retention times

With those numbers I would start to look towards the accuracy of the pump comp. The gradient valve may be going on you...

I am confused. The first analysis time was 4 weeks ago and the second is 6 months after???

Anyways even after 4 weeks and 964 injections on the same column, a ~3% variation in RT would seems reasonable to me.