-
- Posts: 1
- Joined: Thu Mar 01, 2012 7:43 am
I'm working on methods covalidation with a C18 reverse phase YMC ODS AQ 5microm 250x4.6mm column using a water-acetonitrile gradient ; this is a method with which I work since 3 years in an other laboratory (call it Lab A) and I encounter a problem of extra peak in a laboratory B. For information, this is a related substances identification method.
I've never had such a problem in Lab A, where this analyzis is made with 5 different Waters Alliance 2695 with 2487 UV detector or 2998 PDA detector. In lab B we have a e2695 and a 2489 UV detector. In the two labs the solvents are HPLC grade, the water comes from a Milli-Q, the columns are from the same manufacturer.
Parameters of the method
Flow rate ; 0,8ml/min
UV : 240 nm
Column temp : 25°C
Sample temp : 20°C
Injection vol :20 microl
Gradient
0-25min 57%MeCN-43%H2O Milli-Q
25-35min 57to80%MeCN-43to20%H2O
35-70min 80%MeCN-20%H2O
70-75min MeCN to 57%-H2O to 43%
75-90min MeCN 57%-H2O43%
Be sure that the system is good rinsed, washed and the column has a long time to be conditionned.
My problem is the presence of a huge flat toped peak from 65 to 80 min., it doesn't appear at the first injection but at the second and appears in each next run. I monitored the baseline with or without injection, it's always there and its area isn't decreasing even if I monitor 5 times the gradient only, or if I purge the injector several times. I changed the solvents, washed and reconditionned the column and monitored the baseline ; chromatogram perfect, baseline very stable, no pressure change (1100 psi at 57MeCN-43H2O).
I restarted a new sequence and washed the column and the system with methanol then acetonitrile smoothly and I arrived at 43-57% MeCN-H2O, same "mountain" between 65 and 80 min. in the second injection, and which appears in all the following chromatograms. I decided to try another column but the results are the same. I tried to vary the injections volumes to see if the area varies, no, it's still huge and not decreasing at all, monitoring ten gradient without injections, the contamination is still there, injecting ten times pure methanol, ten times pure acetonitrile ; no change.
So, I decided to flush all the lines, needle wash, seal wash,... with water, isopropopanol, dichloromethan, isopropanol, wate MeOH, and then try to see if the contamination could appear with another test lasting less time (column Waters Spherisorb 5*100mm*4.6mm, isocratic 77-23 MeOH-H2O, run 6 minutes), I've made 30 injections for specificty, linearity,... all the results were good.
Seeing that, I thought the system was ready and clean to obtain good results so I've began one more time and the same chromatograms appeared, first injection of MeOH no problem but the mountain still appears during the monitoring of the baseline with the gradient after that.
Could it be the injector ? It seems to be an interference which cames after an injection but doesn't vary with time, I don't know where it comes from but when it's in the system, it stays for a long time.
This is my first topic on this forum so I hope that I give enough informations. If not don't hesitate to ask
I'm not a beginner in HPLC troubleshooting but this situation is very very strange, perhaps I forgot something basic or it is a very complicated problem ; in the two cases I don't know what to do, and I need to discuss about that to have other opinions and ideas because it's possible that I come in the wrong way forgeting something evident.
Thank you in advance,
Damien ROOSA
Belgium