Gradient Baseline UNUSUAL Drift in PAHs analysis
Posted: Sun May 05, 2013 3:28 pm
Dear All,
It is me again!!
Again I am struggling since few weeks with my HPLC. I am running a gradient with the following conditions:
0-5 min: Isocratic at 40%ACN/Water
5-25 min: Linear Gradient 40% ACN/Water to 100% ACN
25-35 min: Isocratic at 100%ACN
35-45 min: Isocratic at 40%ACN (Re-Equilibration)
Low-pressure mixing pump, Flow = 1.5 ml/min, UV Detector at 254nm, Temp.=30 oC, C18 column 250x4.6mm 5um with a guard column, online degasser, autoinjector set at 20 ul injection. Sample solvent is ACN and samples have low levels of analytes.
In a previous post, I discussed a problem with a strange peak on the 1st of Jan this year, found here:
viewtopic.php?f=1&t=21608&hilit=hplc.chem&start=0
The PROBLEM this time is a DRIFT in the baseline at the end of the run as shown in the chromatograms below:
I am analyzing PAHs in Soil extracts (cleaned up by silica column) and I noticed the drift for the first time during the analysis of the first batch of samples (114 injections of which 72 soil extracts). The baseline started to drift sharper than usual, and the gradient ended at higher absorbance levels from the normal level at about 0.0005V to a level above 0.004V, even with acetonitrile injections!
I suspected that some contaminating compounds are accumulating in the column or guard so, I back flushed the column with 100%ACN for at least 1h @ 1.5 ml/min. I also, cleaned the injector by injecting ACN at least ten injections.
After this cleaning, I got the normal level of absorbance for the blank gradient. However, I ran the 2nd batch of extracts and I got the same effect again. So, I did similar cleaning and I got back the good absorbance level.
I then used the instrument for several weeks analyzing only standards and spiked solutions for recovery experiments. During this period everything was OK.
Then I back again to the dirty samples with a third batch of extracts BUT I feel GUILTY this time! The absorbance at the end of the last run was more than 0.004V!
I did the same cleaning procedure but was not successful this time! I tested the level of absorbance by injecting replicates of acetonitrile but I get good absorbance only at the first run! In the rest, absorbance at the end of the run is increasing with a similar pattern to what I noticed in the soil extracts, but with no clear trend between successive injections.
I then changed the pump's filter, new guard, back flushed the column again but no success this time!
I am now feeling GUILTY
because I believe that I KILEED the column! However, I am not sure about this conclusion and I do not want to install a new column in case there is another source of this problem that might kill the new column as well!
Any suggestions from your experience are very appreciated.
Many thanks in advance.
It is me again!!
Again I am struggling since few weeks with my HPLC. I am running a gradient with the following conditions:
0-5 min: Isocratic at 40%ACN/Water
5-25 min: Linear Gradient 40% ACN/Water to 100% ACN
25-35 min: Isocratic at 100%ACN
35-45 min: Isocratic at 40%ACN (Re-Equilibration)
Low-pressure mixing pump, Flow = 1.5 ml/min, UV Detector at 254nm, Temp.=30 oC, C18 column 250x4.6mm 5um with a guard column, online degasser, autoinjector set at 20 ul injection. Sample solvent is ACN and samples have low levels of analytes.
In a previous post, I discussed a problem with a strange peak on the 1st of Jan this year, found here:
viewtopic.php?f=1&t=21608&hilit=hplc.chem&start=0
The PROBLEM this time is a DRIFT in the baseline at the end of the run as shown in the chromatograms below:
I am analyzing PAHs in Soil extracts (cleaned up by silica column) and I noticed the drift for the first time during the analysis of the first batch of samples (114 injections of which 72 soil extracts). The baseline started to drift sharper than usual, and the gradient ended at higher absorbance levels from the normal level at about 0.0005V to a level above 0.004V, even with acetonitrile injections!
I suspected that some contaminating compounds are accumulating in the column or guard so, I back flushed the column with 100%ACN for at least 1h @ 1.5 ml/min. I also, cleaned the injector by injecting ACN at least ten injections.
After this cleaning, I got the normal level of absorbance for the blank gradient. However, I ran the 2nd batch of extracts and I got the same effect again. So, I did similar cleaning and I got back the good absorbance level.
I then used the instrument for several weeks analyzing only standards and spiked solutions for recovery experiments. During this period everything was OK.
Then I back again to the dirty samples with a third batch of extracts BUT I feel GUILTY this time! The absorbance at the end of the last run was more than 0.004V!
I did the same cleaning procedure but was not successful this time! I tested the level of absorbance by injecting replicates of acetonitrile but I get good absorbance only at the first run! In the rest, absorbance at the end of the run is increasing with a similar pattern to what I noticed in the soil extracts, but with no clear trend between successive injections.
I then changed the pump's filter, new guard, back flushed the column again but no success this time!
I am now feeling GUILTY
because I believe that I KILEED the column! However, I am not sure about this conclusion and I do not want to install a new column in case there is another source of this problem that might kill the new column as well!Any suggestions from your experience are very appreciated.
Many thanks in advance.