I am doing experiments with HPLC. I prepared 3 vials from same sample and put them to rack of the hplc. Before that i took the sample from cool batch and i am suspecting that the temperature difference can effect on the peaks? I completed all the preparation in 15 minutes. The peaks are in the image. How can they be different like this? Same behaviour but with different height.
It looks like your peaks have comparable responses, but the baseline is all over the place. Is this a UV detector? If so, what wavelength are you using and what is the mobile phase solvent system? It looks like one of the chromatograms has a stable baseline whereas the others show some drift. My initial thought would be that this is either lack of equilibration, or a loss of equilibrium during the analysis. This looks like more of an instrument/method issue rather than a sample effect.
The wavelength is 254 nm. Mobile Phase is mix of diluted MES and NaCl.
Details;
Chromatography Type: Ion Exchange
Column: Dionex WCX-10 ProPac (10.4 x 250 mm) (Dionex 054993)
Flow Rate: 0.8 ml/min (Gradient)
Column is not thermostatted.
In addition to Steven's comments and by elimination, use of gradient is not a cause since the retention time remains fairly constant. My take would be influence of room temperature since the column is not thermostatted. You may also consider flushing the flow cell with 60% MeOH at a flow rate of 0.1ml/min overnight.
I would highly recommend, and perhaps you have already attempted this:
Show us a plot of when you inject from the same vial 3 times in succession. We must work on the variables one at a time. Thanks. More details the better.
Samples injected were unlikely to be the culprit for such behavior. The same baseline change pattern could have been seen without injecting anything in such situation.
The likely causes, as pointed out already by others: (1) lack of sufficient flushing/purging of the system, (2) temperature drift, and (3) degraded UV lamp or detector performance.
It may not be easy to reproduce the same problematic pattern. Anyhow, sufficient flushing and equilibrating are essential for achieving consistent baseline, particularly for the first several injections, and that’s why many people inject a few blanks before actual samples to warmup and stabilize the system.
I would eliminate one issue at the time. Is it the sample related, by injecting air say 1 uL or mobile phase three times to see if it has the same pictures, then go from there. If it is bad, it is not the sample and you need to fix the system e.g. temperature control, blah...blah...blah...
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