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Topic: Continually decreasing retention times in hplc  (Read 6619 times)

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Offline agros

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Continually decreasing retention times in hplc
« on: April 03, 2009, 12:01:26 PM »
Hi,

I'm running a Phenomenex ODS 250 x 4.6, 5 micron column, mobile phase is gradient of acetonitrile and 10%/5%/1% acetic acid, acetonitrile, and H3PO4 in water. 

My problem is that although my separations look good, the RTs continually decrease over the course of a day, e.g. 7.10 min to 7.05 to 7.01 to 6.96 to 6.92 to 6.88 to 6.86 to 6.81 over subsequent runs of the same sample.  I haven't run more than 9 runs in a row, so I don't know if it plateaus at some point.  If I let the pump run overnight at a .1 ml/min, and then do more runs, the RTs are back up to 7.10 in the morning and decrease over the day again.   

I have tried tweaking the degassing, pressurized He blanket, temperature.  I've checked flow rate and replaced inlet filters.  I am beginning to wonder if there is some flaw with the pump not delivering a repeatable  gradient.  If I remove the inlet filters from the solvent bottles, the retention times become more precise over multiple runs.  Does anyone have experience with hplc pumps and can advise me on what other things to check? 


Offline Arkcon

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Re: Continually decreasing retention times in hplc
« Reply #1 on: April 03, 2009, 12:38:13 PM »
There are a couple of things you can do, now that you've thoroughly checked your system for instrument flaws.  (Caution, when trying to pin down something like this, you can sometimes fail to be thorough, and miss steps you've thought you've done well.  Anything you've already done, may be suspect, because you've done so much.  That's just a warning for you, not a nitpick.)

Determine at what % eluent mix you product comes out, and run isocratically.  Now, you may get a poorer separation, at it may take a longer runtime.  But I'm not suggesting that you always run isocratically.  We're just taking the gardiet mixer out of the equation, so to speak.  So we can what sort of drift there is, run to run.   You may be inadequetly re-equilibrating your runs, 'tho that does seem unlikely to me, as you're not using an ion pair reagent.

You can do a couple of things to test your systems ability to make a gradient.  You can just put two 100 ml graduated cylinders of eluent in place of the reservoir, and run them 50/50.  They should both drain at the same rate.  Another trick is to run, with or without a column, two bottles of degassed methanol, one of which is spiked with 0.1% acetone A UV detector will show you the gradient as it forms, and you can check it for intermittent failures.  (or 0.01% acetone, as it absorbs quite strongly at short wavelengths). 
« Last Edit: April 03, 2009, 12:50:51 PM by Arkcon »
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Offline agros

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Re: Continually decreasing retention times in hplc
« Reply #2 on: April 03, 2009, 05:17:08 PM »
There are a couple of things you can do, now that you've thoroughly checked your system for instrument flaws.  (Caution, when trying to pin down something like this, you can sometimes fail to be thorough, and miss steps you've thought you've done well.  Anything you've already done, may be suspect, because you've done so much.  That's just a warning for you, not a nitpick.)

Thanks for the warning - I have been thinking that with so many tests, it is easy to mix up what happened when. 

Determine at what % eluent mix you product comes out, and run isocratically.  Now, you may get a poorer separation, at it may take a longer runtime.  But I'm not suggesting that you always run isocratically.  We're just taking the gardiet mixer out of the equation, so to speak.  So we can what sort of drift there is, run to run.   You may be inadequetly re-equilibrating your runs, 'tho that does seem unlikely to me, as you're not using an ion pair reagent.

I did experiment early on with lengthening the equilibration time by 50 and 100% and even changing the "clean up" step that increases the % ACN before the equilibration time.  None of those steps changed the decreasing RTs.

You can do a couple of things to test your systems ability to make a gradient.  You can just put two 100 ml graduated cylinders of eluent in place of the reservoir, and run them 50/50.  They should both drain at the same rate.  Another trick is to run, with or without a column, two bottles of degassed methanol, one of which is spiked with 0.1% acetone A UV detector will show you the gradient as it forms, and you can check it for intermittent failures.  (or 0.01% acetone, as it absorbs quite strongly at short wavelengths). 

I did do a gradient step test similar to what you suggested, only I tested water (A) and 0.1% acetone in water (B) increasing in steps.  My test was changing by 10% increments from 100%A to 20%A.  I did find that the step between 50%A and 40%A was about half of what it should have been.  Doing a 5% step test, I found that the step between 50% A and 45% A was < a quarter of what it should be. 

My PE technician did not seem to take this very seriously over the phone and instead suggested switching the inlet filters.  Perhaps he (and you) are right and I should retry the step test with the new filters.  I'll try that next week along with the isocratic runs - I think the first peak elutes at about 7%A and the last before 17%A.  Should I strive for somewhere in-between, do you think? 

Offline Arkcon

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Re: Continually decreasing retention times in hplc
« Reply #3 on: April 03, 2009, 11:11:39 PM »
Yeah, I'd try something like 15% A, and hope your latter peak doesn't take too long to come out.  I'd expect that the isocratic run will wreck retention of your first peak, if it does, you might as well try 17%, anyway.  You're just trying to see, with multiple injections, flowing just one eluent, the column, in effect, being constantly equilibrated, give better RT reproducibility.  Weird things can cause shifts in retention times -- barely leaky hardware, a poor quality solid phase, column voids that aren't severe enough to cause split peaks, etc.  We're just trying to answer the equilibration question here.
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

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