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By Heidi Kula on Tuesday, August 24, 2004 - 07:19 am:
I detected shikimic acid (in the range 1ppm):Aminex HPX-87H Organic Acids Analysis column, 5 mM sulphuric acid as a mobile phase, flow rate 0.6ml min-1 (2000psi) was provided by a Waters 515HPLC pump and the column eluate was monitored at 215nm using Waters 2487 Dual l Absorb, temp.30oC. I had to exchange pre- and column and now I can detect shikimate only in 1000ppm concentration, and the pressure is also lower-1000psi? What could be wrong that sensitivity is so low, right now?
Thank you!
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By Ann on Tuesday, August 24, 2004 - 07:33 am:
The drop in pressure may be explained by you replacing the pre-column. Quite possibly, your original pre-column had become contimated with impurities/particulates (which, after all, is what we use them for!). This was probably causing the higher back pressure. Now you have replaced this with a (new?) pre-column, the system back pressue is much lower.
The loss of sensitivity is a little trickier...unless the quantification of your analyte has been confounded by something (your analyte, something that co-elutes) washing off your original pre-column. Not very likely perhaps but just a thought...
Have you checked all the 'obvious' things? Is the autosampler injecting the correct volume of sample? Is your detector sensitivity setting correct? Have you re-made your standard solution of analyte (in case of weighing/pipetting error)?
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By Heidi on Tuesday, August 24, 2004 - 08:07 am:
Thanks a lot.
I've checked obvious things, and re-made my standard and it can be nothing to co-eluate because it's just fresh water solution...
Have you got any idea what to do to have higer sensitivity?
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By Ann on Tuesday, August 24, 2004 - 08:20 am:
What is the baseline noise like? Has it recently got worse i.e. has your signal to noise ratio decreased? Just wondering whether anything has happened to your detector - is the lamp old? (although lamp failure would normally be fairly unmistakeable)
Although your peak is smaller, does the chromatography 'look' the same (same RT, for example)? Is the new pre-column exactly the same as the old one? If not, could the RT of your peak have shifted slightly (or significantly!) A longer RT might mean that your peak is eluting outside of your data acquisition collection period)
Have you changed anything else recently - different brand of autosampler vial and/or cap, for example?
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By Ann on Tuesday, August 24, 2004 - 08:25 am:
Another thought: could there be a leak somewhere (rheodyne or somewhere between the injector and the column?) Might explain loss of pressure and loss of sensitivity!
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By Heidi on Tuesday, August 24, 2004 - 08:58 am:
Baseline noise: no changes
Detector: the lamp is 60 days old
Peaks: no peak at the concentration below 1000ppm
RT: the same (for 1000ppm = 12,3min but the peak is smaller than before)
pre-column: extacly the same
and no leak.
What about temp.? try higher?
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By Ann on Tuesday, August 24, 2004 - 09:33 am:
Hmmm..not sure that changing the temperature will help you. It might sharpen up your peak and slightly increasing s/n ratio but it's not going to compensate for a 1000 fold reduction in sensitivity, as I'm sure you know.
I think it sounds like either your autosampler is playing up (not actually injecting anything, or very little) or your detector is faulty.
Autosampler (do you have an autosampler or are you manually injecting?)
If autosampler - try getting it to inject something harmless but highly coloured (potassium permangenate or a food colouring solution for example). Disconnect your column first to protect it and run the tubing (from autosampler to column) to waste. When you inject, you should be able to see the coloured liquid emerging (a quick and easy way to check the autosampler is basically functioning).
We had a UV detector (not a Waters) that suddenly lost it's wavelength calibration (dodgy back-up battery) and it stopped detecting the peak (because even though it was physically set to the correct wavelength, internally, it wasn't).
Good luck, hope we get to the bottom of this!
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By Ann on Tuesday, August 24, 2004 - 09:37 am:
P.S. Have you got another UV detector that you could run in series with your Waters detector? Or could you try manually injecting instead of using your autosampler (if you are)?
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By Jaime on Tuesday, August 24, 2004 - 02:05 pm:
I run organic acids in a Brownlee ion exclusion column 25 cmX 7 mm. Eluent is 0.002 N H2SO4. Flow 0.6 ml/min. This column is not suppose to go more than 1000 PSI.My pressure was only around 700 PSI. Check with your column manufacturer if there are pressure limitations to your column. Mine had instructions on pressure limitation.
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By Jaime on Thursday, August 26, 2004 - 11:07 am:
I called BioRad Laboratories Tech Support. The Max pressure for this column type is 1500 psi. Above that, it crushes the resins and you will decrease sensitivity.
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By Chris Pohl on Friday, August 27, 2004 - 08:55 am:
Heidi,
You haven't said anything about other organic acids. Is this sensitivity problem unique to shikimic acid? Or is the problem general and you only mention shikimic acid because it is your analytical target? Three possibilities come to mind: (1). Your fresh column set may be leaching oligomer which are creating a substantial background at that wavelength. You can check this by bypassing the column so as to send the eluent directly to the detector from the injection valve, zeroing the detector and then reconnecting the column. If the effluent coming from the column causes a significant rise in background, then you're columns are likely at fault. (2). Your new columns are contaminated with a metal capable of complexing shikimic acid. There is no straightforward way to prove or disprove this hypothesis, but if you pass 100 millimolar oxalic acid over your column it should strip off any metal contaminants. The problem with this approach, though, is that it could be a while until you get a good baseline again if you use this approach since oxalic acid may be difficult to completely clear from your system. (3). You have an obstruction somewhere in your system which is compromising system performance and somehow this is also affecting your detection sensitivity (maybe there's a blockage in your flowcell?). I agree with the others above that the pressure you report does not seem normal for a column of this sort at the flow rate and temperature you mentioned (although I doubt that the pressure you report is sufficient to damage the column in spite of the manufactures recommendations to the contrary). You might want to make sure that your pressure is reasonably low when you bypass the column set. If not, look for an obstruction somewhere in the flow path.
I detected shikimic acid (in the range 1ppm):Aminex HPX-87H Organic Acids Analysis column, 5 mM sulphuric acid as a mobile phase, flow rate 0.6ml min-1 (2000psi) was provided by a Waters 515HPLC pump and the column eluate was monitored at 215nm using Waters 2487 Dual l Absorb, temp.30oC. I had to exchange pre- and column and now I can detect shikimate only in 1000ppm concentration, and the pressure is also lower-1000psi? What could be wrong that sensitivity is so low, right now?
Thank you!
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 07:33 am:
The drop in pressure may be explained by you replacing the pre-column. Quite possibly, your original pre-column had become contimated with impurities/particulates (which, after all, is what we use them for!). This was probably causing the higher back pressure. Now you have replaced this with a (new?) pre-column, the system back pressue is much lower.
The loss of sensitivity is a little trickier...unless the quantification of your analyte has been confounded by something (your analyte, something that co-elutes) washing off your original pre-column. Not very likely perhaps but just a thought...
Have you checked all the 'obvious' things? Is the autosampler injecting the correct volume of sample? Is your detector sensitivity setting correct? Have you re-made your standard solution of analyte (in case of weighing/pipetting error)?
-------------------------------------------------------------------------------------------------------
By Heidi on Tuesday, August 24, 2004 - 08:07 am:
Thanks a lot.
I've checked obvious things, and re-made my standard and it can be nothing to co-eluate because it's just fresh water solution...
Have you got any idea what to do to have higer sensitivity?
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 08:20 am:
What is the baseline noise like? Has it recently got worse i.e. has your signal to noise ratio decreased? Just wondering whether anything has happened to your detector - is the lamp old? (although lamp failure would normally be fairly unmistakeable)
Although your peak is smaller, does the chromatography 'look' the same (same RT, for example)? Is the new pre-column exactly the same as the old one? If not, could the RT of your peak have shifted slightly (or significantly!) A longer RT might mean that your peak is eluting outside of your data acquisition collection period)
Have you changed anything else recently - different brand of autosampler vial and/or cap, for example?
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 08:25 am:
Another thought: could there be a leak somewhere (rheodyne or somewhere between the injector and the column?) Might explain loss of pressure and loss of sensitivity!
-------------------------------------------------------------------------------------------------------
By Heidi on Tuesday, August 24, 2004 - 08:58 am:
Baseline noise: no changes
Detector: the lamp is 60 days old
Peaks: no peak at the concentration below 1000ppm
RT: the same (for 1000ppm = 12,3min but the peak is smaller than before)
pre-column: extacly the same
and no leak.
What about temp.? try higher?
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 09:33 am:
Hmmm..not sure that changing the temperature will help you. It might sharpen up your peak and slightly increasing s/n ratio but it's not going to compensate for a 1000 fold reduction in sensitivity, as I'm sure you know.
I think it sounds like either your autosampler is playing up (not actually injecting anything, or very little) or your detector is faulty.
Autosampler (do you have an autosampler or are you manually injecting?)
If autosampler - try getting it to inject something harmless but highly coloured (potassium permangenate or a food colouring solution for example). Disconnect your column first to protect it and run the tubing (from autosampler to column) to waste. When you inject, you should be able to see the coloured liquid emerging (a quick and easy way to check the autosampler is basically functioning).
We had a UV detector (not a Waters) that suddenly lost it's wavelength calibration (dodgy back-up battery) and it stopped detecting the peak (because even though it was physically set to the correct wavelength, internally, it wasn't).
Good luck, hope we get to the bottom of this!
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 09:37 am:
P.S. Have you got another UV detector that you could run in series with your Waters detector? Or could you try manually injecting instead of using your autosampler (if you are)?
-------------------------------------------------------------------------------------------------------
By Jaime on Tuesday, August 24, 2004 - 02:05 pm:
I run organic acids in a Brownlee ion exclusion column 25 cmX 7 mm. Eluent is 0.002 N H2SO4. Flow 0.6 ml/min. This column is not suppose to go more than 1000 PSI.My pressure was only around 700 PSI. Check with your column manufacturer if there are pressure limitations to your column. Mine had instructions on pressure limitation.
-------------------------------------------------------------------------------------------------------
By Jaime on Thursday, August 26, 2004 - 11:07 am:
I called BioRad Laboratories Tech Support. The Max pressure for this column type is 1500 psi. Above that, it crushes the resins and you will decrease sensitivity.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Friday, August 27, 2004 - 08:55 am:
Heidi,
You haven't said anything about other organic acids. Is this sensitivity problem unique to shikimic acid? Or is the problem general and you only mention shikimic acid because it is your analytical target? Three possibilities come to mind: (1). Your fresh column set may be leaching oligomer which are creating a substantial background at that wavelength. You can check this by bypassing the column so as to send the eluent directly to the detector from the injection valve, zeroing the detector and then reconnecting the column. If the effluent coming from the column causes a significant rise in background, then you're columns are likely at fault. (2). Your new columns are contaminated with a metal capable of complexing shikimic acid. There is no straightforward way to prove or disprove this hypothesis, but if you pass 100 millimolar oxalic acid over your column it should strip off any metal contaminants. The problem with this approach, though, is that it could be a while until you get a good baseline again if you use this approach since oxalic acid may be difficult to completely clear from your system. (3). You have an obstruction somewhere in your system which is compromising system performance and somehow this is also affecting your detection sensitivity (maybe there's a blockage in your flowcell?). I agree with the others above that the pressure you report does not seem normal for a column of this sort at the flow rate and temperature you mentioned (although I doubt that the pressure you report is sufficient to damage the column in spite of the manufactures recommendations to the contrary). You might want to make sure that your pressure is reasonably low when you bypass the column set. If not, look for an obstruction somewhere in the flow path.
