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By Greg on Friday, June 25, 2004 - 08:51 am:
I am using an Allure C18, 100 mm x 3.2 mm, 5 um LC column. In addition we have a C18 guard column in the system. I am running nitroaromatic compounds in a 0.004 M CaCl2 salt solution. For the last year we have had relatively few problems. However, recently, the peak intensity started to drop. My mobile phase is a 60:40 mixture of ACN:Water. I was advised to reverse the column and pump a 90:10 Water:ACN mixture with 1-2 mL acetic acid per L of water, (so the 90% water is acidified). I did this for 24 hours at a constant, steady flow rate. I was then advised (by tech support for the column manuf.) to run the same mobile phase through without the acetic acid. I did this for about 6 hours. I then switched to 100% ACN for 1 hour before returning to my current mobile phase (60:40, ACN:water). I let the instrument equilibrate with the 60:40 mixture for 12 hours.
When I re-run the standards (0.1, 1, 10, 50 ppm) in a matrix of 0.004 CaCl2, after doing this rinse, my peak instensities are still way down to what it was a month ago. However, my r2 for my standards is 0.999. I'm seeing about a 10x decrease in peak intensity because the 1 ppm peak is now about the same height and area of what my 0.1 ppm peak was.
Tech support for the column manf. is stumped, and I'm not sure if the column has become denatured or if it is something else. Any help would be appreciated and I can provide further info if needed.
Thanks in advance to anyone who takes the time to read this and respond.
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By Anonymous on Friday, June 25, 2004 - 10:31 am:
I have a hard time believing that the column is causing that consistent of a decrease over that wide of a range. Have there been any changes in peak shape or retention time?
Is there anything that has recently been changed/repaired (routine maintentance or otherwise) on the system? Particularly the injector, but also the detector.
Do you have access to a second system to verify the results? More information on the system and control could also be useful.
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By RH on Friday, June 25, 2004 - 12:38 pm:
I agree with anon that this sounds a bit like a
detector problem. Did you check the UV lamp, flow
cell etc.?
Typical column problems concerning peak area
happen when unstable derivatives of analytes are
chromatographed on columns that have seen many
samples.
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By greg on Sunday, June 27, 2004 - 11:13 pm:
Just a thought
I would also look into the accuracy of the auto injector if you are using one. Perhaps the volume you think you are injecting is out by about 10, which would explain your results. Weigh a vial before and after a 200uL injection and look for a 0.2g difference before and after injection.
Good luck!
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By Anonymous on Monday, June 28, 2004 - 01:07 am:
Maybe it is your solvent that has changed, so the background abs. is starting to eat up the peaksignal. Try to change solvent, and do not use recycle.
I am using an Allure C18, 100 mm x 3.2 mm, 5 um LC column. In addition we have a C18 guard column in the system. I am running nitroaromatic compounds in a 0.004 M CaCl2 salt solution. For the last year we have had relatively few problems. However, recently, the peak intensity started to drop. My mobile phase is a 60:40 mixture of ACN:Water. I was advised to reverse the column and pump a 90:10 Water:ACN mixture with 1-2 mL acetic acid per L of water, (so the 90% water is acidified). I did this for 24 hours at a constant, steady flow rate. I was then advised (by tech support for the column manuf.) to run the same mobile phase through without the acetic acid. I did this for about 6 hours. I then switched to 100% ACN for 1 hour before returning to my current mobile phase (60:40, ACN:water). I let the instrument equilibrate with the 60:40 mixture for 12 hours.
When I re-run the standards (0.1, 1, 10, 50 ppm) in a matrix of 0.004 CaCl2, after doing this rinse, my peak instensities are still way down to what it was a month ago. However, my r2 for my standards is 0.999. I'm seeing about a 10x decrease in peak intensity because the 1 ppm peak is now about the same height and area of what my 0.1 ppm peak was.
Tech support for the column manf. is stumped, and I'm not sure if the column has become denatured or if it is something else. Any help would be appreciated and I can provide further info if needed.
Thanks in advance to anyone who takes the time to read this and respond.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 25, 2004 - 10:31 am:
I have a hard time believing that the column is causing that consistent of a decrease over that wide of a range. Have there been any changes in peak shape or retention time?
Is there anything that has recently been changed/repaired (routine maintentance or otherwise) on the system? Particularly the injector, but also the detector.
Do you have access to a second system to verify the results? More information on the system and control could also be useful.
-------------------------------------------------------------------------------------------------------
By RH on Friday, June 25, 2004 - 12:38 pm:
I agree with anon that this sounds a bit like a
detector problem. Did you check the UV lamp, flow
cell etc.?
Typical column problems concerning peak area
happen when unstable derivatives of analytes are
chromatographed on columns that have seen many
samples.
-------------------------------------------------------------------------------------------------------
By greg on Sunday, June 27, 2004 - 11:13 pm:
Just a thought
I would also look into the accuracy of the auto injector if you are using one. Perhaps the volume you think you are injecting is out by about 10, which would explain your results. Weigh a vial before and after a 200uL injection and look for a 0.2g difference before and after injection.
Good luck!
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 28, 2004 - 01:07 am:
Maybe it is your solvent that has changed, so the background abs. is starting to eat up the peaksignal. Try to change solvent, and do not use recycle.
