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By Anonymous on Friday, January 16, 2004 - 04:16 pm:
Dear All,
I am trying to separate mixture of catecholamines. I tried couple of C18 columns and cannot separate norepinephrine and epinephrine. Any suggestions? Column type and mobile phase?
Thank you in advance
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By Anonymous on Saturday, January 17, 2004 - 06:27 am:
HAve you done any reading? Try search the liturature, I am fairly sure you could dig up thousands of references. I'll even get you started with an older one.
Reversed phase HPLC separation of plasma norepinephrine, epinephrine, and dopamine, with three-electrode coulometric detection
Musso, N.R.; Vergassola, C.; Pende, A.; Lotti, G.
Clin. Chem., 1989, 35(9)
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By AllsepTech on Saturday, January 17, 2004 - 06:56 am:
Here are couple of references
http://www.alltechweb.com/productinfo/t ... u28916.pdf
http://www.sdk.co.jp/shodex/english/dc050303.htm
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By Anonymous on Sunday, January 18, 2004 - 06:16 am:
Yes I've done some reading, but what ever I tried have very ugly picks and no base line resolution for norepinephrine and epinephrine and I do not want to use ion-pairing reagent plus being a grad student limits me with equipment time and column selection
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By Anonymous on Sunday, January 18, 2004 - 06:40 am:
If you truly have done some reading, then you know that these compounds are often done isocraticly using ion-pairing agents (often OSA) on longer C18 columns with electrochemical detection. It is important in forums like this one to say what you have tried and not tried, it comes across a little better that simply saying "how do I do it".
The fact that you have few columns to choose from, dont want to use ion pairs, and have little instrument time, leaves you with a significant challange. Perhaps you could enlighten us on the resources you have availble so maybe we could offer a few suggestions.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Monday, January 19, 2004 - 02:59 pm:
For the catecholamine pathway (6 compounds)you might want to consider the following method on Primesep 200 column. No ion-pairing reagent, isocratic method, LC/MS and preparative chromatography compatible mobile phase.
http://allsep.com/makeChr.php?chr=Chr_044
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By Anonymous on Tuesday, April 27, 2004 - 10:44 am:
Or you could go the simple method and use some of the commercially available immunoassays that have 0 interferances and a very simple extraction step.
-------------------------------------------------------------------------------------------------------
By MD on Friday, April 30, 2004 - 12:42 pm:
Contact a company that actually does and supports such assays: ESA, Bioanalytical Systems (BAS) or Antec Leydan.
-------------------------------------------------------------------------------------------------------
By martin on Thursday, July 22, 2004 - 12:23 pm:
Hi all, I've been reading some stuff and a lot of it is helpful. However I have a question on HPLC of catecholamines using electrochemical detection. We have a shimadzu set up, detector, oven pumps, degasser, and our column is a 100x3mm catecholamine cartridge. We followed all the steps prior to running a standards sample; like passivation leak checks etc. It was shut down for about 2 months and then upon reactivation we had a baseline problem (no baseline) that was resolved, but upon injection of the standards we got the first peak at the right time but the second peak was off by 8 minutes. Furthermore on blank injections ( just mobile phase) we would see these same peaks, spiked samples resulted in low level peaks as well. Now we've tried passivation a second time, carbon plate replacement and cell cleaning and we still obtained the same results. We then took the manual injector out cleaned and sonicated the relative parts and I'm waiting on it passing the leak check. Thus the question is, during the cell cleaning oxidation and corrosion was detected at the outflow portion, our system is set up in a room that has a window to salt water (i.e. ocean/bay) could there be an issue with this? Did moisture or water get into the column? Thanks for your help.
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By Chris Pohl on Sunday, July 25, 2004 - 04:12 pm:
Martin,
If one of your analytes is eluting at the wrong retention time, there's no point in spending time cleaning the cell. Your problem must be with your column and/or your eluent. Have you tried replacing the column? How did you "store" your column during the 2 months of inactivity? Your shutdown protocol is much more likely the issue than tiny amounts of salt in the air. Your eluent probably caused the corrosion problem you observed. What is your eluent system? What solutions were left in the instrument during shutdown? Did you turn off the cell prior to shutdown?
Dear All,
I am trying to separate mixture of catecholamines. I tried couple of C18 columns and cannot separate norepinephrine and epinephrine. Any suggestions? Column type and mobile phase?
Thank you in advance
-------------------------------------------------------------------------------------------------------
By Anonymous on Saturday, January 17, 2004 - 06:27 am:
HAve you done any reading? Try search the liturature, I am fairly sure you could dig up thousands of references. I'll even get you started with an older one.
Reversed phase HPLC separation of plasma norepinephrine, epinephrine, and dopamine, with three-electrode coulometric detection
Musso, N.R.; Vergassola, C.; Pende, A.; Lotti, G.
Clin. Chem., 1989, 35(9)
-------------------------------------------------------------------------------------------------------
By AllsepTech on Saturday, January 17, 2004 - 06:56 am:
Here are couple of references
http://www.alltechweb.com/productinfo/t ... u28916.pdf
http://www.sdk.co.jp/shodex/english/dc050303.htm
-------------------------------------------------------------------------------------------------------
By Anonymous on Sunday, January 18, 2004 - 06:16 am:
Yes I've done some reading, but what ever I tried have very ugly picks and no base line resolution for norepinephrine and epinephrine and I do not want to use ion-pairing reagent plus being a grad student limits me with equipment time and column selection
-------------------------------------------------------------------------------------------------------
By Anonymous on Sunday, January 18, 2004 - 06:40 am:
If you truly have done some reading, then you know that these compounds are often done isocraticly using ion-pairing agents (often OSA) on longer C18 columns with electrochemical detection. It is important in forums like this one to say what you have tried and not tried, it comes across a little better that simply saying "how do I do it".
The fact that you have few columns to choose from, dont want to use ion pairs, and have little instrument time, leaves you with a significant challange. Perhaps you could enlighten us on the resources you have availble so maybe we could offer a few suggestions.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Monday, January 19, 2004 - 02:59 pm:
For the catecholamine pathway (6 compounds)you might want to consider the following method on Primesep 200 column. No ion-pairing reagent, isocratic method, LC/MS and preparative chromatography compatible mobile phase.
http://allsep.com/makeChr.php?chr=Chr_044
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, April 27, 2004 - 10:44 am:
Or you could go the simple method and use some of the commercially available immunoassays that have 0 interferances and a very simple extraction step.
-------------------------------------------------------------------------------------------------------
By MD on Friday, April 30, 2004 - 12:42 pm:
Contact a company that actually does and supports such assays: ESA, Bioanalytical Systems (BAS) or Antec Leydan.
-------------------------------------------------------------------------------------------------------
By martin on Thursday, July 22, 2004 - 12:23 pm:
Hi all, I've been reading some stuff and a lot of it is helpful. However I have a question on HPLC of catecholamines using electrochemical detection. We have a shimadzu set up, detector, oven pumps, degasser, and our column is a 100x3mm catecholamine cartridge. We followed all the steps prior to running a standards sample; like passivation leak checks etc. It was shut down for about 2 months and then upon reactivation we had a baseline problem (no baseline) that was resolved, but upon injection of the standards we got the first peak at the right time but the second peak was off by 8 minutes. Furthermore on blank injections ( just mobile phase) we would see these same peaks, spiked samples resulted in low level peaks as well. Now we've tried passivation a second time, carbon plate replacement and cell cleaning and we still obtained the same results. We then took the manual injector out cleaned and sonicated the relative parts and I'm waiting on it passing the leak check. Thus the question is, during the cell cleaning oxidation and corrosion was detected at the outflow portion, our system is set up in a room that has a window to salt water (i.e. ocean/bay) could there be an issue with this? Did moisture or water get into the column? Thanks for your help.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Sunday, July 25, 2004 - 04:12 pm:
Martin,
If one of your analytes is eluting at the wrong retention time, there's no point in spending time cleaning the cell. Your problem must be with your column and/or your eluent. Have you tried replacing the column? How did you "store" your column during the 2 months of inactivity? Your shutdown protocol is much more likely the issue than tiny amounts of salt in the air. Your eluent probably caused the corrosion problem you observed. What is your eluent system? What solutions were left in the instrument during shutdown? Did you turn off the cell prior to shutdown?
