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By Anonymous on Thursday, June 10, 2004 - 06:21 pm:
Hello,
For degradation products or impurities quantification, should I consider the peaks with retention time smaller than that of unretained compound(t0)( void volume)?
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By A.Mouse on Thursday, June 10, 2004 - 06:50 pm:
I think you need to consider all peaks for either of these studies.
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By HW Mueller on Friday, June 11, 2004 - 12:58 am:
A.Mouse,
assuming that Anon has a MS wouldn´t it be better not to use HPLC at all if he can´t do better? He wouldn´t pick up all kinds of complications created by the chromatography itself. In other words, is it not nearly essential to do good chromatography even if you have a LCMS?
Also, when I have to do this kind of thing I usually have Gamma emitters which allow for a simple mass balance. Wouldn´t one have to do TLC (in addition to HPLC) to get a mass balance (in absence of radioactive samples)? (Or estimate detector responses if possible?)
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By bernd Mischke on Friday, June 11, 2004 - 03:54 am:
Hi Anon,
peak faster than to = how You will breake chromatographic rules?
to is the fastest peak who is possible.
Any baselinedrift before is an error or instrument fault (ghost peak also possible)
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By Anonymous on Friday, June 11, 2004 - 05:13 am:
Hello Bernd Mischke
retention times smaller than t0 are possible not only in GPC.
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By Anonymous on Friday, June 11, 2004 - 10:44 am:
Indeed
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By A.Mouse on Friday, June 11, 2004 - 02:15 pm:
HW Mueller:
Let me ask you the question back! If he has peaks eluting at and before t0, wouldn't you maximize the ion-suppression in LC/MS especially with the popular electrospray interface. (In addition, there is an article in RCM that shows serious ion-suppression with APcI as well.) If you want to do analysis on an unretained peak via LC/MS, my recommendation would be to save the money for the equipment and buy a set of dice to make up the numbers. It is cheaper this way, and much simpler.
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By Anonymous on Friday, June 11, 2004 - 03:00 pm:
Hello HW Mueller,
I'm sorry I didn't mention, I have only HPLC.
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By Anonymous on Friday, June 11, 2004 - 04:55 pm:
HW Mueller / June 11 / 12:58 ??Maybee you want to clarify??
With respect to the original question. The general protocol for this type of thing is to quantitate everything that you don't see in the blank. But I think one should not include peaks (or apparent peaks) before the void. I agree with the statement above that in theory it should be impossible to have a peak before the t0 peak. unless it's not the true t0.
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By Anonymous on Friday, June 11, 2004 - 06:48 pm:
If it is a peak, it is part of the sample, and it needs to be defined and quantitated, ESPECIALLY for degradation products and impurities.
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By Anonymous on Friday, June 11, 2004 - 08:13 pm:
Protonated amine analized on the column containing residual amines (amide based columns)will repell the protonated amines (amines, aminoacids, amidine, etc.). All of this compound will come before the void if they are not hudrophobic enough to be retained by RP mechanism. The phenomena is known as repulsion. You must change your method to retain all of the compounds or you have to include the one before void.
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By HW Mueller on Monday, June 14, 2004 - 01:17 am:
A.Mouse,
it could be that your first statement was misunderstood (I thought you suggested to first Anon to go ahead and treat this pre-tm peak like any other peak).
It seems that we are actually pulling in the same direction, so I am not sure that I understand your counter-question. The ion suppression is just one more reason for not using HPLC if you can not get a better chromtogramm.
First Anon,
MS or not, that early peak is a problem, you have to keep working to get a better analysis, possibly even use TLC in addition.
Anon, June 11, 4:55pm,
others have detailed the occurrence of pre-tm peaks, one more thing: Don´t forget that if you do a serial injection you may get a peak there from a previous injection.
Further clarification: Because of the special effects near tm and because things can get hung up on the column it is very difficult, if not impossible, to get a mass balance with HPLC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 14, 2004 - 02:08 pm:
Hello,
Thank you for yours halpfull answers.
First Anon
Hello,
For degradation products or impurities quantification, should I consider the peaks with retention time smaller than that of unretained compound(t0)( void volume)?
-------------------------------------------------------------------------------------------------------
By A.Mouse on Thursday, June 10, 2004 - 06:50 pm:
I think you need to consider all peaks for either of these studies.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Friday, June 11, 2004 - 12:58 am:
A.Mouse,
assuming that Anon has a MS wouldn´t it be better not to use HPLC at all if he can´t do better? He wouldn´t pick up all kinds of complications created by the chromatography itself. In other words, is it not nearly essential to do good chromatography even if you have a LCMS?
Also, when I have to do this kind of thing I usually have Gamma emitters which allow for a simple mass balance. Wouldn´t one have to do TLC (in addition to HPLC) to get a mass balance (in absence of radioactive samples)? (Or estimate detector responses if possible?)
-------------------------------------------------------------------------------------------------------
By bernd Mischke on Friday, June 11, 2004 - 03:54 am:
Hi Anon,
peak faster than to = how You will breake chromatographic rules?
to is the fastest peak who is possible.
Any baselinedrift before is an error or instrument fault (ghost peak also possible)
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 05:13 am:
Hello Bernd Mischke
retention times smaller than t0 are possible not only in GPC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 10:44 am:
Indeed
-------------------------------------------------------------------------------------------------------
By A.Mouse on Friday, June 11, 2004 - 02:15 pm:
HW Mueller:
Let me ask you the question back! If he has peaks eluting at and before t0, wouldn't you maximize the ion-suppression in LC/MS especially with the popular electrospray interface. (In addition, there is an article in RCM that shows serious ion-suppression with APcI as well.) If you want to do analysis on an unretained peak via LC/MS, my recommendation would be to save the money for the equipment and buy a set of dice to make up the numbers. It is cheaper this way, and much simpler.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 03:00 pm:
Hello HW Mueller,
I'm sorry I didn't mention, I have only HPLC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 04:55 pm:
HW Mueller / June 11 / 12:58 ??Maybee you want to clarify??
With respect to the original question. The general protocol for this type of thing is to quantitate everything that you don't see in the blank. But I think one should not include peaks (or apparent peaks) before the void. I agree with the statement above that in theory it should be impossible to have a peak before the t0 peak. unless it's not the true t0.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 06:48 pm:
If it is a peak, it is part of the sample, and it needs to be defined and quantitated, ESPECIALLY for degradation products and impurities.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 08:13 pm:
Protonated amine analized on the column containing residual amines (amide based columns)will repell the protonated amines (amines, aminoacids, amidine, etc.). All of this compound will come before the void if they are not hudrophobic enough to be retained by RP mechanism. The phenomena is known as repulsion. You must change your method to retain all of the compounds or you have to include the one before void.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Monday, June 14, 2004 - 01:17 am:
A.Mouse,
it could be that your first statement was misunderstood (I thought you suggested to first Anon to go ahead and treat this pre-tm peak like any other peak).
It seems that we are actually pulling in the same direction, so I am not sure that I understand your counter-question. The ion suppression is just one more reason for not using HPLC if you can not get a better chromtogramm.
First Anon,
MS or not, that early peak is a problem, you have to keep working to get a better analysis, possibly even use TLC in addition.
Anon, June 11, 4:55pm,
others have detailed the occurrence of pre-tm peaks, one more thing: Don´t forget that if you do a serial injection you may get a peak there from a previous injection.
Further clarification: Because of the special effects near tm and because things can get hung up on the column it is very difficult, if not impossible, to get a mass balance with HPLC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 14, 2004 - 02:08 pm:
Hello,
Thank you for yours halpfull answers.
First Anon
