Retention time shortening in ion-pair [August 13, 2004]
Posted: Sun Aug 29, 2004 6:52 pm
By Isabella on Friday, August 13, 2004 - 09:30 am:
We observed a gradual shortening of RT for all peaks of interest (related structures) after extended equilibration time. It happens from run to run and also within one sequence. Premixed mobile phase contains water/ACN/sodium lauryl sulfate/Triethylamine, pH=2.5, column temperature controlled. Column not old, stable at pH 1.5-10.0. Peak responses very reproducible, resolution not affected. Is it possible that TEA is interfering with the ion pair over time, causing less retention? Thank you for any ideas.
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By Mark on Friday, August 13, 2004 - 11:15 am:
Isabella,
Possibly the column is still equilibrating. Using IP reagents these take a very long time to fully equilibrate on reversed phase systems. Also if you are in the habit of flushing your column after a sequence then the column will have to equilibrate all over again.
Regards,
Mark
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By Isabella on Friday, August 13, 2004 - 12:35 pm:
Thank you, Mark.
I thought run times would be longer when it is fully equilibrated with ion-pairing, not shorter. We do not wash after each run, and it is still getting shorter. It is equilibrated after wash (every 2-3 days) and also in between when fresh mobile phase is prepared. How long do you recommend to equilibrate?
Is it true that TEA attaches to the column permanently? TEA is my main suspect.
Regards, Isabella
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By Zelechonok on Friday, August 13, 2004 - 03:32 pm:
What is pH of the mobile phase?
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By Zelechonok on Friday, August 13, 2004 - 03:41 pm:
pH = 2.5, I see it now.
Column probably degraded. It may start to degrade at higher pH with TEA because manufacturer test most likely was performed at different conditions than yours.
Also your sample may have non-eluting components that change the column performance.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, August 13, 2004 - 03:56 pm:
There is no reason that TEA will degrade a standard C18 at this pH. Equilibration can occasionally take as much as 500 mL of mobile phase in ion-pair separations. My recommendation would be to stop the washing completely.
There are also other possibilities. Does your sample contain other ingredients besides the analytes, such as matrix components? Maybe the problem is there...
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By Zelechonok on Saturday, August 14, 2004 - 03:34 am:
It is easy to exclude one reason or another. Make one injection, then continue to wash column with the same mobile phase for few hrs even over night and make another injection. Compare changes of retention between these two injections separated by several hrs of wash with two usual injections following one after another. If retention changes are the same than most likely you have irreversibly retained component in your sample. If retention change is bigger in two injections separated by long wash hrs then you may experience column degradation.
It is all true if your column is fully equilibrated with MP.
Uwe is right equilibration may take long time. If you want to eliminate long time equilibration problem use columns with embedded ion-pairing reagent such as Primesep.
-------------------------------------------------------------------------------------------------------
By Kostas Petritis on Saturday, August 14, 2004 - 01:40 pm:
Isabella,
Another reason for retention loss in ion-pairing chromatography is if your injected analytes have been dissolved in something different than your mobile phase (especially in the case of large injection volumes and/or if the injection solvent is much different than your mobile phase).
As Uwe mentioned matrix components can be another reason as these components might compete for the sites occupied by your ion-pairing reagents.
In any of the above cases things can be improved if you decrease your injection volumes.
-------------------------------------------------------------------------------------------------------
By Isabella on Monday, August 16, 2004 - 06:39 am:
Thank you very much, dear friends, for all your suggestions, I appreciate it.
Uwe, what do you mean by "stop the washing completely"? Do not equilibrate overnight? Then RT changes will occur during the long run making it unacceptable.
Is it possible for TEA to ion-pair with SDS and reduce its effect over time?
My standard is dissolved in 50%ACN/50%water and neat sample is 95% aqueous (can't be further diluted due to low analyte amount) and has excipients. Injection volumn is 10 and can't be lowered for the same reason. I am not sure what to do next. Changing column is not an option. Thank you,
Isabella
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By Uwe Neue on Monday, August 16, 2004 - 06:51 pm:
Isabella,
Ion-pair chromatography has usually slow equilibration times. To see exactly what is going on, I would need to know the concentrations of your ion-pair reagents and of TEA. Since I do not know these, I made a guess....
The common first problem in ion-pair chromatography is a slow equilibration with the reagents. It may take a large volume of mobile phase... several 100 column volumes is not uncommon. I suspected that this might be your problem. However, after some period of time, the ion-pair will usually be in equilibrium with the stationary phase, and retention will become constant. On the other hand, if - at this point in time or earlier - you "wash" the column (and thus remove the ion-pair reagent), the whole cycle starts over again. This was the reason why I recommended to stop "washing" the column.
Unless I know the concentrations of your ion-pair reagents and of the TEA, I do not know if this guess was reasonable or not. Pleas provide this information, together with the column dimensions, the run time over which the shift in retention is happening, and the flow rate. These items are necessary to help me to see if my guess was right.
Uwe
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By Isabella on Tuesday, August 17, 2004 - 06:28 am:
Hi, again,
Sorry for my bad English. By the "run" I meant sample set. Within sample set no changes occur, but from one sample set to another it gets shorter regardless of wherther column was washed and reequilibrated or not. Biggest changes happened between the very first 2 sequences (about 10%)after which column was washed and left for a week. Upon reequilibration and injection RT got shorter another 5-7% and now it decreases very slowly from one sample set to another (not washed). Washing does not restore the column to the original longer RT.Probably it needs long initial aquaintance with this mobile phase.
Thank you so much,
Isabella
We observed a gradual shortening of RT for all peaks of interest (related structures) after extended equilibration time. It happens from run to run and also within one sequence. Premixed mobile phase contains water/ACN/sodium lauryl sulfate/Triethylamine, pH=2.5, column temperature controlled. Column not old, stable at pH 1.5-10.0. Peak responses very reproducible, resolution not affected. Is it possible that TEA is interfering with the ion pair over time, causing less retention? Thank you for any ideas.
-------------------------------------------------------------------------------------------------------
By Mark on Friday, August 13, 2004 - 11:15 am:
Isabella,
Possibly the column is still equilibrating. Using IP reagents these take a very long time to fully equilibrate on reversed phase systems. Also if you are in the habit of flushing your column after a sequence then the column will have to equilibrate all over again.
Regards,
Mark
-------------------------------------------------------------------------------------------------------
By Isabella on Friday, August 13, 2004 - 12:35 pm:
Thank you, Mark.
I thought run times would be longer when it is fully equilibrated with ion-pairing, not shorter. We do not wash after each run, and it is still getting shorter. It is equilibrated after wash (every 2-3 days) and also in between when fresh mobile phase is prepared. How long do you recommend to equilibrate?
Is it true that TEA attaches to the column permanently? TEA is my main suspect.
Regards, Isabella
-------------------------------------------------------------------------------------------------------
By Zelechonok on Friday, August 13, 2004 - 03:32 pm:
What is pH of the mobile phase?
-------------------------------------------------------------------------------------------------------
By Zelechonok on Friday, August 13, 2004 - 03:41 pm:
pH = 2.5, I see it now.
Column probably degraded. It may start to degrade at higher pH with TEA because manufacturer test most likely was performed at different conditions than yours.
Also your sample may have non-eluting components that change the column performance.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, August 13, 2004 - 03:56 pm:
There is no reason that TEA will degrade a standard C18 at this pH. Equilibration can occasionally take as much as 500 mL of mobile phase in ion-pair separations. My recommendation would be to stop the washing completely.
There are also other possibilities. Does your sample contain other ingredients besides the analytes, such as matrix components? Maybe the problem is there...
-------------------------------------------------------------------------------------------------------
By Zelechonok on Saturday, August 14, 2004 - 03:34 am:
It is easy to exclude one reason or another. Make one injection, then continue to wash column with the same mobile phase for few hrs even over night and make another injection. Compare changes of retention between these two injections separated by several hrs of wash with two usual injections following one after another. If retention changes are the same than most likely you have irreversibly retained component in your sample. If retention change is bigger in two injections separated by long wash hrs then you may experience column degradation.
It is all true if your column is fully equilibrated with MP.
Uwe is right equilibration may take long time. If you want to eliminate long time equilibration problem use columns with embedded ion-pairing reagent such as Primesep.
-------------------------------------------------------------------------------------------------------
By Kostas Petritis on Saturday, August 14, 2004 - 01:40 pm:
Isabella,
Another reason for retention loss in ion-pairing chromatography is if your injected analytes have been dissolved in something different than your mobile phase (especially in the case of large injection volumes and/or if the injection solvent is much different than your mobile phase).
As Uwe mentioned matrix components can be another reason as these components might compete for the sites occupied by your ion-pairing reagents.
In any of the above cases things can be improved if you decrease your injection volumes.
-------------------------------------------------------------------------------------------------------
By Isabella on Monday, August 16, 2004 - 06:39 am:
Thank you very much, dear friends, for all your suggestions, I appreciate it.
Uwe, what do you mean by "stop the washing completely"? Do not equilibrate overnight? Then RT changes will occur during the long run making it unacceptable.
Is it possible for TEA to ion-pair with SDS and reduce its effect over time?
My standard is dissolved in 50%ACN/50%water and neat sample is 95% aqueous (can't be further diluted due to low analyte amount) and has excipients. Injection volumn is 10 and can't be lowered for the same reason. I am not sure what to do next. Changing column is not an option. Thank you,
Isabella
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, August 16, 2004 - 06:51 pm:
Isabella,
Ion-pair chromatography has usually slow equilibration times. To see exactly what is going on, I would need to know the concentrations of your ion-pair reagents and of TEA. Since I do not know these, I made a guess....
The common first problem in ion-pair chromatography is a slow equilibration with the reagents. It may take a large volume of mobile phase... several 100 column volumes is not uncommon. I suspected that this might be your problem. However, after some period of time, the ion-pair will usually be in equilibrium with the stationary phase, and retention will become constant. On the other hand, if - at this point in time or earlier - you "wash" the column (and thus remove the ion-pair reagent), the whole cycle starts over again. This was the reason why I recommended to stop "washing" the column.
Unless I know the concentrations of your ion-pair reagents and of the TEA, I do not know if this guess was reasonable or not. Pleas provide this information, together with the column dimensions, the run time over which the shift in retention is happening, and the flow rate. These items are necessary to help me to see if my guess was right.
Uwe
-------------------------------------------------------------------------------------------------------
By Isabella on Tuesday, August 17, 2004 - 06:28 am:
Hi, again,
Sorry for my bad English. By the "run" I meant sample set. Within sample set no changes occur, but from one sample set to another it gets shorter regardless of wherther column was washed and reequilibrated or not. Biggest changes happened between the very first 2 sequences (about 10%)after which column was washed and left for a week. Upon reequilibration and injection RT got shorter another 5-7% and now it decreases very slowly from one sample set to another (not washed). Washing does not restore the column to the original longer RT.Probably it needs long initial aquaintance with this mobile phase.
Thank you so much,
Isabella