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By mpaciolla on Monday, June 16, 2003 - 09:25 am:
I recently observed that the type of acid used to pH a buffer greatly affects the retention of ionic compounds on amide columns. For example, we observed a decrease in retention with decreasing buffer concentration for a compound containing an amine group. The pH of the buffer was titrated using HCl. However, the retention was even less (1 minute) when titrating to the desired pH with H3PO4. All other conditions where the same. Would someone mind explaining this?
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By bill tindall on Monday, June 16, 2003 - 05:04 pm:
I will make the assumption that the buffer pH was adjusted in an aqueous solvent while the separation was done in a partially aqueous solvent in reversed phase mode. (Your didn't fully describe your separation so my assumption may be wrong and the following explanation irrelevant to what your are really doing.)
In this case the hydrogen ion activity (the value that really governs ionization in the partially aqueous mobile phase) of the mobile phase will not be the same as it was when it was adjusted without organic solvent, and the change in hydrogen ion activity upon adding the organic solvent is not the same for all acids and bases.
Repeat the experiment and measure the pH of the various mobile phases after addition of the organic solvent. (If a gradient is used, then estimate the composition where the sample elutes.) See if this pH correlates with your observed retention time.
For more on this subject read LCGC November, december 2002 and Jan 2003.
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By mpaciolla on Tuesday, June 17, 2003 - 07:16 am:
Bill,
The mobile phase is 100% buffer and we didn't use a gradient. Thanks for the refernce.
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By bill on Tuesday, June 17, 2003 - 02:38 pm:
Well I'm stumped if this is a reversed phase separation.
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By HW Mueller on Tuesday, June 17, 2003 - 11:37 pm:
It would be surprising if this is due to the difference in ionic strength, therefore, one can suspect that you have a problem with ascertaining your pH, and/or with buffering.
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By Anonymous on Friday, June 20, 2003 - 05:15 pm:
What is your "ionic compound"? A base?
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By jt on Tuesday, June 24, 2003 - 10:24 am:
was you amine compound in charged state under the separation conditions? then you may be seeing the "counter ion effect". what was the buffer, why would you use two different acid to adjust the pH? at lower buffer concentration, the ionizationof the chloride or phosphate salt of your compound may shift toward more completion (the compound spent more time in charged state and eluted earlier). I would guess the solubilities of the chloride and phosphate salt of you compound are different.
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By mpaciolla on Wednesday, June 25, 2003 - 06:31 am:
Since we are weighing the salt and using a pH meter that is calibrated before each use, I doubt the problem lies with an unreliable pH reading or the concentration of the buffer. The compound is a hydrchloride salt of an amine with a pKa of 9.0. The pH of our mobile phase was adjusted to pH 2.4. Hydrochloric acid was used to simply lower the pH. We didn't think it would matter but we were obvioulsy wrong.
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By Andreas Neumaier on Wednesday, June 25, 2003 - 08:02 am:
I don't have that much experience with buffered mobile phases, but I have a assumption to your problem:
Let me guess the retention time shift is related to the different degree of hydrolisation of the two acids you have used. HCl is a strong acid and hydrolyse to nearly 100%. H3PO4 is a weaker acid and more of this acid is needed for pH adjustment (compared to HCl).
Which means the overall concentration (of the adjusted buffer) is lower with HCl and higher when H3PO4 is used.
The higher the concentration of the buffer the lower the retention time.
If this assumption is right, you should observe the same shorter retention when using sulfuric acid (half of the concentration c of HCl; the same n as HCl) instead of HCl.
Nethertheless I also remember a special chromatographic problem which showed that different acids had different influence to the seperation.
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By Chris Pohl on Wednesday, June 25, 2003 - 08:25 am:
Although this phenomenon is not well documented in the literature, I believe the effect you report is connected to the weakly basic character of the amide functional group. We have observed modest increases in asymmetry for acidic compounds at low pH on prototype amide materials. Furthermore, we find that this affect diminishes dramatically as the pH is increased. In addition, however, many of the commercially available amide phases are synthesized in a manner which introduces anion exchange sites. You can distinguish between the two phenomena comparing the effect of pH on peak asymmetry of acidic solutes. At pH 4 there is that essentially no peak asymmetry in the case of pure amide phases, whereas in the case of phases containing significant contamination with anion exchange sites, the effect persists even at pH 6.5.
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By mpaciolla on Wednesday, June 25, 2003 - 08:56 am:
Andreas,
Your assumption does seem to make sense. However, the retention time of the basic analyte continues to increase as we increase the concentration of the buffer when using HCl to titrate to pH 2.4.
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By Andreas Neumaier on Thursday, June 26, 2003 - 06:02 am:
Sorry mpaciolla,
I've missed this point (english's not so easy for me as I sometimes think).
My assumption:
When increasing your buffer concentration the balance of interaction between stationary phase and buffer is shiftet toward stationary phase.
(This could be caused by decreasing solubility of the compound at increasing ionic concentration - but I'm not sure if this assumption's right.)
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By Anonymous on Thursday, June 26, 2003 - 08:03 am:
Perhaps "Effect of the counter-anion type and concentration on the liquid chromatography retention of B-blockers" Journal of Chromatography A, 964 (2002) 179-187, holds the answer.
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By Uwe Neue on Thursday, June 26, 2003 - 03:59 pm:
You said that the retention decreases with decreasing buffer concentration, but you did not tell us how low the total ion concentration was. There is nothing wrong with this phenomenon, if the ion concentration is very low. Under these circumstances, you may get exclusion from the pores and from the surface of a nicely hydrophobic C18 column. At pH 2.4, you do not have a lot hydrochloric acid, I presume. 4 mM, I guess. Try to add neutral salt to increase retention!
What was the buffer that you started off with? Type and concentration?
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By HW Mueller on Friday, June 27, 2003 - 04:15 am:
You still have not divulged crucial data on your buffer, not even what buffer you use. It would also be interesting to know how much dilution the HCl or H3PO4 produces, and how consistent that is.
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By mpaciolla on Monday, June 30, 2003 - 09:37 am:
Hello Everyone,
First, I would like to thank all of you for your insight. The above reference may explain what we are observing. We are using a 40mM Ammonium Phosphate Buffer that is titrated to pH 2.4 with HCl. Therefore, the approximate concentration of the HCl is 22mM. We tried using phosphoric acid to pH the buffer. The retention time was slightly less but the peak shape was poor (excessive tailing). We also tried using NaCl with the NH4H2PO4/H3PO4 buffer. The retention of the amine increased and the peak shape was slightly better but not nearly as good when using HCl to pH the buffer. Someone recently told me that HCl at that concentration in our mobile phase could damage the stainless steel column and tubing. Would someone please care to comment on this as well. One other note. The retention for the amine is decreasing as the colunm ages but the neutral compound is not.
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By Anonymous on Tuesday, July 1, 2003 - 01:48 am:
Uwe Neue-why should you get exclusion from the pores of a nicely hydrophobic column when the ionic concentration is low?
Thanks for your advice!
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By HW Mueller on Tuesday, July 1, 2003 - 03:56 am:
Your concentrations should be in the range (ionic strength, if my estimations are not way off) where practically all salts are chaotropic (NH4HxPO4 is considered highly lyotropic at higher concentrations). Therefore, I still think that ionic strength should not play much of a role here. Now, to be more consistent one may prepare two ammonium phosphate solutions of the same concentration, but with pH bracketing the desired one. These are then mixed together until the desired pH obtains.
On the HCl: It´s a matter of pH and concentration, it´s doubtful you will have a problem, but to make sure: take a piece of stainless capillary and leave it in your solution for some time. If you polish a section of the capillary with Ajax it is very easy to see wether there is tarnishing. But it would be preferable if you are consistent by mixing the buffer as mentioned.
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By Anonymous on Thursday, March 25, 2004 - 12:44 am:
1. would u explain to me how is the ph effect on chromatogram profile?
2. would u explain how is the effect of the kind of buffer on chromatogram profile?
3. what is the advantages of methanol between acetonitril(ACN) as mobile phase in HPLC?
4.what is the function of internal standard on the research? what's make's we choose the internal standard? and why?
5.to get a good resolution, what must we do for up grade on chromatography?
6. to get a good peak, what must we do for up grade on chromatography?
thanks u. i'm sorry if my english is very bad. i hope u understand with the question i have to ask
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By tom jupille on Thursday, March 25, 2004 - 10:32 am:
We have a free on-line course which covers the answers to most of your questions:
http://www.lcresources.com/resources/reslinksform.html
By the way, your english is quite understandable.
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By Uwe Neue on Thursday, March 25, 2004 - 03:42 pm:
All your questions require a lengthy answer. Therefore I agree with Tom: you will be better off looking at the course, and afterwards come back with specific questions.
I recently observed that the type of acid used to pH a buffer greatly affects the retention of ionic compounds on amide columns. For example, we observed a decrease in retention with decreasing buffer concentration for a compound containing an amine group. The pH of the buffer was titrated using HCl. However, the retention was even less (1 minute) when titrating to the desired pH with H3PO4. All other conditions where the same. Would someone mind explaining this?
--------------------------------------------------------------------------------------------------------
By bill tindall on Monday, June 16, 2003 - 05:04 pm:
I will make the assumption that the buffer pH was adjusted in an aqueous solvent while the separation was done in a partially aqueous solvent in reversed phase mode. (Your didn't fully describe your separation so my assumption may be wrong and the following explanation irrelevant to what your are really doing.)
In this case the hydrogen ion activity (the value that really governs ionization in the partially aqueous mobile phase) of the mobile phase will not be the same as it was when it was adjusted without organic solvent, and the change in hydrogen ion activity upon adding the organic solvent is not the same for all acids and bases.
Repeat the experiment and measure the pH of the various mobile phases after addition of the organic solvent. (If a gradient is used, then estimate the composition where the sample elutes.) See if this pH correlates with your observed retention time.
For more on this subject read LCGC November, december 2002 and Jan 2003.
--------------------------------------------------------------------------------------------------------
By mpaciolla on Tuesday, June 17, 2003 - 07:16 am:
Bill,
The mobile phase is 100% buffer and we didn't use a gradient. Thanks for the refernce.
--------------------------------------------------------------------------------------------------------
By bill on Tuesday, June 17, 2003 - 02:38 pm:
Well I'm stumped if this is a reversed phase separation.
--------------------------------------------------------------------------------------------------------
By HW Mueller on Tuesday, June 17, 2003 - 11:37 pm:
It would be surprising if this is due to the difference in ionic strength, therefore, one can suspect that you have a problem with ascertaining your pH, and/or with buffering.
--------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 20, 2003 - 05:15 pm:
What is your "ionic compound"? A base?
--------------------------------------------------------------------------------------------------------
By jt on Tuesday, June 24, 2003 - 10:24 am:
was you amine compound in charged state under the separation conditions? then you may be seeing the "counter ion effect". what was the buffer, why would you use two different acid to adjust the pH? at lower buffer concentration, the ionizationof the chloride or phosphate salt of your compound may shift toward more completion (the compound spent more time in charged state and eluted earlier). I would guess the solubilities of the chloride and phosphate salt of you compound are different.
--------------------------------------------------------------------------------------------------------
By mpaciolla on Wednesday, June 25, 2003 - 06:31 am:
Since we are weighing the salt and using a pH meter that is calibrated before each use, I doubt the problem lies with an unreliable pH reading or the concentration of the buffer. The compound is a hydrchloride salt of an amine with a pKa of 9.0. The pH of our mobile phase was adjusted to pH 2.4. Hydrochloric acid was used to simply lower the pH. We didn't think it would matter but we were obvioulsy wrong.
--------------------------------------------------------------------------------------------------------
By Andreas Neumaier on Wednesday, June 25, 2003 - 08:02 am:
I don't have that much experience with buffered mobile phases, but I have a assumption to your problem:
Let me guess the retention time shift is related to the different degree of hydrolisation of the two acids you have used. HCl is a strong acid and hydrolyse to nearly 100%. H3PO4 is a weaker acid and more of this acid is needed for pH adjustment (compared to HCl).
Which means the overall concentration (of the adjusted buffer) is lower with HCl and higher when H3PO4 is used.
The higher the concentration of the buffer the lower the retention time.
If this assumption is right, you should observe the same shorter retention when using sulfuric acid (half of the concentration c of HCl; the same n as HCl) instead of HCl.
Nethertheless I also remember a special chromatographic problem which showed that different acids had different influence to the seperation.
--------------------------------------------------------------------------------------------------------
By Chris Pohl on Wednesday, June 25, 2003 - 08:25 am:
Although this phenomenon is not well documented in the literature, I believe the effect you report is connected to the weakly basic character of the amide functional group. We have observed modest increases in asymmetry for acidic compounds at low pH on prototype amide materials. Furthermore, we find that this affect diminishes dramatically as the pH is increased. In addition, however, many of the commercially available amide phases are synthesized in a manner which introduces anion exchange sites. You can distinguish between the two phenomena comparing the effect of pH on peak asymmetry of acidic solutes. At pH 4 there is that essentially no peak asymmetry in the case of pure amide phases, whereas in the case of phases containing significant contamination with anion exchange sites, the effect persists even at pH 6.5.
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By mpaciolla on Wednesday, June 25, 2003 - 08:56 am:
Andreas,
Your assumption does seem to make sense. However, the retention time of the basic analyte continues to increase as we increase the concentration of the buffer when using HCl to titrate to pH 2.4.
--------------------------------------------------------------------------------------------------------
By Andreas Neumaier on Thursday, June 26, 2003 - 06:02 am:
Sorry mpaciolla,
I've missed this point (english's not so easy for me as I sometimes think).
My assumption:
When increasing your buffer concentration the balance of interaction between stationary phase and buffer is shiftet toward stationary phase.
(This could be caused by decreasing solubility of the compound at increasing ionic concentration - but I'm not sure if this assumption's right.)
--------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 26, 2003 - 08:03 am:
Perhaps "Effect of the counter-anion type and concentration on the liquid chromatography retention of B-blockers" Journal of Chromatography A, 964 (2002) 179-187, holds the answer.
--------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, June 26, 2003 - 03:59 pm:
You said that the retention decreases with decreasing buffer concentration, but you did not tell us how low the total ion concentration was. There is nothing wrong with this phenomenon, if the ion concentration is very low. Under these circumstances, you may get exclusion from the pores and from the surface of a nicely hydrophobic C18 column. At pH 2.4, you do not have a lot hydrochloric acid, I presume. 4 mM, I guess. Try to add neutral salt to increase retention!
What was the buffer that you started off with? Type and concentration?
--------------------------------------------------------------------------------------------------------
By HW Mueller on Friday, June 27, 2003 - 04:15 am:
You still have not divulged crucial data on your buffer, not even what buffer you use. It would also be interesting to know how much dilution the HCl or H3PO4 produces, and how consistent that is.
--------------------------------------------------------------------------------------------------------
By mpaciolla on Monday, June 30, 2003 - 09:37 am:
Hello Everyone,
First, I would like to thank all of you for your insight. The above reference may explain what we are observing. We are using a 40mM Ammonium Phosphate Buffer that is titrated to pH 2.4 with HCl. Therefore, the approximate concentration of the HCl is 22mM. We tried using phosphoric acid to pH the buffer. The retention time was slightly less but the peak shape was poor (excessive tailing). We also tried using NaCl with the NH4H2PO4/H3PO4 buffer. The retention of the amine increased and the peak shape was slightly better but not nearly as good when using HCl to pH the buffer. Someone recently told me that HCl at that concentration in our mobile phase could damage the stainless steel column and tubing. Would someone please care to comment on this as well. One other note. The retention for the amine is decreasing as the colunm ages but the neutral compound is not.
--------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, July 1, 2003 - 01:48 am:
Uwe Neue-why should you get exclusion from the pores of a nicely hydrophobic column when the ionic concentration is low?
Thanks for your advice!
--------------------------------------------------------------------------------------------------------
By HW Mueller on Tuesday, July 1, 2003 - 03:56 am:
Your concentrations should be in the range (ionic strength, if my estimations are not way off) where practically all salts are chaotropic (NH4HxPO4 is considered highly lyotropic at higher concentrations). Therefore, I still think that ionic strength should not play much of a role here. Now, to be more consistent one may prepare two ammonium phosphate solutions of the same concentration, but with pH bracketing the desired one. These are then mixed together until the desired pH obtains.
On the HCl: It´s a matter of pH and concentration, it´s doubtful you will have a problem, but to make sure: take a piece of stainless capillary and leave it in your solution for some time. If you polish a section of the capillary with Ajax it is very easy to see wether there is tarnishing. But it would be preferable if you are consistent by mixing the buffer as mentioned.
--------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, March 25, 2004 - 12:44 am:
1. would u explain to me how is the ph effect on chromatogram profile?
2. would u explain how is the effect of the kind of buffer on chromatogram profile?
3. what is the advantages of methanol between acetonitril(ACN) as mobile phase in HPLC?
4.what is the function of internal standard on the research? what's make's we choose the internal standard? and why?
5.to get a good resolution, what must we do for up grade on chromatography?
6. to get a good peak, what must we do for up grade on chromatography?
thanks u. i'm sorry if my english is very bad. i hope u understand with the question i have to ask
--------------------------------------------------------------------------------------------------------
By tom jupille on Thursday, March 25, 2004 - 10:32 am:
We have a free on-line course which covers the answers to most of your questions:
http://www.lcresources.com/resources/reslinksform.html
By the way, your english is quite understandable.
--------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, March 25, 2004 - 03:42 pm:
All your questions require a lengthy answer. Therefore I agree with Tom: you will be better off looking at the course, and afterwards come back with specific questions.
