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What are the possible causes of peak shift and broadening in HPLC?
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+ Hplc analysis
+ High-performance liquid chromatography
+ High pressure liquid chromatography
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Lin Lev
What are the possible causes of peak shift and broadening in HPLC?
Not sure if the chemical of interest is charged, but columns generally have negatively charged carboxiclic acid groups on the surface (although manufacturers try to limit this as much as possible). At pH <3 these are protonated. The higher the pH, the more charged groups remain on the surface of the column. This can impact the retention greatly, especially if the chemical of interest is charged. cations will be retained partly by ion-interaction at pH 7 for example, but not at pH 3 because the acid groups are neutralised. Neutral chemicals will be retained stronger at low pH because there are no ions at the surface to repel them a bit.
Not sure if the chemical of interest is charged, but columns generally have negatively charged carboxiclic acid groups on the surface (although manufacturers try to limit this as much as possible). At pH <3 these are protonated. The higher the pH, the more charged groups remain on the surface of the column. This can impact the retention greatly, especially if the chemical of interest is charged. cations will be retained partly by ion-interaction at pH 7 for example, but not at pH 3 because the acid groups are neutralised. Neutral chemicals will be retained stronger at low pH because there are no ions at the surface to repel them a bit.
Thanks William, that seems interesting. @Stephen with all respect, it really doesn't matter, whether you mix acid and base or acid and salt if you're in the right pH range. The equilibrium will be always the same (except I have 15 mM formate anion, while you'd have 30 mM formate). I'll check the equilibration, but the method is still the same, so it shouldn't change.However, shouldn't it cause frontal tailing?
Thanks William, that seems interesting. @Stephen with all respect, it really doesn't matter, whether you mix acid and base or acid and salt if you're in the right pH range. The equilibrium will be always the same (except I have 15 mM formate anion, while you'd have 30 mM formate). I'll check the equilibration, but the method is still the same, so it shouldn't change.However, shouldn't it cause frontal tailing?
It looks like you're just pH adjusting and not buffering. You might be better served having 15 mM formic acid + 15 mM ammonium formate and adjusting to your pH's to ensure your pH stays constant. I find that my shifts are due primarily to insufficient equilibrium time after my gradient returns to starting composition. If you're just looking for 1 peak, find an isocratic method that gives you good S/N, and then really push the peak through with as high of flow as your LC will tolerate to minimize broadening. If your injections are dirty enough that you want to start with high water for rinsing the column and junk away first, then eluting with high organic to clean the column of organics, you'll need a long equilibration for good T(r) precision. I'll usually return back to initial composition as quickly as I can after my peak of interest comes of to minimize time, but it does depend on the concoction you're injecting. The greater the gradient difference, the longer the equilibration time. Choose as short, narrow-bore, and small particle size of a column as you can to minimize this exchange volume. Monitor your LC pressure profile. Make sure the pressure curve is smooth and returns to the starting pressure during your re-equilibration.
It looks like you're just pH adjusting and not buffering. You might be better served having 15 mM formic acid + 15 mM ammonium formate and adjusting to your pH's to ensure your pH stays constant. I find that my shifts are due primarily to insufficient equilibrium time after my gradient returns to starting composition. If you're just looking for 1 peak, find an isocratic method that gives you good S/N, and then really push the peak through with as high of flow as your LC will tolerate to minimize broadening. If your injections are dirty enough that you want to start with high water for rinsing the column and junk away first, then eluting with high organic to clean the column of organics, you'll need a long equilibration for good T(r) precision. I'll usually return back to initial composition as quickly as I can after my peak of interest comes of to minimize time, but it does depend on the concoction you're injecting. The greater the gradient difference, the longer the equilibration time. Choose as short, narrow-bore, and small particle size of a column as you can to minimize this exchange volume. Monitor your LC pressure profile. Make sure the pressure curve is smooth and returns to the starting pressure during your re-equilibration.
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention. There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention. There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention. There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention. There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
Careful pH adjustment, stability of pH and sufficient equilibration may be crucial in certain methods as William and Stephen already pointed out. Other than that, column aging can cause peak broadening worsening by time up to peak splitting with a method that has previously worked. You can try a fresh column of the same kind or run the column backwards to rule it out.
Careful pH adjustment, stability of pH and sufficient equilibration may be crucial in certain methods as William and Stephen already pointed out. Other than that, column aging can cause peak broadening worsening by time up to peak splitting with a method that has previously worked. You can try a fresh column of the same kind or run the column backwards to rule it out.
Not sure if the chemical of interest is charged, but columns generally have negatively charged carboxiclic acid groups on the surface (although manufacturers try to limit this as much as possible). At pH <3 these are protonated. The higher the pH, the more charged groups remain on the surface of the column. This can impact the retention greatly, especially if the chemical of interest is charged. cations will be retained partly by ion-interaction at pH 7 for example, but not at pH 3 because the acid groups are neutralised. Neutral chemicals will be retained stronger at low pH because there are no ions at the surface to repel them a bit.
Not sure if the chemical of interest is charged, but columns generally have negatively charged carboxiclic acid groups on the surface (although manufacturers try to limit this as much as possible). At pH <3 these are protonated. The higher the pH, the more charged groups remain on the surface of the column. This can impact the retention greatly, especially if the chemical of interest is charged. cations will be retained partly by ion-interaction at pH 7 for example, but not at pH 3 because the acid groups are neutralised. Neutral chemicals will be retained stronger at low pH because there are no ions at the surface to repel them a bit.
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Thanks William, that seems interesting.
@Stephen with all respect, it really doesn't matter, whether you mix acid and base or acid and salt if you're in the right pH range. The equilibrium will be always the same (except I have 15 mM formate anion, while you'd have 30 mM formate).
I'll check the equilibration, but the method is still the same, so it shouldn't change.However, shouldn't it cause frontal tailing?
Thanks William, that seems interesting.
@Stephen with all respect, it really doesn't matter, whether you mix acid and base or acid and salt if you're in the right pH range. The equilibrium will be always the same (except I have 15 mM formate anion, while you'd have 30 mM formate).
I'll check the equilibration, but the method is still the same, so it shouldn't change.However, shouldn't it cause frontal tailing?
More
VOTE
It looks like you're just pH adjusting and not buffering. You might be better served having 15 mM formic acid + 15 mM ammonium formate and adjusting to your pH's to ensure your pH stays constant.
I find that my shifts are due primarily to insufficient equilibrium time after my gradient returns to starting composition. If you're just looking for 1 peak, find an isocratic method that gives you good S/N, and then really push the peak through with as high of flow as your LC will tolerate to minimize broadening. If your injections are dirty enough that you want to start with high water for rinsing the column and junk away first, then eluting with high organic to clean the column of organics, you'll need a long equilibration for good T(r) precision. I'll usually return back to initial composition as quickly as I can after my peak of interest comes of to minimize time, but it does depend on the concoction you're injecting. The greater the gradient difference, the longer the equilibration time. Choose as short, narrow-bore, and small particle size of a column as you can to minimize this exchange volume.
Monitor your LC pressure profile. Make sure the pressure curve is smooth and returns to the starting pressure during your re-equilibration.
It looks like you're just pH adjusting and not buffering. You might be better served having 15 mM formic acid + 15 mM ammonium formate and adjusting to your pH's to ensure your pH stays constant.
I find that my shifts are due primarily to insufficient equilibrium time after my gradient returns to starting composition. If you're just looking for 1 peak, find an isocratic method that gives you good S/N, and then really push the peak through with as high of flow as your LC will tolerate to minimize broadening. If your injections are dirty enough that you want to start with high water for rinsing the column and junk away first, then eluting with high organic to clean the column of organics, you'll need a long equilibration for good T(r) precision. I'll usually return back to initial composition as quickly as I can after my peak of interest comes of to minimize time, but it does depend on the concoction you're injecting. The greater the gradient difference, the longer the equilibration time. Choose as short, narrow-bore, and small particle size of a column as you can to minimize this exchange volume.
Monitor your LC pressure profile. Make sure the pressure curve is smooth and returns to the starting pressure during your re-equilibration.
More
VOTE
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention.
There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention.
There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
More
VOTE
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention.
There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention.
There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
More
VOTE
Careful pH adjustment, stability of pH and sufficient equilibration may be crucial in certain methods as William and Stephen already pointed out. Other than that, column aging can cause peak broadening worsening by time up to peak splitting with a method that has previously worked. You can try a fresh column of the same kind or run the column backwards to rule it out.
Careful pH adjustment, stability of pH and sufficient equilibration may be crucial in certain methods as William and Stephen already pointed out. Other than that, column aging can cause peak broadening worsening by time up to peak splitting with a method that has previously worked. You can try a fresh column of the same kind or run the column backwards to rule it out.
More
VOTE