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What cause the peak splitting in LC-MS?
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+ Flavonoids
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Marla Robinson
What cause the peak splitting in LC-MS?
Ke Jin: *"Post updated 4-3-22" with more info. Please speak with someone at your school who has been trained to use the LC-MS system. They can help you to develop an HPLC method for use with the MS (ESI) system for your sample. The method shown does not appear to follow good chromatography fundamentals.
You stated that the flow rate for your polymeric 4.6 x 150mm, 5u column is 300 ul/min (~ 1.0 mL/min is approx linear flow rate for a 4.6 mm ID, but may be reduced for some polymeric columns). Your Tzero time of 0.72 min (est from your chromatograms "blip", but it also may be 6 min too) vs ~1.6 min (the expected estimated time) at 1.000 mL/min (or ~ 6 min if really running at 300 ul/min, which your chromatograms also appear to show as well, but with remaining sample from previous runs being visible). So something is very wrong with your described set up. *e.g. Analysis appears to have started before samples have all eluted off the column with peaks eluting before Tzero. Please confirm actual column flow rate.
Scan range is set to start at 70 so the MS is picking up just about everything. Baseline shows noise and signal at 90. This needs to be balanced out.
You are using a novel column type (SeQuant® ZIC®-pHILIC Polymeric) which may or may not be stable at pH 9.1. We have seen other clients use this same column with a guard column in-place and have experienced problems with dissolved silica ending up in the flow path and detector (because some of the guard columns are based on SILICA, which breaks down at basic pH) so do NOT use any silica columns with this setup.
Gradient program is questionable. Peaks do not appear to elute until around 7 min (which is evidence that you may be running at 300ul/min vs 1 mL/min, too low resulting in diffusion). Your gradient and analysis end at 95% ACN (too high, try 90% max to prevent precipitation) and you are ending the method right when the gradient hits 95% at 15min,with NO HOLD TIME at all. This is not acceptable as the mobile phase has not even reached the column yet and good chromatography practices require that you include an appropriate hold time to allow peaks to elute off the column. Failure to do so may lead to sample eluting out on the next run and column fouling (*both of which appear to be happening in your chromatograms). The HPLC method requires re-development.
Peak shapes are poor. Reasons for this could be due to a number of things including: column sample overloading / fouling; injection solution is different from mobile phase (it should be the same); insolubility of samples; gradient program not optimized; MS signal not optimized, etc.
Have someone local with HPLC and LC-MS experience help you to first develop a good quality HPLC method suitable for use with MS (ESI), then follow good chromatography fundamentals to optimize the separation. This will lead to success.
Ke Jin: *"Post updated 4-3-22" with more info. Please speak with someone at your school who has been trained to use the LC-MS system. They can help you to develop an HPLC method for use with the MS (ESI) system for your sample. The method shown does not appear to follow good chromatography fundamentals.
You stated that the flow rate for your polymeric 4.6 x 150mm, 5u column is 300 ul/min (~ 1.0 mL/min is approx linear flow rate for a 4.6 mm ID, but may be reduced for some polymeric columns). Your Tzero time of 0.72 min (est from your chromatograms "blip", but it also may be 6 min too) vs ~1.6 min (the expected estimated time) at 1.000 mL/min (or ~ 6 min if really running at 300 ul/min, which your chromatograms also appear to show as well, but with remaining sample from previous runs being visible). So something is very wrong with your described set up. *e.g. Analysis appears to have started before samples have all eluted off the column with peaks eluting before Tzero. Please confirm actual column flow rate.
Scan range is set to start at 70 so the MS is picking up just about everything. Baseline shows noise and signal at 90. This needs to be balanced out.
You are using a novel column type (SeQuant® ZIC®-pHILIC Polymeric) which may or may not be stable at pH 9.1. We have seen other clients use this same column with a guard column in-place and have experienced problems with dissolved silica ending up in the flow path and detector (because some of the guard columns are based on SILICA, which breaks down at basic pH) so do NOT use any silica columns with this setup.
Gradient program is questionable. Peaks do not appear to elute until around 7 min (which is evidence that you may be running at 300ul/min vs 1 mL/min, too low resulting in diffusion). Your gradient and analysis end at 95% ACN (too high, try 90% max to prevent precipitation) and you are ending the method right when the gradient hits 95% at 15min,with NO HOLD TIME at all. This is not acceptable as the mobile phase has not even reached the column yet and good chromatography practices require that you include an appropriate hold time to allow peaks to elute off the column. Failure to do so may lead to sample eluting out on the next run and column fouling (*both of which appear to be happening in your chromatograms). The HPLC method requires re-development.
Peak shapes are poor. Reasons for this could be due to a number of things including: column sample overloading / fouling; injection solution is different from mobile phase (it should be the same); insolubility of samples; gradient program not optimized; MS signal not optimized, etc.
Have someone local with HPLC and LC-MS experience help you to first develop a good quality HPLC method suitable for use with MS (ESI), then follow good chromatography fundamentals to optimize the separation. This will lead to success.
Ke Jin: *"Post updated 4-3-22" with more info.
Please speak with someone at your school who has been trained to use the LC-MS system. They can help you to develop an HPLC method for use with the MS (ESI) system for your sample. The method shown does not appear to follow good chromatography fundamentals.
- You stated that the flow rate for your polymeric 4.6 x 150mm, 5u column is 300 ul/min (~ 1.0 mL/min is approx linear flow rate for a 4.6 mm ID, but may be reduced for some polymeric columns). Your Tzero time of 0.72 min (est from your chromatograms "blip", but it also may be 6 min too) vs ~1.6 min (the expected estimated time) at 1.000 mL/min (or ~ 6 min if really running at 300 ul/min, which your chromatograms also appear to show as well, but with remaining sample from previous runs being visible). So something is very wrong with your described set up. *e.g. Analysis appears to have started before samples have all eluted off the column with peaks eluting before Tzero. Please confirm actual column flow rate.
- Scan range is set to start at 70 so the MS is picking up just about everything. Baseline shows noise and signal at 90. This needs to be balanced out.
- You are using a novel column type (SeQuant® ZIC®-pHILIC Polymeric) which may or may not be stable at pH 9.1. We have seen other clients use this same column with a guard column in-place and have experienced problems with dissolved silica ending up in the flow path and detector (because some of the guard columns are based on SILICA, which breaks down at basic pH) so do NOT use any silica columns with this setup.
- Gradient program is questionable. Peaks do not appear to elute until around 7 min (which is evidence that you may be running at 300ul/min vs 1 mL/min, too low resulting in diffusion). Your gradient and analysis end at 95% ACN (too high, try 90% max to prevent precipitation) and you are ending the method right when the gradient hits 95% at 15min, with NO HOLD TIME at all. This is not acceptable as the mobile phase has not even reached the column yet and good chromatography practices require that you include an appropriate hold time to allow peaks to elute off the column. Failure to do so may lead to sample eluting out on the next run and column fouling (*both of which appear to be happening in your chromatograms). The HPLC method requires re-development.
- Peak shapes are poor. Reasons for this could be due to a number of things including: column sample overloading / fouling; injection solution is different from mobile phase (it should be the same); insolubility of samples; gradient program not optimized; MS signal not optimized, etc.
Have someone local with HPLC and LC-MS experience help you to first develop a good quality HPLC method suitable for use with MS (ESI), then follow good chromatography fundamentals to optimize the separation. This will lead to success.Ke Jin: *"Post updated 4-3-22" with more info.
Please speak with someone at your school who has been trained to use the LC-MS system. They can help you to develop an HPLC method for use with the MS (ESI) system for your sample. The method shown does not appear to follow good chromatography fundamentals.
- You stated that the flow rate for your polymeric 4.6 x 150mm, 5u column is 300 ul/min (~ 1.0 mL/min is approx linear flow rate for a 4.6 mm ID, but may be reduced for some polymeric columns). Your Tzero time of 0.72 min (est from your chromatograms "blip", but it also may be 6 min too) vs ~1.6 min (the expected estimated time) at 1.000 mL/min (or ~ 6 min if really running at 300 ul/min, which your chromatograms also appear to show as well, but with remaining sample from previous runs being visible). So something is very wrong with your described set up. *e.g. Analysis appears to have started before samples have all eluted off the column with peaks eluting before Tzero. Please confirm actual column flow rate.
- Scan range is set to start at 70 so the MS is picking up just about everything. Baseline shows noise and signal at 90. This needs to be balanced out.
- You are using a novel column type (SeQuant® ZIC®-pHILIC Polymeric) which may or may not be stable at pH 9.1. We have seen other clients use this same column with a guard column in-place and have experienced problems with dissolved silica ending up in the flow path and detector (because some of the guard columns are based on SILICA, which breaks down at basic pH) so do NOT use any silica columns with this setup.
- Gradient program is questionable. Peaks do not appear to elute until around 7 min (which is evidence that you may be running at 300ul/min vs 1 mL/min, too low resulting in diffusion). Your gradient and analysis end at 95% ACN (too high, try 90% max to prevent precipitation) and you are ending the method right when the gradient hits 95% at 15min, with NO HOLD TIME at all. This is not acceptable as the mobile phase has not even reached the column yet and good chromatography practices require that you include an appropriate hold time to allow peaks to elute off the column. Failure to do so may lead to sample eluting out on the next run and column fouling (*both of which appear to be happening in your chromatograms). The HPLC method requires re-development.
- Peak shapes are poor. Reasons for this could be due to a number of things including: column sample overloading / fouling; injection solution is different from mobile phase (it should be the same); insolubility of samples; gradient program not optimized; MS signal not optimized, etc.
Have someone local with HPLC and LC-MS experience help you to first develop a good quality HPLC method suitable for use with MS (ESI), then follow good chromatography fundamentals to optimize the separation. This will lead to success.More
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pH of mobile phase and column are a major culprit for peak splitting.
pH of mobile phase and column are a major culprit for peak splitting.
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"HPLC Peak Splitting. Common Reasons For It"; https://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html
"HPLC Peak Splitting. Common Reasons For It"; https://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html
More
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"Do your HPLC Methods Meet Basic Good Chromatography Fundamentals? HPLC Training: RETAIN, SEPARATE and RESOLVE"; https://hplctips.blogspot.com/2019/12/do-your-hplc-methods-meet-basic.html
"Do your HPLC Methods Meet Basic Good Chromatography Fundamentals? HPLC Training: RETAIN, SEPARATE and RESOLVE"; https://hplctips.blogspot.com/2019/12/do-your-hplc-methods-meet-basic.html
More
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