Lysine was dissolved in mobile phase and injected in HPLC but there are two peaks. One for positive at 2.1 min and another for negative at 2.8 min? Contents of moilbe phase are ACN:Water:TFA = 80:20:0.1 and i can not find the reason why. Please help me.
*Plus ; when i inject mobile phase it shows smooth baseline. So I thought negative peak is not related to solvent. Also the solution was detected at 210 nm. Only UV detection was used in analysis.
Please have someone at your school who has training in HPLC assist you. HPLC takes many years of training just to have a basic level of proficiency and your questions imply you may be using it for the first time and/or without someone experienced to guide you. You did not mention what column (type and dimensions) you are using or the flow rate so it is impossible to put your observations or results in scientific context.
Seeing small positive and negative peaks at the beginning of a run may be normal (at T0), but the scale that they are observed at is important (and we do not know what yours is). The chromatogram 'Y' scale of the two peaks would be useful to known.
*Park wrote: "Contents of moilbe phase are ACN:Water:TFA = 80:20:0.1".
80% ACN is A VERY HIGH PERCENTAGE of organic for a RP method to retain Lysine. We do not know the details of your method, but in many situations it would be expected to precipitate out or not be retained at all and elute at T0.
CAN you attach a chromatogram with scales shown? This would help.
Basic Questions: What is the T0 (Tzero) of your HPLC method (*this is something that you would need to know to run the method)? What is the K prime of your peaks? What is the injection volume? What is the flow rate? Which column was used and what are the dimensions? What is the amplitude of the two peaks (+/-)? When you injected just mobile phase, you should have also observed a double peak (+/-) too. Did you view this run on the same scale as the one with your sample? What are you trying to resolve Lysine from? Where did you acquire this method from?
Please have someone at your school who has training in HPLC assist you. HPLC takes many years of training just to have a basic level of proficiency and your questions imply you may be using it for the first time and/or without someone experienced to guide you. You did not mention what column (type and dimensions) you are using or the flow rate so it is impossible to put your observations or results in scientific context.
Seeing small positive and negative peaks at the beginning of a run may be normal (at T0), but the scale that they are observed at is important (and we do not know what yours is). The chromatogram 'Y' scale of the two peaks would be useful to known.
*Park wrote: "Contents of moilbe phase are ACN:Water:TFA = 80:20:0.1".
80% ACN is A VERY HIGH PERCENTAGE of organic for a RP method to retain Lysine. We do not know the details of your method, but in many situations it would be expected to precipitate out or not be retained at all and elute at T0.
CAN you attach a chromatogram with scales shown? This would help.
Basic Questions: What is the T0 (Tzero) of your HPLC method (*this is something that you would need to know to run the method)? What is the K prime of your peaks? What is the injection volume? What is the flow rate? Which column was used and what are the dimensions? What is the amplitude of the two peaks (+/-)? When you injected just mobile phase, you should have also observed a double peak (+/-) too. Did you view this run on the same scale as the one with your sample? What are you trying to resolve Lysine from? Where did you acquire this method from?
Even a UV detector is not immune from 'refractive index' differences which you would see at the solvent front due to the solvent. But that is not your case since this peak is well after the solvent front AND is negative. I have no idea? How big is this peak? Can you ignore it since it is not relevant? As I recall Lysine can be either an R or S configuration.
Find out which peak is your analyte by injecting x and 10 x concentration on the same solvent. Pick the one that is x and 10 x in response and see if the negative peak is the same. As long as you can integrate you analyte, ignore the negtative one and move on.
I apologize to all for late reply. I really appreciate to all answers you gave to me and they really help me. Actually positive peak shows the pattern which Mr. Narong comment on me and I just decide to ignore negative peak as it does not affect positive peak at all. Thank you for all your comments again.
- Park In Woo wrote; "I just decide to ignore negative peak as it does not affect positive peak at all." ????
- Narong Chamkasem wrote: "As long as you can integrate you analyte, ignore the negtative one and move on." ????
Park, based on the limited info you provided us, it sounds like your method is invalid. You asked for free advice, but decided not to provide any details or answer questions. Instead, you have decided to just ignore the issue (a poor choice) and not worry about following the scientific method or basic HPLC guidelines. Good luck with that approach.
As we know lysine is known as alpha-amino acid and all the known compounds or chemicals have a particular method, wavelength, and solvent ratio (A & B) for mobile phase and column size and their flow rate. It also depends on the purity of your compound, as far I know Lysine is >95% pure in HPLC analytical standard. some times you may get more than two peaks due to impurities of the compounds but impure peaks will appear as very small in size which is negligible. Secondly, it also depends on the polarity and non-polarity of the compounds. If the polarity of the stationary phase and compound are similar, the retention time increases because the compound interacts stronger with the stationary phase. As a result, polar compounds have long retention times on polar stationary phases and shorter retention times on non-polar columns using the same temperature. Chiral stationary phases are based on amino acid derivative and we know that Lysine is a polar amino acid. I hope this explanation might help you to choose which one is your compound peak.
Adding one more option to think about above all expert responses and help provided here... The age and health of the HPLC column matters as well. Please try priming before next use and allow sufficient time to get the column free from micro-sized bubbles, etc. and let it be conditioned with mobile phase (blank) for at least an hour before injecting the standard and sample solutions prepared. If still get the negative peak, not to worry. Check with the column manufacturer on how to regenerate the column and use it carefully again. Hope this will help a bit to get to the root cause of the negative peak.
Is it a void peak? Please also confirm pressure graph if you have any pressure drift due to air trapped on system.
It can be connected with injection valve and pressure fluctuation during injection.
But as already mentioned it not acting on results.
Negative peak relates to the elution of a compound that generates less detector response than the mobile-phase baseline. You may read some articles written by John Dolan - an expert in HPLC (www. lcgceurope.com and find “LC TROUBLESHOOTING”). Hope you will find it helpful.
Possibly may be due to vacancy chromatography effect.
Kindly read the interesting research article by Scott et al., 1972
Some Aspects of Liquid-Solid Vacancy Chromatography
Article Liquid-solid vacancy chromatography
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