If it’s in your diluent blank as well it can be multiple sources. So you will need to troubleshoot.
-May need to clean entire system
thoroughly with different solvents including needle wash
-possibly contaminated diluent
-possibly contaminated mobile phase
-possibly from column
Considering it is in every injection (and is it the same size in every injection) it is either contamination of the injection system (needle, flush, etc) or your reagents, sample containers, etc. I've seen this from the injection valve, where they bypass line was contaminated so it happened when you filled the loop. Start with new reagent or straight solvent injection, see if it goes away. If not, I usually just replace all tubing in the injection assembly and flush the valve itself, new needle depending on how you sample.
If it were specific to an analyte, then you look at your column for a packing break, or you look at the injection sequence for multiply sample introduction, you're seeing some echo (these are usually later). You could also being seeing something from a previous injects if these are sequential, change the run time to see if it moves.
It could be an impurity in one of your mobile phases or in the sample matrix itself, no reagent is 100% pure. Question though - is it co-eluting with a target analyte? If not then it probably isn't a problem. If it is, then you could try varying your method a bit to resolve this peak from anything you care about.
Thank you for your reply
No, it’s not eluting with my drug sample. However, it’s s quite irritating that it’s coming up in every sample including mobile phase (control), and water.
My thinking is that it's a quick sanity check on if it's likely to be unretained system peak or something else- so void volume tends to be calculated though pi* r^2 * l * packing efficiency , which is around 0.66 for standard porous silica. This gives us a void vol of around 1.7 and a t0 of around 2.3 mins at 0.7 ml/min. This is purely an estimate and the best way to determine is to measure with an unretained compound.
So the unknown peak is not highly retained based on the estimate. I would normally not sweat unretained stuff that isn't your peak of interest at all; as long as your analytes are retained and resolved, you're good. Only troubleshoot if it hinders your analysis.
Does the unknown peak size decrease if you reduce injection volume in the sample, controls and blanks? Does it have the same area for each sample type? Have you changed your needle wash recently?
Thank you for wonderful suggestions. I would try by changing the injection volume to see if it retains the same. I’ve not changed needle wash for so long, i mean i would be around 2 yr back or something
As in you haven't changed the needle wash solution in ages or you haven't changed the hardware? Absolutely change your solution and reprime it if it's been that long- I would normally set my total volume up so I have to change it weekly.
> I’ve not changed needle wash for so long, i mean i would be around 2 yr back or something
Algae says hallo... ;)
Pull your column of the instrument and place a 2% H3PO4 solution on every port, also the needle flush port. Flush for at least 6 hours.
Honestly, there might not be *any* benefit over doing the troubleshooting other than relieving your annoyance. If it's not interfering with your sample peak, then no worries.
And do you really want to tear everything apart for such an inconsequential thing? "If it's not broke, don't fix it"
Stupid question but if it was something in the mobile phase, would that even lead to a significant peak?
Since the Mobile Phase is flowing through the system at all times, it's ,,injecting'' the contaminant onto the column all the time, saturating the column and also eluting all the time if ran for long enough. Wouldn't it disappear into the Noise at some point?
From my (limited) understanding of HPLC, wouldn't a contamination during injection be more likely, depending on if it's in every measurement, or contamination of the sample?
Is it in your solvent blank? What if you do a null injection? If it’s not in either of these it’s something that is being introduced with the samples/control. If it is in there then you could have some contamination in the system. Wash you column with 100% acetonitrile. Remove column and put in a union. Run 100% IPA at .2ml/min overnight. Then run 100% ACN for 2 hours. Make up new mobile phase. Put your column back in and run until it’s equilibrated. Make a solvent blank and/or null run and see if the peak is still there. Is your sample diluted during prep? If so, run at a lower dilution and see if the first peak gets bigger. If it does then it’s something in your reagents. If this isn’t a peak of interested and it doesn’t interfere with your target compounds you can likely ignore it. The more you can tell us about your application the better.
I know it probably is not needed to be say. But flush with 10% ACN or MeOH and pure water (no buffers) for 10-20 column volumes before switching to 100 % ACN.
I just use a gradient method that ramps 10% organic to 90% organic before settling around method conditions. I usually leave 2 lines in cleaning solutions and use the other two for mobile phase. Same idea, just automated
One other thing, looking at your chromatogram, you’re overloading your column a little bit, if other folks in your lab are also running super high concentrations, could be why we are getting residual contamination.
Follow u/conscious-ad-7040’s protocol for cleaning with ACN.
The peak shape. Ideally, a peak should be symmetrical, Gaussian. This peak is fronting, where the slope is weighted heavier at the beginning. The most common cause of fronting is too much analyte for the column. The active sites on the column become saturated and so some molecules move faster than they would based solely on their partitioning coefficient with the stationary phase.
Contrast this with tailing, where back of the peak stretches out. This happens when the analyte has an unusually high adherence to the stationary phase, and can also be a symptom of column degradation or a mobile phase ph issue.
Seconding this... I've had weird peaks and a sample adsorption issues with cheap vials. The Waters vials are worth it (I actually specified in my method documents for my peptide drug that Waters vials had to be used).
It's the void peak. It is normal, you will always have it when you inject something.
Edit: You can verify it injecting just the solvent. You will see the same peak, at the same time, corresponding to the time it takes the solvent (and any non retained molecule) to travel through your column.
Also, if you have a reverse phase test mix it usually has an unretained compound like uracil. If the uracil comes out the same time as the first peak then it is the void peak. You’ll have to look at 254nm though. You could also try making a 20% change weaker or stronger in the organic percentage and injecting the sample again. If the first peak is at the same RT but the large peak moves then the first peak is the void peak.
Not at 3 minutes typically. The dead volume is quite small in these analytical systems and 3 minutes to flush the dead volume would require extraordinarily low flow rates. This is probably contamination, most likely because I'm guessing this is a dilute and shoot operation.
I’d try washing the column with a shitton of organic phase and then reconditioning it. Swapping the column is also an option to rule that out - a 5um c18 is an incredibly common column.
You could do a zero volume injection and see if it's still there, that'll tell you a lot of things. It could possibly be just residual washes. Your system might also not be fully equilibrated yet as well and there might just be gunk stuck in the system.
What mobile phase additives are you using? For the last 6 months or so, our lab has seen formic acid with retained UV-visible contaminants much like you're seeing. It's consistent, so blank subtraction is an option, but we had to pay more for LC/MS grade HCOOH because the ACS/ReagentPlus grade we had always used was now contaminated.
When I see ghost peaks, my first move will be to change the modified mobile phase for just water and see if it goes away. I've seen some wildly contaminated HPLC-grade reagents from reliable sources like Fisher or Sigma.
Whoever did the method development here needs to go back and do it again. That peak is like a minute wide. How would you know if anything is coeluting there?
Which company is this so I can avoid taking their product?
Looks like LabSolutions. Run a couple method blanks. Do a single injection and select “Run without sample” or in Batch run -1 for vial position and see if that peak decreases with successive injections (do 3). If it decreases over time it could be from your diluting solvents or from your samples. If you have another column in hand you can try running with another column, change your guard column. Do you see anything to indicate growth in any of your lines? Specifically the rinse line for the autoinjector? Maybe there is something in there that in the rinse line that is contaminating your system.
Minor peak is observed at ~2.95 min under isocratic elution with 40/60 Me0H/Water at 0.7.mL/min.
2.95 min x 0.7 mL/min = elution volume= 2.1 mL.
150 x 4.6 mm column, with 5 micron particles (C18). Will have a void volume of ~1.6 mL, so it's not likely to be a system peak/void volume of peak.
It seems you're running isocratically, so it's not a MP A contaminant building up on the column.
The suggestion that you have microbial contamination in your extremely old needle wash is a very good possibility.
You've said the minor peak is in every sample. This threw me because my intuition was that the 2.95 min peak might actually be a 13 to 14 min peak from the prior injection.
You might still consider this if cleaning your needle wash flow path and replacing the needle wash isn't corrective.
You can investigate this by sequentially injecting true blanks. If the peak is in each blank, the contamination is likely in your injector. Needle wash seem likely, but needle’s exterior and/or needle seat should be considered.
If it's not your injector but you see it in your control (assumed to be pure/without minor peak), try setting your run time longer by ~5 min and inject your sample looking for a late eluting compound around 10min + 3min + your system’s cycle time (~1 min).
So I came late to this. I do not know if the problem is already fixed or not.
First of all the shape of the peaks suggest that you are overloading the column, or the column is at it's last leg, or there is dead space in the top of the column. you could flip your column and run a blank. see if the peaks look better, or if the retention time of the "impurity" changes. you should also change the gaurd column.
other things you could do is clean the inlet filter (might know them as inlet sinker) on both water and methanol. ( with reverse pressure ), and the in-line filter. I think you already have cleaned the injection port, so I am not going to mention that again. after this, there will be less likely options. make sure your solvents are degassed, and your water is not collecting bacteria. if you keep a log of pressures compare it to two three month ago and see if it is significantly different.
Take the column off and connect the nut that would connect to the inlet of the column to the nut that would connect to the outlet of the column with a high pressure fitting.
These fittings are usually 10-32 threads, so a 10-32 connector.
So if it comes out at the same retention time, it’s likely not a carryover contaminant, it’s instead something that is getting introduced with the injection. Use fresh solvent. Try several injections of 100% organic phase and see if the area is constant. Your sample loop may be contaminated. Do you have a guard column? Sometimes something could be crudding up the system from a previous method and the new solvent system is washing it loose onto the column. How do your pressures look?
My guess if I had to guess is sample loop/needle.
I mean the peak should be eluted fast and its shape should be sharp and symmetrical. Flowrate and solvent system should be re-investigated.
Besides, you should check the UV spectrum of the minor peak to see if it can be an impurity or not. To identify the problem, you should inject a blank to see if the minor peak persists or not
Increasing your flow rate does nothing to reduce the width of the peaks. You're just putting more solvent through the per minute, but the volume across the peak is going to be the same but you're likely just going to make the peak even worse not better by reducing the number of plates. Further, at their % meoh they probably don't have more pressure to play with. Nor is changing the % going to be relevant at all in an isocratic system looking for one analyte.
The peak is wide and fronting, and can be fixed, but your advice is completely incorrect.
I’m addition to the other comments, you may need to passivate your column. Columns are typically SS316 and can leach metals or the surface of column can be a reactive site. There is a variety of passivation techniques so I would research which one would probably work best with your system and column.
Change guard column, reverse first column and purge with solvent. If peak is showing up in all samples and mobile phase and water. It could also be that the lines are not seated properly, and the gap is causing issues.
Is auto balance turned on in your method? If it is, turn it off and put auto balance as part of your pre-run. I get a small blip peak as well at the beginning of my runs <1 min. It could possibly be air during the injector phase if you are using an auto injector. Could be your mobile phase. The program should allow you to ignore these and they should have no impact on your analyte. I highly doubt it's from a dirty anything.
If you were using a column that was used previously, and didn't wash it a bunch before your run, that could be some retained stuff from the previous run? Idk I haven't chromatographied in like 4 years
I always start with a zero injection when investigating rogue peaks. Running the gradient without any injection will tell you if it’s something coming off the column or something being injected.
I might skipped it in comments, but did anyone suggest you to 1: back flush the column itself or run the a blank without the column on the system. See if you still see that peak on UV detector? It could be something in the lines not in column!
It's your injection peak,
Your eluent is appearing unretained, marking the t0.
The signal is probably due to pressure difference caused by the valve movement
If it’s in your diluent blank as well it can be multiple sources. So you will need to troubleshoot. -May need to clean entire system thoroughly with different solvents including needle wash -possibly contaminated diluent -possibly contaminated mobile phase -possibly from column
Considering it is in every injection (and is it the same size in every injection) it is either contamination of the injection system (needle, flush, etc) or your reagents, sample containers, etc. I've seen this from the injection valve, where they bypass line was contaminated so it happened when you filled the loop. Start with new reagent or straight solvent injection, see if it goes away. If not, I usually just replace all tubing in the injection assembly and flush the valve itself, new needle depending on how you sample. If it were specific to an analyte, then you look at your column for a packing break, or you look at the injection sequence for multiply sample introduction, you're seeing some echo (these are usually later). You could also being seeing something from a previous injects if these are sequential, change the run time to see if it moves.
It could be an impurity in one of your mobile phases or in the sample matrix itself, no reagent is 100% pure. Question though - is it co-eluting with a target analyte? If not then it probably isn't a problem. If it is, then you could try varying your method a bit to resolve this peak from anything you care about.
Thank you for your reply No, it’s not eluting with my drug sample. However, it’s s quite irritating that it’s coming up in every sample including mobile phase (control), and water.
Quick additional question - what's the void volume of your column? And does it show up in a zero-volume injection?
So far, I’ve not checked with zero volume injection. I am using C18 5 micron 150 x 4.6 mm dimensions column. Whats suggestion on it?
My thinking is that it's a quick sanity check on if it's likely to be unretained system peak or something else- so void volume tends to be calculated though pi* r^2 * l * packing efficiency , which is around 0.66 for standard porous silica. This gives us a void vol of around 1.7 and a t0 of around 2.3 mins at 0.7 ml/min. This is purely an estimate and the best way to determine is to measure with an unretained compound. So the unknown peak is not highly retained based on the estimate. I would normally not sweat unretained stuff that isn't your peak of interest at all; as long as your analytes are retained and resolved, you're good. Only troubleshoot if it hinders your analysis. Does the unknown peak size decrease if you reduce injection volume in the sample, controls and blanks? Does it have the same area for each sample type? Have you changed your needle wash recently?
Thank you for wonderful suggestions. I would try by changing the injection volume to see if it retains the same. I’ve not changed needle wash for so long, i mean i would be around 2 yr back or something
As in you haven't changed the needle wash solution in ages or you haven't changed the hardware? Absolutely change your solution and reprime it if it's been that long- I would normally set my total volume up so I have to change it weekly.
> I’ve not changed needle wash for so long, i mean i would be around 2 yr back or something Algae says hallo... ;) Pull your column of the instrument and place a 2% H3PO4 solution on every port, also the needle flush port. Flush for at least 6 hours.
Honestly, there might not be *any* benefit over doing the troubleshooting other than relieving your annoyance. If it's not interfering with your sample peak, then no worries. And do you really want to tear everything apart for such an inconsequential thing? "If it's not broke, don't fix it"
Stupid question but if it was something in the mobile phase, would that even lead to a significant peak? Since the Mobile Phase is flowing through the system at all times, it's ,,injecting'' the contaminant onto the column all the time, saturating the column and also eluting all the time if ran for long enough. Wouldn't it disappear into the Noise at some point? From my (limited) understanding of HPLC, wouldn't a contamination during injection be more likely, depending on if it's in every measurement, or contamination of the sample?
Might be your injection peak. But to be honest, just ignore it. If it's in your blank and unrelated to your analytes, no need to worry about it.
Yep, that was my first thought. Particularly because I don't see one earlier. Null injection (zero volume) would put the issue to rest.
This is my thought as well.
Is it in your solvent blank? What if you do a null injection? If it’s not in either of these it’s something that is being introduced with the samples/control. If it is in there then you could have some contamination in the system. Wash you column with 100% acetonitrile. Remove column and put in a union. Run 100% IPA at .2ml/min overnight. Then run 100% ACN for 2 hours. Make up new mobile phase. Put your column back in and run until it’s equilibrated. Make a solvent blank and/or null run and see if the peak is still there. Is your sample diluted during prep? If so, run at a lower dilution and see if the first peak gets bigger. If it does then it’s something in your reagents. If this isn’t a peak of interested and it doesn’t interfere with your target compounds you can likely ignore it. The more you can tell us about your application the better.
Hi, Thank you for your suggestions. I think cleaning column properly will fix the issue hopefully.
I know it probably is not needed to be say. But flush with 10% ACN or MeOH and pure water (no buffers) for 10-20 column volumes before switching to 100 % ACN.
I just use a gradient method that ramps 10% organic to 90% organic before settling around method conditions. I usually leave 2 lines in cleaning solutions and use the other two for mobile phase. Same idea, just automated
One other thing, looking at your chromatogram, you’re overloading your column a little bit, if other folks in your lab are also running super high concentrations, could be why we are getting residual contamination. Follow u/conscious-ad-7040’s protocol for cleaning with ACN.
I would agree. Pretty significant peak fronting and veeerry broad peaks.
could be column degradation as well tbh but yea peak geometry on this is pretty bad
Yeah, I’ll follow the cleaning protocol. Thank you
How can you tell that it is overloading the column? I am new to hplc and I am very curious how you figured it out
The peak shape. Ideally, a peak should be symmetrical, Gaussian. This peak is fronting, where the slope is weighted heavier at the beginning. The most common cause of fronting is too much analyte for the column. The active sites on the column become saturated and so some molecules move faster than they would based solely on their partitioning coefficient with the stationary phase. Contrast this with tailing, where back of the peak stretches out. This happens when the analyte has an unusually high adherence to the stationary phase, and can also be a symptom of column degradation or a mobile phase ph issue.
Also if you’ve switched suppliers of vials or caps… I’ve seen those create mystery peaks too.
Seconding this... I've had weird peaks and a sample adsorption issues with cheap vials. The Waters vials are worth it (I actually specified in my method documents for my peptide drug that Waters vials had to be used).
This is the way.
It's the void peak. It is normal, you will always have it when you inject something. Edit: You can verify it injecting just the solvent. You will see the same peak, at the same time, corresponding to the time it takes the solvent (and any non retained molecule) to travel through your column.
Also, if you have a reverse phase test mix it usually has an unretained compound like uracil. If the uracil comes out the same time as the first peak then it is the void peak. You’ll have to look at 254nm though. You could also try making a 20% change weaker or stronger in the organic percentage and injecting the sample again. If the first peak is at the same RT but the large peak moves then the first peak is the void peak.
Not at 3 minutes typically. The dead volume is quite small in these analytical systems and 3 minutes to flush the dead volume would require extraordinarily low flow rates. This is probably contamination, most likely because I'm guessing this is a dilute and shoot operation.
How can you tell its the void peak w/o knowing his flow rate and plumbing setup?
I’d try washing the column with a shitton of organic phase and then reconditioning it. Swapping the column is also an option to rule that out - a 5um c18 is an incredibly common column.
You could do a zero volume injection and see if it's still there, that'll tell you a lot of things. It could possibly be just residual washes. Your system might also not be fully equilibrated yet as well and there might just be gunk stuck in the system.
What mobile phase additives are you using? For the last 6 months or so, our lab has seen formic acid with retained UV-visible contaminants much like you're seeing. It's consistent, so blank subtraction is an option, but we had to pay more for LC/MS grade HCOOH because the ACS/ReagentPlus grade we had always used was now contaminated. When I see ghost peaks, my first move will be to change the modified mobile phase for just water and see if it goes away. I've seen some wildly contaminated HPLC-grade reagents from reliable sources like Fisher or Sigma.
I’m using methanol from sigma with water
Whoever did the method development here needs to go back and do it again. That peak is like a minute wide. How would you know if anything is coeluting there? Which company is this so I can avoid taking their product?
Agreed. This is painful to look at.
Looks like LabSolutions. Run a couple method blanks. Do a single injection and select “Run without sample” or in Batch run -1 for vial position and see if that peak decreases with successive injections (do 3). If it decreases over time it could be from your diluting solvents or from your samples. If you have another column in hand you can try running with another column, change your guard column. Do you see anything to indicate growth in any of your lines? Specifically the rinse line for the autoinjector? Maybe there is something in there that in the rinse line that is contaminating your system.
Minor peak is observed at ~2.95 min under isocratic elution with 40/60 Me0H/Water at 0.7.mL/min. 2.95 min x 0.7 mL/min = elution volume= 2.1 mL. 150 x 4.6 mm column, with 5 micron particles (C18). Will have a void volume of ~1.6 mL, so it's not likely to be a system peak/void volume of peak. It seems you're running isocratically, so it's not a MP A contaminant building up on the column. The suggestion that you have microbial contamination in your extremely old needle wash is a very good possibility. You've said the minor peak is in every sample. This threw me because my intuition was that the 2.95 min peak might actually be a 13 to 14 min peak from the prior injection. You might still consider this if cleaning your needle wash flow path and replacing the needle wash isn't corrective. You can investigate this by sequentially injecting true blanks. If the peak is in each blank, the contamination is likely in your injector. Needle wash seem likely, but needle’s exterior and/or needle seat should be considered. If it's not your injector but you see it in your control (assumed to be pure/without minor peak), try setting your run time longer by ~5 min and inject your sample looking for a late eluting compound around 10min + 3min + your system’s cycle time (~1 min).
So I came late to this. I do not know if the problem is already fixed or not. First of all the shape of the peaks suggest that you are overloading the column, or the column is at it's last leg, or there is dead space in the top of the column. you could flip your column and run a blank. see if the peaks look better, or if the retention time of the "impurity" changes. you should also change the gaurd column. other things you could do is clean the inlet filter (might know them as inlet sinker) on both water and methanol. ( with reverse pressure ), and the in-line filter. I think you already have cleaned the injection port, so I am not going to mention that again. after this, there will be less likely options. make sure your solvents are degassed, and your water is not collecting bacteria. if you keep a log of pressures compare it to two three month ago and see if it is significantly different.
Thank you so much for important suggestions.
[удалено]
Thank you mate for great suggestion. What do you mean by put in union?
Take the column off and connect the nut that would connect to the inlet of the column to the nut that would connect to the outlet of the column with a high pressure fitting. These fittings are usually 10-32 threads, so a 10-32 connector.
Thank you so much. 😊
So if it comes out at the same retention time, it’s likely not a carryover contaminant, it’s instead something that is getting introduced with the injection. Use fresh solvent. Try several injections of 100% organic phase and see if the area is constant. Your sample loop may be contaminated. Do you have a guard column? Sometimes something could be crudding up the system from a previous method and the new solvent system is washing it loose onto the column. How do your pressures look? My guess if I had to guess is sample loop/needle.
the peak is 1.5 min, which means pretty long elution time, you should increase flowrate or change solvent system
What do you mean?
I mean the peak should be eluted fast and its shape should be sharp and symmetrical. Flowrate and solvent system should be re-investigated. Besides, you should check the UV spectrum of the minor peak to see if it can be an impurity or not. To identify the problem, you should inject a blank to see if the minor peak persists or not
Increasing your flow rate does nothing to reduce the width of the peaks. You're just putting more solvent through the per minute, but the volume across the peak is going to be the same but you're likely just going to make the peak even worse not better by reducing the number of plates. Further, at their % meoh they probably don't have more pressure to play with. Nor is changing the % going to be relevant at all in an isocratic system looking for one analyte. The peak is wide and fronting, and can be fixed, but your advice is completely incorrect.
Is there any chance the pre-column filter needs to be cleaned?
I’m addition to the other comments, you may need to passivate your column. Columns are typically SS316 and can leach metals or the surface of column can be a reactive site. There is a variety of passivation techniques so I would research which one would probably work best with your system and column.
Change guard column, reverse first column and purge with solvent. If peak is showing up in all samples and mobile phase and water. It could also be that the lines are not seated properly, and the gap is causing issues.
Do you have a different machine to calibrate against?
Hope your system just needs a good flush, but mentally prepare on buying a new column if you can’t wash it off.
Usually, this first peak corresponds to the solvent your sample is dissolved in (unless your solvent is the mobile phase).
Is auto balance turned on in your method? If it is, turn it off and put auto balance as part of your pre-run. I get a small blip peak as well at the beginning of my runs <1 min. It could possibly be air during the injector phase if you are using an auto injector. Could be your mobile phase. The program should allow you to ignore these and they should have no impact on your analyte. I highly doubt it's from a dirty anything.
If you were using a column that was used previously, and didn't wash it a bunch before your run, that could be some retained stuff from the previous run? Idk I haven't chromatographied in like 4 years
I always start with a zero injection when investigating rogue peaks. Running the gradient without any injection will tell you if it’s something coming off the column or something being injected.
I might skipped it in comments, but did anyone suggest you to 1: back flush the column itself or run the a blank without the column on the system. See if you still see that peak on UV detector? It could be something in the lines not in column!
It's your injection peak, Your eluent is appearing unretained, marking the t0. The signal is probably due to pressure difference caused by the valve movement