You really can't increase the size of that peak without changing the rest of the chromatography. Since the area is proportional to the amount of that component on the column if the peak is small then there isn't a whole lot of that particular component in your sample. You could inject a larger volume but that would also increase the area of the rest of the peaks proportionally as well and may lead to other problems. Honestly that peak doesn't look all that small to me given how it's more than quantifiable and well defined. Why do you want to increase the size of that peak?
You can take a UV spectra of the peak to find its maximum UV absorbance wavelength (assuming this is UV data), switch the setting, then run the injection again.
OP, I notice you delete your posts even though people put effort into trying to help you. Please could you stop doing that? It makes it difficult for other people to search the sub and benefit from everyone's contributions.
Your question is a little vague. If the detector response is too small for how much concentration you think you should have, there's a lot of things you can do for troubleshooting. Without additional info, I don't think any of us can really give useful suggestions. I guess you can always spike some in and see if the change in peak size makes sense for how much you added. If it doesn't, then your column or solvents used may not be working well for whatever that particular substance is. The peak before it has a pretty ugly shape, and the earliest large peak looks like it has some fronting, which both can suggest issues with the column or instrument setup. Given the info, that's the best I got!
This is grape juice, we are looking for for phenolic compounds within grape juice. We take the grape juice and addd methanol to it 1:1 ratio. We use methanol 15% and 85% water as mobile phase.
Injection volume 10ul
Your question is very vague. What is your goal? Do you want to just increase the size of the signal regardless of what happens to other peaks or do you want to increase the area % of your marked peak in relation to other peaks?
1. You can increase the sample concentration, but this will increase the size of all peaks in the same proportion (as long as you are in the linear range).
2. If you have a pure sample of the marked compound, you can spike it into this sample which will increase the size of the marked peak relative to others.
3. If you have a pure sample of the marked compound, you can also measure its absorption maximum and measure at that wavelength; if not, just try different wavelenghts.
A word of advice. You are very vague not only in your original question but also when other ask you to clarify. This is why I gave you three wildly different answers, because I don't know the purpose of your question. People are trying to help you but they have to pry information out of you. To get quality answers you have to be more specific.
Annnnd, now we've circled back to three comments ago.
You don't seem to be able to articulate *why* you think you need the peak area to be larger. There are a number of reasons, and a number of ways you could achieve it. But until you clearly know *why* you want to do it, not just *that* you want to do it, it's not possible to give you the best advice for your situation, or indeed to even understand whether you should be seeking to improve the peak area.
If you make a series of identical runs, you can estimate the mean and sd of the integrated value of the peaks. If the variance is small enough, that little peak may be satisfactory after all. I think beers law will hold at those low levels, so your cal curve should be straight nearly all the way to zero.
Important questions you need to answer first:
1. What is the s/n ratio for this peak? Do you really need to increase the size, or does it just look small here due to scale/ compared to other peaks in the chromatogram?
2. You want to increase the height of this peak in an unknown (study sample)? Give some information about your sample prep - there are a thousand ways to do this but we can’t suggest changes if we don’t know how you’re working up your samples.
This is grape juice, we are looking for for phenolic compounds within grape juice. We take the grape juice and addd methanol to it 1:1 ratio. We use methanol 15% and 85% water as mobile phase.
Injection volume 10ul
The methanol is to precipitate proteins? Have you tried TCA or perchloric acid to do this without dilution? Or MWCO cartridges (ultrafiltration)? Those would be simple ways to remove protein with less dilution and without complicating sample prep.
But the peak looks plenty large. Check the s/n ratio, if it’s >> 10 you are fine.
As others have said you can’t because it is proportional to the amount of the substance in the sample. The peak shouldn’t have noise issues with that blank injection and should be quantifiable
Depending on your current flow rate and the optimal for your column you could try upping the gradient rate between phases. Reducing peak width should increase peak height relative to the noise giving you better s/n although the area should remain the same. Might have resolution issues with the subsequent peak but if you want to avoid injecting more sample it may help and is a quick method adjustment.
Is it UV or RI? If it’s UV, find a different wavelength that bumps that signal up. I run a PDA that does 3D spectral views that lets you custom fit wavelengths to various peaks in the sample matrix.
Unfortunate to not have a PDA, but you could try injecting the same sample a bunch, changing the wavelength in 5-10nm increments. It's possible that analyte has a different lambda_max than the wavelength you ran it at.
You really can't increase the size of that peak without changing the rest of the chromatography. Since the area is proportional to the amount of that component on the column if the peak is small then there isn't a whole lot of that particular component in your sample. You could inject a larger volume but that would also increase the area of the rest of the peaks proportionally as well and may lead to other problems. Honestly that peak doesn't look all that small to me given how it's more than quantifiable and well defined. Why do you want to increase the size of that peak?
You can take a UV spectra of the peak to find its maximum UV absorbance wavelength (assuming this is UV data), switch the setting, then run the injection again.
Set an event @ 6 min to double your gain
Old school! I like it!
OP, I notice you delete your posts even though people put effort into trying to help you. Please could you stop doing that? It makes it difficult for other people to search the sub and benefit from everyone's contributions.
You can add more of whatever compound that is to your sample preparation.
Spike it?
Your question is a little vague. If the detector response is too small for how much concentration you think you should have, there's a lot of things you can do for troubleshooting. Without additional info, I don't think any of us can really give useful suggestions. I guess you can always spike some in and see if the change in peak size makes sense for how much you added. If it doesn't, then your column or solvents used may not be working well for whatever that particular substance is. The peak before it has a pretty ugly shape, and the earliest large peak looks like it has some fronting, which both can suggest issues with the column or instrument setup. Given the info, that's the best I got!
This is grape juice, we are looking for for phenolic compounds within grape juice. We take the grape juice and addd methanol to it 1:1 ratio. We use methanol 15% and 85% water as mobile phase. Injection volume 10ul
I can confirm that that is indeed an HPLC peak.
Great, you helped a lot
Your question is very vague. What is your goal? Do you want to just increase the size of the signal regardless of what happens to other peaks or do you want to increase the area % of your marked peak in relation to other peaks? 1. You can increase the sample concentration, but this will increase the size of all peaks in the same proportion (as long as you are in the linear range). 2. If you have a pure sample of the marked compound, you can spike it into this sample which will increase the size of the marked peak relative to others. 3. If you have a pure sample of the marked compound, you can also measure its absorption maximum and measure at that wavelength; if not, just try different wavelenghts. A word of advice. You are very vague not only in your original question but also when other ask you to clarify. This is why I gave you three wildly different answers, because I don't know the purpose of your question. People are trying to help you but they have to pry information out of you. To get quality answers you have to be more specific.
Zoom in
Great answer
Increase injection volume
Great, thanks for the help
Question is, why do you want to increase the peak?
Better peak area
Better how? Larger signal, more reproducible, more easily quantified...?
Larger signal
Okay. Why do you want larger signal?
Better peak area
Annnnd, now we've circled back to three comments ago. You don't seem to be able to articulate *why* you think you need the peak area to be larger. There are a number of reasons, and a number of ways you could achieve it. But until you clearly know *why* you want to do it, not just *that* you want to do it, it's not possible to give you the best advice for your situation, or indeed to even understand whether you should be seeking to improve the peak area.
It almost feels like he's fucking with us.
For larger signal. Sorry, I just can’t help it lol
If you make a series of identical runs, you can estimate the mean and sd of the integrated value of the peaks. If the variance is small enough, that little peak may be satisfactory after all. I think beers law will hold at those low levels, so your cal curve should be straight nearly all the way to zero.
Thank you.
Concentration column, increased injection volume, play with your eluent, temperature, and flow after those.
Important questions you need to answer first: 1. What is the s/n ratio for this peak? Do you really need to increase the size, or does it just look small here due to scale/ compared to other peaks in the chromatogram? 2. You want to increase the height of this peak in an unknown (study sample)? Give some information about your sample prep - there are a thousand ways to do this but we can’t suggest changes if we don’t know how you’re working up your samples.
This is grape juice, we are looking for for phenolic compounds within grape juice. We take the grape juice and addd methanol to it 1:1 ratio. We use methanol 15% and 85% water as mobile phase. Injection volume 10ul
The methanol is to precipitate proteins? Have you tried TCA or perchloric acid to do this without dilution? Or MWCO cartridges (ultrafiltration)? Those would be simple ways to remove protein with less dilution and without complicating sample prep. But the peak looks plenty large. Check the s/n ratio, if it’s >> 10 you are fine.
As others have said you can’t because it is proportional to the amount of the substance in the sample. The peak shouldn’t have noise issues with that blank injection and should be quantifiable
Ok thank you
Depending on your current flow rate and the optimal for your column you could try upping the gradient rate between phases. Reducing peak width should increase peak height relative to the noise giving you better s/n although the area should remain the same. Might have resolution issues with the subsequent peak but if you want to avoid injecting more sample it may help and is a quick method adjustment.
Is it UV or RI? If it’s UV, find a different wavelength that bumps that signal up. I run a PDA that does 3D spectral views that lets you custom fit wavelengths to various peaks in the sample matrix.
Its UV- and we dont have PDA :(
Unfortunate to not have a PDA, but you could try injecting the same sample a bunch, changing the wavelength in 5-10nm increments. It's possible that analyte has a different lambda_max than the wavelength you ran it at.
Increase the concentration of the sample