Need Help ! HPLC detection

[steve123]

Hi,
I am using HPLC gradient mobile phase(ACN:water::0-100:100-0) for 35 minute run for my new peptide sample. I am getting peaks at nearly first 3-5 minutes. Unsure, the parameters are correct or not. Also, 1micro molar concentration is not detecting. Could anyone please guide me.

Thanks in Advance.

[B.Biwi]

You want that the peaks are more spread?, then i would recommend to make the gradient more flat...for example starting 20:80 and going up to 80:20 ACN:Water in 30 minutes, then the last 5 minutes 100 % ACN. Maybe this helps.

Do you usually detect 1 micro molar of your peptide?, or is this your first try and you have no experience?

Regards,
Biwi

[Kreall]

Hello,
In order to find solution to your problem you need to give people here more details about you method.
Please write what column do you use, specify your sample - is it long short peptide?; Is it more polar or non polar? What detector your are using (I assume you use UV/DAD detector, so if you have problem with limit of detection please write what wavelenght you use). Is you gradient linear for whole 35 min, or is it have different slopes?

From what you wrote I can only advise to not use gradient 0-100% ACN, even if the other component is water. Better option would be propably to use 5-95% ACN gradient.

[steve123]

Hi Folks,

Sorry for incomplete details.

Sample is a short 12 amino acid cyclic peptide. It is polar. Regarding detector, yes, its UV detector. I am using wavelength of 215nm. Currently I am following this parameter:
0-5 min- 15
5-10 min- 15
10-15 min- 30
15-20 min-50
20-23 min- 50
23-28 min-100
28-32 min-100
32-35 min- 50

Furthermore, I am getting good peak at 3-4 minutes. However, my concern is limit of detection, as I have to do stability studies for that I need in nano molar range. And I am not getting peak at 1 microM.

Waiting for reply.

Thank you.

regards,
Steve

[Kreall]

Hey,
You wrote in your first post that your peaks comes in 3-5 min, so why you are doing a 30 min gradient?

If you want to raise sensitivity there is some things you can do:
1. Change the wavelength - to be exact lowering the wavelength. As you wrote you have gradient water:ACN, ACN have cut off about 190 nm so beside more steeper base line of gradient or little more noise there should be no other disadvantages.
2. Changing the particle size in your column, thus making the peak of for peptide more narrow so making the LOD much lower. Go for column < 3um, if you dont have UPLC try using a core shell particle columns like Kinetex.
3. Trying to use derivatisation on your peptide. You need to check if there is possibility of derivatiosation reaction with free amino groups in for peptide. If there is multiple amino gruops that can undergo derivatisation process, there is always a question would they all react?. This would be the hard solution, there is much more work need to be done. But this is an other option you could consider.

[steve123]

Hi,

Peak is coming at 3-5 minute. However, peaks are coming adjacent to the negative injection peak. How can I change that..

[tom jupille]

Start at a lower %B. Your original post implied you were starting at 0%, but the more detailed profile you gave started at 15%.

Are you following an existing method or starting de novo? If you are developing your own method it's best to begin with a linear gradient from 0% (or 5% if you're using a C18 column) up to high enough to elute everything from the column (for peptides, generally 60% or so will be adequate). 1%/minute is typical. Depending on what you see, you can then tweak the gradient profile as necessary.