Peptides cover a wide range of polarity. If you synthesized them, you will probably have a good idea about when they will elute. Some peptides have a weak UV absorbance because they have amino acids without chromophores; the only absorbance for these will be the amide linkage at ~210 nm

I tend to use water/acetonitrile as a solvent system, both containing 0.1% TFA. If you can use a mass spectrometer with your UPLC, that would be helpful since you can more easily identify the desired peptide.

I would initially run a gradient from 10 to 100% acetonitrile to see approximately where my peptide elutes, then adjust the gradient to improve resolution from impurities.

After your are done with your analysis, wash your column with water/acetonitrile with no TFA as I feel my columns last longer with this wash.

Hi Maghesree,

5% TFA is a good solution to keep the peptides in.  Before you carry out HPLC analysis, you will need to dilute it down to about 0.5-0.1% TFA with MQ water.  Otherwise, the high TFA will cause the peptides not to completely bind to your column.

HPLC solvents are typically prepared as follows:

Buffer A: 0.1% TFA in HPLC grade or MilliQ fileterd water

Buffer B: 90% Acetonitrile in 0.1 % TFA .

You need to equilibrate your HPLC with Buffer A and depending on the column lenght , flow rate and column internal diameter, this will vary.  After equilibration, you can introduce (inject) the peptides, wash off any small Mr additives in the peptide solution by keeping the system in 100% Buffer A.

The peptides are eluted using an organic solvent gradient.  0-50% B over 50 min is a good start.  Then, 50-100% over 20 min will clean and help elute some of the more hydrophobic peptides.  Finally, use 100%B for 10 min to clean the column and go back to 0% B and keep it there for the time you need to equilibrate they column/system

You may need to play with the acetonitrile (B) gradient to resolve or separated closly eluting peptides (eg., try 0-50% Acetonitrile for 30 or 40 minutes)

Best,

Hediye

Peptides cover a wide range of polarity. If you synthesized them, you will probably have a good idea about when they will elute. Some peptides have a weak UV absorbance because they have amino acids without chromophores; the only absorbance for these will be the amide linkage at ~210 nm

I tend to use water/acetonitrile as a solvent system, both containing 0.1% TFA. If you can use a mass spectrometer with your UPLC, that would be helpful since you can more easily identify the desired peptide.

I would initially run a gradient from 10 to 100% acetonitrile to see approximately where my peptide elutes, then adjust the gradient to improve resolution from impurities.

After your are done with your analysis, wash your column with water/acetonitrile with no TFA as I feel my columns last longer with this wash.

Thank you !