Not enough details are provided.

1. The C18 grafting density could be different.

2. TFA helps more in retention if several amino groups exist on the peptide.

3. "0–30% ACN or a 2–30% ACN gradient over 12 minutes", which condition was used for the analytical column?

I agree with Omar Gonzalez-Ortega. And I assume loading solvents for the peptide are the same for both columns.

1. I'm concerned about the differences between C18 grafting on your analytical versus prep columns.

2. If you don't mind sharing your AA content, it would help us advise you. Basic and acidic residues behave differently on RPHPLC, especially with different stationary phase end-capping.

In my experience, sometimes extremely hydrophilic peptides (especially small ones) need to be desalted for prep RPHPLC. You may be able to do that on a simple spin column in 1% HOAc, and then dry down.

I assume that you have injected your peptide dissolved in a strong solvent and not in water or a highly aqueous solvent mixture. In the case that you have equilibrated your column with 0 % ACN, phase collapse may also occur if you have not used a column specifically designed for this purpose. In this case, no compound will show retention. These columns often have the designation Aqua or Hydro in their name.

Thank you very much, Dr. Michael G. Weller, for your insights.

In my case, the peptides are fully water-soluble and were dissolved in 100% water—no organic solvent was used for injection. The starting mobile phase was also 0% ACN, so the injection conditions matched the initial gradient.

However, I hadn’t considered the possibility of phase collapse. The preparative column I used (Agilent Prep 100Å C18, 24% carbon load, endcapped) does not have an “Aqua” or “Hydro” designation. Could phase collapse still occur even though the column is equilibrated in 0% ACN and the sample is injected in water? If so, would using a more polar-compatible C18 phase (e.g., with polar-embedded groups or specifically designed for aqueous conditions) be advisable?

Appreciate your thoughts on this.

Thank you, Dr. John Carter, for your valuable input.

Yes, the loading solvent was 100% water for both analytical and prep columns, matching the starting mobile phase (0% ACN), so injection conditions were consistent.

You're right—the analytical column (9% carbon load, double endcapped) differs significantly from the prep column (24% carbon load, endcapped), which may affect retention of highly polar peptides like mine.

The peptide is ~2.5 kDa, highly hydrophilic (~10% hydrophobicity), and rich in charged/polar residues. I can share the sequence if helpful.

Thanks also for the desalting tip using a spin column in 1% HOAc—I’ll try that before the prep run to see if it improves retention.

Most peptide separations are performed in acidic conditions, with addition of TFA or formic acid to the mobile phase and the samples. This also influences the retention of the analytes.

You may use your column, if you need this separation not too frequently. You would have to equibrate the column with a high concentration of ACN, e.g. 80%, switch to 0% ACN until the baseline is stable, and then inject the sample. If after the run, an analytical amount of a standard peptide is still retained properly, then everything went well. It cannot be predicted, when the phase collaps will occur. However, you can repeat the regeneration as often as you like.

In water analysis, a similar approach is used for the extraction of water with C18 cartridges. Here, this step is known as "conditioning".

The higher carbon load could significantly reduce retention if there is no endcapping on the analytical column, especially your peptide is basic. This is because the peptide interacts with the silica substrate in addition to the carbon grafting. As Michael G. Weller suggested, you probably should try your analytical separation on the prep column.

Again, if you can share your peptide AA content (preferably sequence) we can better understand this challenge. Do you have access to counter-current distribution liquid phase chromatography?

Phase collapse might indeed be the cause of no retention. Use proper degassing of the eluent (especially the water): He degassing in combination with He pressure in the eluent bottles. Or in line degassing of sufficient capacity for preparative separation (higher flow rates!). Start with 100% ACN to reverse the phase collapse and use subsequently a shallow gradient to your initial condions (preferably with 2% ACN or somewhat more).