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I began working in a newly setup lab, and I'm having a dilysine (K2) peptide synthesized by solid phase method. I plan to cleave the peptides from the resin with a cleavage cocktail of TFA. Unfortunately, our freeze-drier is missing a part and not working then. We also don't have a rotary evaporator yet. We have our HPLC setup, but do you think it is a good idea to take the resulting solution of cleavage (after filtration to remove beads), which contains Boc, Fmoc, TFA, and possibly other impurities, directly to the HPLC?

I've read that HPLC sample should be dissolved in initial composition of my HPLC solvents, but it is in the case of having a crude dry sample.

It is my first time doing a peptide synthesis so I appreciate your ideas.

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If you are a using reversed-phase (RP) column, it is okay to directly inject your crude sample in the running HPLC solvent system. But, make sure to filter the injecting sample through a membrane filter to remove minute particles (from glass beats, etc.) from the original sample after dissolving it in suitable chosen solvent. Please also note that your chosen solvent for peptide separation usually contain small percentage of TFA. Therefore, you may need at least a rotary evaporator for final concentration.

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  • $\begingroup$ The "dissolving" part is a bit confusing to me because I already have a cleaved peptide + TFA solution, is dissolving just the same as mixing with water? (since my initial solvent composition is going to be water, linearly increasing acetonitrile over time). $\endgroup$ – Mo Samani May 15 at 5:13
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    $\begingroup$ I believe you don't have dry sample. Usually you dissolve your dry sample in HPLC mobile phase you supposed to use. In this case I'd add enough water to dilute TFA if needed. Sample should be clear solution (do not let it precipitate when adding water). Since you are using RP, using water to inject did not effect the separation much (volume should be small as possible). $\endgroup$ – Mathew Mahindaratne May 15 at 15:46

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