How to remove TFA from synthetic peptides using HCl? | LifeTein Peptide Blog

Purified peptides must be free of Trifluoroacetate (TFA) because TFA could alter the results of downstream biological assays.

Synthetic peptides are manufactured using solid-phase procedures. TFA is usually used for cleavage and purification steps. TFA binds to the free amino termini and side chains of positively charged amino acids. The TFA counterions could change the secondary structure, mass, and solubility of peptides or the results of in vivo studies.

All LifeTein peptides are lyophilized to easily remove excess and unbound TFA. However, HPLC and salt exchange are required to remove the TFA counterions that are binding to the positively charged peptide residues.

The most adapted method is to replace TFA counterions with a stronger acid such as hydrochloric acid (HCl).

How to remove TFA from synthetic peptides using HCl?

Dissolve the peptide in distilled water at 1 mg (weight) per 1 mL of solvent. Phosphate buffer (50mM phosphate and 100mM NaCl) can be used instead of water.

Add 100 mM HCl to the peptide solution for a final HCl concentration between 2 mM and 10 mM. HCl concentration below 2 mM or higher than 10 mM may result in incomplete TFA exchange or modified peptides.

Allow the solution to stand at room temperature for at least a minute.

Freeze the solution at -20,-80, or preferably in liquid nitrogen.

Lyophilize overnight to remove all liquid.

Re-dissolve the lyophilized powder in an HCl solution.

Freeze the solution again and then lyophilize overnight.

Repeat steps 6 to 7 at least two times.

After the final lyophilization step, re-dissolve the peptide in water or your desired buffer at around 2 mg (weight) per 1 mL of solvent.