Peptides have a range of properties which the following guidelines may not address. If you require more information concerning a specific peptide, please call our technical staff at 800.227.0627 or contact us here.
Use only a small amount of peptide to test for solubility. Only when the peptide has been fully dissolved should be the buffer salts be added and the solution diluted to its final concentration.
NOTE: Always add the peptide into a stirring solution of appropriate solvents, not the other way around.
- The main problem associated with the dissolution of a peptide is the formation of aggregated secondary structures. Although secondary structures are more pronounced with hydrophobic peptides, they are liable to occur with all except the shortest peptides, regardless of polarity. Therefore, the first rule is to try to dissolve the peptide in sterile, distilled or deionized (and, if possible, oxygen-free) water.
- Bacterial degradation can be a problem with peptide solutions. To avoid this, peptides should be dissolve in sterile water or have the peptide solution sterile filtered using a 0.45 cm micron or 0.2 micron filter.
- Peptides containing cysteine, methionine, or tryptophan are particularly susceptible to oxidation and should be dissolved in oxygen free water. This can be prepared by degassing under reduced pressure and replacing with an inert gas such as nitrogen, helium or argon.
If the peptide is insoluble in pure water, sonication may help break up any particles and increase the rate of dissolution.
CAUTION: Sonication can cause warming of the solution and degradation of the peptide.
- If the peptide contains many basic amino acids, use an aqueous acetic acid (1 to 10%) solution, with or without sonication. For every hydrophobic peptides, use a 50% aqueous acetic acid.
- If the peptide has many acidic amino acids, use an aqueous ammonia (1 to 10%) solution, or a volatile basic buffer (up to pH8) such as N-ethylmorpholine acetate or bicarbonate with or without sonication. The pH may have to be adjusted before chromatography.
- Propanol and acetonitrile can dissolved some medium-size peptides. If the peptide is to be injected onto a solumn, the amount of organic solvent, especially propanol, must be kept small, or retention time will be greatly affected.
- If the peptide is highly hydrophobic with aromatic or hydrocarbon-like side chains, such as Val, Leu, Ile, Met, Phe, Tyr, Ala, or if the peptide is neutral, use a chaotropic agent such as DMF or DMSO.
- High concentrations of chaotropic salts help to dissolve the peptide by breaking up the secondary structures.
- Chaotropic agents are suitable for preparing solutions for analysis, but may interfere with a biological system used for the study of the peptide.
- The best agent is DMF (up to 30%), added drop by drop, until the peptide dissolves.
- On reverse-phase chromatography, the DMF will elute with the buffer front. The UV absorbance can be very high, depending on howmuch is injected. Most peptides are retained longer than the few minutes it takes for DMF to elute. If the peptide is very small and elutes early, the introduction of the organic solvent component of the mobile phase should be delayed following injection.