Purity versus net peptide content
A figure of 99% by HPLC describes the peptide fraction of a powder, not the powder itself. A lyophilized peptide powder also holds counter-ions, bound water and sometimes inorganic salts, so peptide is commonly reported as 70 to 90% by weight, and net peptide content is a separate measurement.
Reverse-phase HPLC reports the share of detected peptide-related material that is the target sequence, usually as a percentage of peak area at a stated wavelength such as 214 nm. Water, salts and counter-ions fall outside that ratio.
What else is in the powder
A lyophilized peptide with basic residues is usually a salt rather than a pure free base. The common non-peptide components are:
- Trifluoroacetate (TFA, 113 g/mol), carried over from purification by reverse-phase HPLC. Up to one is bound at each basic site: the side chains of lysine, arginine and histidine, and the free N-terminus.
- Acetate (59 g/mol), present when an exchange step has replaced trifluoroacetate. Exchange to chloride is also described in the literature.
- Residual water. Lyophilized cakes are hygroscopic and take up more in humid air.
- Inorganic salts, in some preparations.
A worked example
An illustration with assumed figures: a 10 mg powder of a 2,000 g/mol peptide with four basic sites, as a TFA salt holding 5% water by weight.
| Component | Assumed basis | Mass in 10 mg of powder |
|---|---|---|
| Peptide | 2,000 g/mol (free base) | 7.7 mg (77%) |
| Trifluoroacetate | 4 per peptide, 456 g/mol with protons | 1.8 mg (18%) |
| Water | 5% of powder weight | 0.5 mg (5%) |
| Total | All components | 10.0 mg |
Net peptide content here is about 77%, while a chromatogram of the same powder could still read 99% by HPLC.
How net peptide content is measured
Three methods are common, each with limits.
- Amino acid analysis after acid hydrolysis quantifies the amino acids released from a weighed sample. Hydrolysis in hydrochloric acid at about 110 °C destroys tryptophan and converts asparagine and glutamine to their acids, so recovery depends on the sequence.
- Elemental nitrogen analysis compares measured nitrogen with the amount the sequence predicts. Ammonium salts or nitrogen-containing solvents inflate the result.
- UV absorbance at 280 nm uses an extinction coefficient calculated from tryptophan, tyrosine and cystine content, following Pace and colleagues. It needs those residues, and scattering from aggregates distorts the reading.
Why both numbers matter
Purity states how clean the peptide fraction is. Net peptide content states how much peptide a weighed mass contains. Molar calculations in a laboratory depend on the second.
Solara vials are lyophilized powders labelled with the compound name, the quantity in milligrams, the form, an expiry date and a research-use-only statement. Solara does not publish net peptide content or counter-ion content per lot, and has not published whether the labelled milligram figure is a gross powder weight or a net peptide weight.
What this does not establish
- This article does not state what the milligram quantity on a Solara vial label refers to, because that has not been published.
- A high net peptide content does not imply high purity, or the reverse; the two are measured independently.
- Counter-ion identity is a specification item; this article compares the two salt forms by mass and composition only.
Questions
Does a purity of 99% by HPLC mean the peptide is 99% by weight of the powder?
What is a TFA salt?
How is net peptide content measured?
References
- Roux S et al. Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches. J Pept Sci, 2008.
- Rutherfurd SM, Gilani GS. Amino acid analysis. Curr Protoc Protein Sci, 2009.
- Pace CN et al. How to measure and predict the molar absorption coefficient of a protein. Protein Sci, 1995.
- Andrushchenko VV et al. Optimization of the hydrochloric acid concentration used for trifluoroacetate removal from synthetic peptides. J Pept Sci, 2007.
Every compound on this site is supplied as a lyophilized powder for laboratory research use only. Nothing in this article describes an effect on a person or is a claim about any product.