What HPLC purity measures
An HPLC purity figure is the area of the main peak as a percentage of all peak areas in a chromatogram, usually from reverse-phase HPLC with UV detection near 214 nm. It reports the main peak's share of the integrated UV signal from material that elutes; it does not measure mass, identity or anything absent from the trace.
In reverse-phase HPLC, the peptide in solution passes through a column packed with C18-bonded silica. A gradient from water toward acetonitrile, each solvent holding 0.1% trifluoroacetic acid as an ion-pairing agent, elutes species in order of hydrophobicity, polar ones first, past a UV detector.
The peptide bond itself absorbs near 214 nm, so essentially every peptide gives a signal. Detection at 280 nm sees mainly tryptophan and tyrosine residues, and would miss a peptide that lacks them. The purity figure is then area-percent: the integrated area under the main peak divided by the summed area of all integrated peaks, multiplied by 100.
What the figure includes and leaves out
The figure includes the target and any closely related peptide impurities that separate from it, such as truncated or deletion sequences, incompletely deprotected products and oxidised forms.
It leaves out whatever the detector does not see or the column does not release: water, counter-ions, inorganic salts, and any material that does not absorb at 214 nm or does not elute. A lyophilized powder can therefore read 99% by HPLC and still hold well under 99 g of peptide in every 100 g. That second quantity is net peptide content, covered in Purity versus net peptide content.
Why the method matters
Gradient slope, column chemistry, wavelength and integration settings all move the figure. A shallow gradient can pull apart peaks that a steep one merges. A lower integration threshold counts small impurity peaks that a higher one discards.
A purity number without its method is an adjective. With its method stated in full (column, gradient, wavelength and integration settings), it becomes a record another laboratory can check.
What a chromatogram shows
A trace plots detector signal against retention time. Features, in order of appearance:
- The solvent front: unretained salts and small polar species, normally excluded from the integration.
- Early-eluting peaks ahead of the main peak: more polar impurities, often shorter fragments.
- The main peak: narrow and symmetric, with a flat baseline on both sides.
- Late-eluting peaks after the main peak: more hydrophobic impurities, such as species still carrying protecting groups.
- Shoulders, split tops or tailing on the main peak: possible hidden co-eluting species.
Where Solara's documentation stands
What this does not establish
- Purity by HPLC does not confirm identity, which needs mass spectrometry or another orthogonal method.
- It does not give net peptide content, the mass fraction of the powder that is peptide.
- It says nothing about sterility or endotoxin, which this method does not measure and which Solara does not claim.
- Area-percent assumes every species absorbs equally per unit amount at 214 nm; it is not a molar or mass ratio.
Questions
Why is peptide HPLC run at 214 nm instead of 280 nm?
Can two laboratories get different purity figures for the same vial?
Does a purity of 99% by HPLC mean that 99 g in every 100 g of powder is peptide?
References
- Mant CT et al. HPLC analysis and purification of peptides. Methods Mol Biol, 2007.
- Aguilar MI. HPLC of peptides and proteins: basic theory and methodology. Methods Mol Biol, 2004.
- Kuipers BJ, Gruppen H. Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J Agric Food Chem, 2007.
- D'Hondt M et al. Related impurities in peptide medicines. J Pharm Biomed Anal, 2014.
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.