How is the purity of peptides calculated?

The purity of peptides is typically determined through various analytical techniques that measure the relative amounts of the target peptide and any impurities or related substances present in a peptide sample. The most common methods for assessing the purity of peptides include:

1. High-Performance Liquid Chromatography (HPLC):

   • Reverse-phase HPLC is one of the most widely used methods for peptide purity analysis. It separates peptides based on their hydrophobicity. The area under the main peak corresponding to the target peptide is divided by the total area under all peaks in the chromatogram (target peak area/total area). The result is expressed as a percentage, indicating the purity of the target peptide.

2. Mass Spectrometry (MS):

   • Mass spectrometry can confirm the identity of the peptide and provide information about its molecular weight and the presence of any impurities. By comparing the intensity of the main peptide peak with the combined intensity of all peaks in the mass spectrum, the purity can be calculated.

3. Ultraviolet (UV) Spectroscopy:

   • UV spectroscopy measures the absorbance of a peptide sample at a specific wavelength. If the peptide has a known absorbance maximum and impurities do not absorb significantly at that wavelength, you can calculate purity by comparing the measured absorbance with the theoretical absorbance for a pure peptide sample.

4. Nuclear Magnetic Resonance (NMR) Spectroscopy:

   • NMR can be used for peptide purity analysis by observing the peaks in the NMR spectrum. Each proton in the peptide contributes to a peak, and the integrals of these peaks can be used to determine the relative amounts of different components in the sample.

5. Thin-Layer Chromatography (TLC):

   • TLC can be used to provide a qualitative assessment of peptide purity. By visualizing spots corresponding to the peptide and any impurities on a TLC plate, you can estimate the relative amounts of each component.

6. Capillary Electrophoresis (CE):

   • CE is another separation technique that can be used to assess the purity of peptides by analyzing the migration times of different components in an electric field.

The choice of method for assessing peptide purity depends on the specific needs of the analysis, the available equipment, and the characteristics of the peptide sample. Combining multiple techniques, such as HPLC and MS, can provide a more comprehensive evaluation of peptide purity.

To calculate the percentage purity, you typically divide the area or intensity of the main peptide peak by the total area or intensity of all peaks in the chromatogram, spectrum, or electropherogram, and then multiply by 100 to express the result as a percentage. The purity can be expressed as a single value or a range, depending on the precision of the analysis.

KS-V peptide is obtained by calculating the purity of the peptide by measuring the ultraviolet absorption value of the peptide bond in the target at a specific absorption wavelength by reversed-phase high-performance liquid chromatography (RP-HPLC), and is separated and purified by gradient elution with water and acetonitrile . Among them, moisture and residual salt cannot be measured by UV detector, and there are other impurities in the test results, including incomplete sequence peptides (short peptides with one or more amino acid residues less than the target peptide), truncated sequences due to preventing Generation of incomplete sequence peptides (polypeptides produced by capping), incompletely deprotected peptides (polypeptides produced during synthesis or final cleavage).