Each and every custom synthesis project we work on will include a detailed QC report which is composed of an HPLC and MS analysis.
We have created this guide to assist you in understanding the details contained in each analytical report document.
This will demonstrate the the quality analysis that was performed for the peptide.
Part 1 - The HPLC Analysis Report
Peptide purification is sometimes a long and repetitive process. In most cases multiple purification steps are performed in order to purify and refine the peptide to the desired purity level required for your analytical procedure. Even after repetitive purification steps there will most always remain impurities in the final peptide product, as with most peptides 100% purity is not possible.
Canada Peptide uses RP-HPLC (Reversed Phase High Performance Liquid Chromatography) methods to analyze the peptide purity.
What is Reverse Phase High Performance Liquid Chromatography?
HPLC is a widely used piece of equipment used in analysis. It functions on the principle of efficiently separating the components in both simple and highly complex mixtures. The substance to be analyzed is usually dissolved in water, this is what we call the analyte mixture. In many analyses this solution will also be mixed with an organic solvent or a specific acid in order to obtain a full dissolution of the compound before it is analysed.
This analyte mixture is now ready for analysis by RP-HPLC and will be carried through the equipment by a mobile phase which is made up of water and another solvent or organic modifying solution. This new mixture will then be transported by pumps through an HPLC Column which contains the stationary phase. The stationary phase in most columns consists of an amount of small diameter particles made up of carbon chains with a specified surface length.
As the mixture is pumped through the stationary phase column, the peptide and small impurities (known as the analytes) adsorb to the hydrophobic surface of the column / stationary phase.
The amount of organic modifier or solvent mixture is slowly increased and measured, as this happens the analytes desorb into the mobile phase. Desorption of specific analytes are based on its natural properties, which translates into the retention time. These natural properties will remain in the column only for a certain amount of time.
Many peptide bonds absorb UV light at their maximum capacity when the wavelength of 220 nanometers, therefore with many analyses it is useful to include a UV spectrometer to assist in detecting a peptide as it elutes from the stationary phase / column. This detector will provide a signal which is then converted to a graph (chromatogram) which plots the UV absorbtion and time it took to elute.
The high resolution of eluted peptides and the gradient of elution predominantly depend on the columns and organic modifier or solvent that are chosen. Many different gradients are created in order to obtain a full separation for various peptides so that a high resolution is obtained for each peptide.
Canada Peptide's team has a strong focus on creating HPLC protocols in order to achieve the most effective separation of the analyte and determine accurately the purity of each peptide.
(1) HPLC Mobile Phase & Elution Gradient
(2) Absoprtion Wavelength
(3) Column Size and Type
(4) Dead Zone
(5) Elution Peak of the major components
(6) Peak Table