How does HPLC work?
In HPLC, a small volume of sample is injected into a flowing liquid, called the mobile phase. It takes the sample through a special column packed with a material called the stationary phase.
The stationary face is interacted with to varying degrees by the different components. Some go through the column faster, some slower. This causes the components to separate out as they go through the system.
Retention time of a substance is the time it takes to travel on the column. A chromatogram is prepared which displays peaks and is used to identify the compounds eluting from the column.
Purity of peptide and HPLC
HPLC is suitable and is often used to determine the purity of peptides. Looking at the chromatogram, the major peak is the compound of interest. The other peaks are impurities or other compounds.
The percentage purity can be estimated from the relative areas of the detected peaks. For example, a declared purity of 98% generally indicates that the major component measured by that specific HPLC method is approximately 98% of the material detected.
However, an HPLC purity result is dependent on the column, mobile phase, detection conditions and other method parameters. Different methods may give different results for the same sample.
What is not confirmed by HPLC?
HPLC can give useful information on the composition of a sample, but it cannot answer every quality question. One major peak on a chromatogram does not establish the complete identity or molecular makeup of a peptide by itself.
HPLC can be used in conjunction with other analytical techniques. Mass spectrometry (MS) can be used to confirm molecular mass and to support peptide identity. Other techniques can be used to look at particular features of the sample.
This is why HPLC results are often considered together with information such as molecular weight, peptide sequence and Certificate of Analysis (CoA).
HPLC in a Certificate of Analysis
A peptide’s Certificate of Analysis may include an HPLC purity result for a particular batch. When reviewing this information, it is useful to check the reported percentage along with the test method and other available analytical results.
HPLC is therefore best understood as one part of peptide quality assessment. It provides a practical way to separate and examine the components of a sample, making it particularly useful for monitoring purity and related substances in research materials.
Related terms: High-Performance Liquid Chromatography · Chromatogram · Retention Time · Purity · Impurity · Mass Spectrometry · Molecular Weight · Certificate of Analysis
All products supplied by Prime Peptides UK are intended for laboratory research use only and are not for human consumption.
Research Use Only. This glossary explains terminology used in published research. It is not medical advice and does not describe any product for human or animal use.