Working Principle of Molecular Weight Determination in HPLC Analysis

In the field of biopharmaceuticals, peptides, as important bioactive molecules, have a broad application prospect. In order to determine the structure and properties of peptides, accurate determination of their molecular weight has become an important task. High-Performance Liquid Chromatography (HPLC) analysis technology plays a crucial role in peptide analysis with its high resolution, high sensitivity, and wide applicability. This article will detail the working principle of the molecular weight determination method in high-performance liquid chromatography analysis, revealing its key role in peptide identification.

 

The Basic Principle of High-Performance Liquid Chromatography

High-Performance Liquid Chromatography is a liquid-based chromatography technique. The basic principle is to separate samples based on the distribution behavior between the mobile phase and the stationary phase. In High-Performance Liquid Chromatography, the sample solution passes through the chromatography column, which is filled with the stationary phase. Different components are separated due to their hydrophilicity, molecular weight, and chemical properties through the mobile phase flow. This separation process allows peptides to be separated according to their molecular weight.

 

Working Principle of Molecular Weight Determination Method

In High-Performance Liquid Chromatography analysis, commonly used peptide molecular weight determination methods include approximate molecular weight determination and accurate molecular weight determination. The approximate molecular weight determination determines the peptide's molecular weight range by comparison with a reference of known molecular weight, while precise molecular weight determination obtains more accurate results by measuring the exact molecular weight of the peptide.

 

1. Approximate Molecular Weight Determination

The method of approximate molecular weight determination often uses Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). In this method, the peptide sample and the reference pass through the chromatography column for separation. The molecular weight range of the peptide is estimated by measuring the retention time difference between the peptide peak and the reference peak.

 

2. Accurate Molecular Weight Determination

Accurate molecular weight determination methods often use peak shape quantification method and Tandem Mass Spectrometry (MS/MS). The peak shape quantification method calculates the accurate molecular weight of peptides by measuring the retention time and peak shape parameters of the peptide and combining with the calibration curve. In contrast, Tandem Mass Spectrometry combines the high resolution and high sensitivity of the mass spectrometer to obtain accurate molecular weight information by measuring the peptide's mass-to-charge ratio and mass spectrometry fragments.

 

Application and Importance in Peptide Identification

Molecular weight determination is one of the critical steps in peptide identification. Through the molecular weight determination method in High-Performance Liquid Chromatography analysis, the molecular weight of peptides can be quickly and accurately determined, revealing their structure, activity, and other essential information. This is of great significance for peptide synthesis optimization, drug design, and bioactivity research. The application of High-Performance Liquid Chromatography analysis technology in the field of biopharmaceuticals has also been widely recognized, providing strong support for the development and quality control of peptide drugs.

 

The molecular weight determination method in High-Performance Liquid Chromatography analysis plays a vital role in peptide identification with its accuracy and reliability. By understanding the working principle of the molecular weight determination method, we can better understand the application of High-Performance Liquid Chromatography technology in peptide analysis and provide strong support for research and application in the field of biopharmaceuticals.