Principle of HILIC-UHPLC in N-Glycosidic Bonds Analysis

With the deepening of research into glycoscience, the analysis of N-glycans has garnered increasing attention in the fields of biomedicine, drug development, and biotechnology. N-glycans, as modifications of glycan chains, are widely present in various proteins and lipids, and changes in their structure are closely related to multiple diseases, such as cancer and diabetes. Therefore, developing efficient analytical methods is crucial for uncovering their biological functions and related mechanisms.

 

Principle of HILIC

Hydrophilic interaction chromatography (HILIC) is a chromatographic technique used for separating hydrophilic and polar compounds, primarily based on the interactions between aqueous and organic phases. In HILIC, the stationary phase is typically a material containing hydrophilic groups, such as amino, carboxyl, or other polar groups. Polar molecules in the sample are separated through interactions with the stationary phase (such as hydrogen bonds, electrostatic interactions, etc.) and the organic solvents in the mobile phase.

 

HILIC is particularly suitable for analyzing carbohydrates because sugar molecules exhibit high polarity and hydrophilicity. By controlling the composition and flow rate of the mobile phase, effective separation of different N-glycan structures can be achieved.

 

Principle of UHPLC

Ultra-high-performance liquid chromatography (UHPLC) is an improved liquid chromatography technique that enhances separation efficiency by using smaller diameter chromatographic packing particles and higher system pressures. The main advantages of UHPLC include shorter separation times, higher resolution, and greater sensitivity, making it especially suitable for analyzing target compounds in complex samples.

 

In the analysis of N-glycans, UHPLC combined with HILIC technology allows for rapid and efficient separation and quantification of N-glycans. By optimizing separation conditions, such as the pH, composition, and flow rate of the mobile phase, the reproducibility and accuracy of the analytical results can be significantly improved.

 

Application of HILIC-UHPLC in N-Glycosidic Bond Analysis

In HILIC-UHPLC analysis, the detection and quantification of N-glycosidic bonds typically rely on identifying the characteristic signals of glycosidic chains. This process can be divided into several key steps:

 

1. Sample Preparation

First, N-glycans are extracted and purified from biological samples, often requiring enzyme digestion and solid-phase extraction to remove interfering substances. The extracted N-glycans may need to be appropriately derivatized to enhance their separation efficiency and detection sensitivity in HILIC.

 

2. Separation Process

The samples are then separated through the HILIC-UHPLC system. During this process, polar N-glycan molecules interact with the stationary phase, while the mobile phase assists in eluting the target compounds being analyzed. By adjusting the proportions and gradients of the mobile phase, effective separation of different N-glycan chains can be achieved.

 

3. Detection and Quantification

The separated N-glycans are detected using mass spectrometry or other detectors (such as fluorescence detectors). Mass spectrometry provides molecular weight and structural information, allowing for qualitative and quantitative analysis of N-glycosidic bond compositions and structures.

 

4. Data Processing

Analytical results must be processed and interpreted using specialized software. Typically, standard curves are established to facilitate quantitative analysis. Simultaneously, known N-glycan structures from databases and literature are compared to determine the components of unknown samples.

 

HILIC-UHPLC serves as a powerful tool for researching N-glycosidic bonds. By deeply understanding the separation principles and optimizing analytical conditions, we can better elucidate the functions of N-glycans in biological systems and their relationships with diseases.