Mechanism of HILIC-UHPLC in N-Glycosidic Bond Detection

In biological molecules, carbohydrates play a crucial role as important modifying groups in the functions and structures of proteins. N-glycosidic bonds refer to the connection of carbohydrates to the nitrogen atom of proteins or other biomolecules through glycosidic bonds. Accurate detection and analysis of N-glycosidic bonds is essential for studying glycobiology, glycomics, and related diseases such as diabetes and cancer. Hydrophilic interaction liquid chromatography-ultra-high performance liquid chromatography (HILIC-UHPLC) is an effective separation technique widely used for the detection of N-glycosidic bonds.

 

Basic Principles of HILIC

Hydrophilic interaction liquid chromatography (HILIC) is a chromatographic technique based on hydrophilic interactions. In HILIC, the stationary phase is typically made of hydrophilic materials, while the mobile phase consists mainly of a mixture of organic solvents and a small amount of water. The key to this technique lies in the thin layer of water molecules formed on the surface of the stationary phase, which provides hydrogen bonding interactions that allow effective separation of polar compounds in the samples.

 

In HILIC, the separation of N-glycans mainly relies on the following mechanisms:

1. Hydrogen Bonding Interactions

The hydroxyl groups in N-glycans can form hydrogen bonds with water molecules on the surface of the stationary phase, influencing their retention time.

 

2. Polarity Differences

The differences in polarity among various N-glycans lead to different distributions in the stationary and mobile phases, thus enabling their separation.

 

3. Hydrophobic Interactions

Increasing the proportion of organic solvents in the mobile phase promotes the elution of hydrophobic molecules, allowing the stronger hydrophilic N-glycans to be captured and separated more effectively.

 

Advantages of UHPLC

Ultra-high performance liquid chromatography (UHPLC) is a technique that accelerates the separation process by increasing pressure. Compared to traditional high-performance liquid chromatography (HPLC), UHPLC offers higher separation efficiency and shorter analysis times. The core advantages of UHPLC include:

 

1. Smaller Particle Size of Stationary Phase

UHPLC uses smaller diameter stationary phase particles, providing a larger surface area and thus enhancing separation efficiency.

 

2. Higher Resolution

The optimization of flow rate and separation time in UHPLC allows for higher resolution, especially when analyzing complex samples.

 

3. Rapid Analysis

The fast separation capability of UHPLC enables the acquisition of large amounts of data in a short time, making it suitable for high-throughput screening and analysis.

 

Mechanism of HILIC-UHPLC Combination

The combination of HILIC and UHPLC provides robust technical support for the detection of N-glycosidic bonds. In this process, the samples are first separated through HILIC, followed by the use of UHPLC to further enhance separation efficiency. The specific process of this combination mechanism is as follows:

 

1. Sample Preparation

Before HILIC-UHPLC analysis, samples must undergo appropriate processing, including desalting, concentration, and derivatization, to enhance analytical sensitivity and accuracy.

 

2. Separation Process

(1) Sample Loading

The pre-treated samples are loaded onto the HILIC column. At this stage, the hydrophilic interactions and polarity differences between N-glycans and the stationary phase will determine their retention times.

 

(2) Separation and Elution

As the mobile phase continuously elutes, N-glycans will separate within the column. Changes in the organic solvent ratio in the mobile phase will affect the elution order of N-glycans, achieving separation of different N-glycans.

 

3. Detection and Analysis

The separated samples enter the UHPLC system, where higher efficiency separation and rapid analysis are achieved through high pressure and small particle stationary phases. Combined with highly sensitive detectors (such as mass spectrometers), precise measurement of N-glycan types, structures, and relative abundances can be performed.

 

HILIC-UHPLC provides an efficient and sensitive analytical method for the detection of N-glycosidic bonds. The combination of hydrophilic interaction and ultra-high performance liquid chromatography enables researchers to better understand the biological functions of N-glycans and their roles in diseases. The application of this technique not only promotes the development of glycomics but also provides important tools for biomedical research.