The high performance liquid chromatography is a user‑centric intelligent analytical instrument. It not only possesses all the basic capabilities of a conventional HPLC but is also compatible with both reverse‑controlled liquid chromatography workstations and general‑purpose chromatography workstations. Its novel appearance, combined with higher reliability, stability, and ease of operation, makes it an ideal choice for laboratory analysis.
A high performance liquid chromatography mainly consists of five core parts: the injection system, the fluid delivery system, the separation system, the detection system, and the data processing system. Below, we will explain the functions and characteristics of these five systems in detail.
I. Injection System
The injection system typically uses a septum syringe or a high‑pressure injection valve to introduce the sample, ensuring a high degree of consistency in injection volume. This constant injection method significantly improves the repeatability and accuracy of analytical results, which is particularly important for experiments requiring high‑precision data.
II. Fluid Delivery System
The fluid delivery system comprises a high‑pressure pump, a mobile phase reservoir, and a gradient device. The working pressure range of the high‑pressure pump is typically 1.47‑4.4×10⁷ Pa, delivering an adjustable and stable flow rate. The high‑pressure environment effectively reduces sample diffusion in the column while accelerating sample migration through it. This not only helps improve separation resolution but also better preserves the biological activity and recovery efficiency of the sample. The combined use of the mobile phase reservoir and the gradient device allows the mobile phase to be adjusted according to the properties of the stationary phase and the sample, including changes in eluent polarity, ionic strength, pH, and other parameters.
III. Separation System
The separation system includes key components such as the chromatographic column, connecting tubing, and a thermostat. Columns are typically 10‑50 cm in length (multiple columns can be connected in series using connecting tubing if needed), with an internal diameter of 2‑5 mm, and are made of high‑quality stainless steel, thick‑walled glass tubing, or titanium alloy.
The column is packed with a stationary phase of 5‑10 μm particles, consisting of a matrix and a stationary liquid. The stationary phase matrix is usually made of resin or silica gel with high mechanical strength, characterised by good inertness, high porosity, and large specific surface area. The surface of these matrices can be coated or chemically coupled to attach functional groups such as phosphate, quaternary ammonium, hydroxymethyl, phenyl, amino, or alkyl chains of different lengths.
This refined stationary phase design offers good selectivity for substances with different structures. For example, after coupling pea lectin (PSA) to the surface of porous silica gel, it can specifically separate certain glycoproteins from fibroblasts.
The small particle size of the stationary phase allows the packed bed to be highly uniform and dense, effectively reducing eddy diffusion. At the same time, shallow pores and short mass transfer paths help narrow the spectral band width and improve separation resolution. According to plate theory, the smaller the matrix particle size, the larger the theoretical plate number N, and the higher the separation efficiency. The thermostat can adjust the temperature from room temperature up to 60 °C, shortening analysis time by improving mass transfer speed and further enhancing the separation efficiency of the column.
IV. Detection System
Three types of detectors are commonly used in high performance liquid chromatography:
UV Detector
Suitable for detecting samples that absorb UV or visible light. Its advantages include a wide range of applications (can be used for proteins, nucleic acids, amino acids, nucleotides, peptides, hormones, and many other substances), high sensitivity (detection limit down to 10⁻¹⁰ g/mL), a wide linear range, and insensitivity to temperature and flow rate changes, making it suitable for gradient elution.
Refractive Index Detector
Suitable for detecting any sample component whose refractive index differs from that of the mobile phase; it is especially commonly used for carbohydrate analysis. This detector is versatile and easy to operate, but its sensitivity is relatively low (detection limit around 10⁻⁷ g/mL), and it is sensitive to changes in the mobile phase, so it is not suitable for trace analysis or gradient elution.
Fluorescence Detector
Specifically suitable for detecting organic compounds that exhibit fluorescence, such as polycyclic aromatic hydrocarbons, amino acids, amines, vitamins, and certain proteins. Its greatest advantage is extremely high sensitivity (detection limit as low as 10⁻¹² g/mL to 10⁻¹⁴ g/mL), making it suitable for both trace analysis and gradient elution.
V. Data Processing System
The data processing system is responsible for acquiring, storing, displaying, printing, and further processing the test data, ensuring that sample separation, preparation, or identification work can be carried out accurately and efficiently. Modern data processing systems are typically equipped with intelligent chromatography workstations, offering user‑friendly interfaces and powerful data analysis capabilities, greatly enhancing the convenience of experimental work and the reliability of results.
Through the coordinated work of these five major systems, the high performance liquid chromatography provides reliable and accurate analytical results for the laboratory, meeting a wide variety of complex analytical needs.
