A good explanation of the principles of HPLC

The basic principle is the tray theory, which is explained in detail in many places.

An HPLC instrument generally consists of a solvent delivery system, a sampling system, a separation system (column), a detection system, and a data processing and recording system.

1. Solvent delivery system.

Reservoir: Used to store a sufficient amount of mobile phase that meets the requirements. Equipped with a solvent filter to prevent particles from the mobile phase from entering the pump.

Degasser: The purpose of degassing is to prevent the mobile phase from releasing air bubbles into the detector when it flows out of the column, which can cause noise and fail to detect normally.

Infusion pump: The mobile phase in the reservoir is continuously fed into the liquid system at high pressure, allowing the sample to complete the separation process in the column.

Gradient elution device: It is a device that increases the elution capacity by gradually changing the composition of the mobile phase during the separation process.

2. Sampling system.

Injector: It is a device that feeds the sample into the chromatographic column, and the injection method can be divided into two types: valve injection or automatic injection. Autosampler loading is more common.

3. Separation system.

Column: The lead separation of the sample is the heart of the entire chromatography system, and its quality directly affects the separation effect.

4. Detection system.

Detector: Converts the sample components that flow continuously out of the column into an easy-to-measure electrical signal, which is received by the data system and results in a chromatogram of the sample separation.

5. Data processing and recording system.

Chromatographic data is processed and involved in the automatic control of HPLC instruments.

Principle: The mobile phase in the reservoir is injected into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector, and is loaded into the chromatographic column by the mobile phase (solid phase).

Separated into individual components that sequentially flow out of the column, the sample concentration is converted into an electrical signal and sent to the recorder as it passes through the detector, where the data can be printed out as a map for analysis by the researcher.

HPLC stands for High Performance Liquid Chromatography, and the full name in English is High Performance Liquid Chromatography. This method is used as an important separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commodity inspection and forensic inspection.

Uses: High performance liquid chromatography is more suitable for the separation and analysis of substances with high boiling point, poor thermal stability, physiological activity and relatively large relative molecular weight, so it is widely used in the analysis of nucleic acids, peptides, lactones, fused cyclic aromatic hydrocarbons, polymers, drugs, human metabolites, surfactants, antioxidants, pesticides, herbicides and other substances.

Principle: High performance liquid chromatography uses liquid as the mobile phase, adopts a high-pressure infusion system, pumps a single solvent with different polarities or mixed solvents, buffers and other mobile phases with different polarities into the chromatographic column equipped with stationary phase, and after the components in the column are separated, they enter the detector for detection, so as to realize the analysis and separation of the sample.

Operation method: As shown in the figure below, the mobile phase in the solvent receptacle is sucked in by the pump, mixed and then output by the gradient controller according to a certain gradient, the pressure and flow rate are measured, and the injection valve (device) is introduced into the detector after the protection column and the separation column are tested, the data is processed by the data processing equipment or the chromatogram is recorded by the recorder, the fraction collector collects the fraction, and the waste bottle collects the waste liquid.

HPLC isHigh performance liquid chromatography(High Performance Liquid Chromatography).

High performance liquid chromatography is an important branch of chromatography, using liquid as the mobile phase, using high-pressure infusion system to rent Qiaozhou system, with different polarity of a single solvent or different proportions of mixed solvents, buffers.

The isomobile phase is pumped into the column with the stationary phase attached.

After the components in the column are separated, they enter the detector for detection, so that the sample can be analyzed. This method has become an important application of separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commodity inspection and forensic inspection.

Features of HPLC:

1. High disadvantages: the mobile phase is liquid, and when it flows through the column, it is subject to greater resistance, and in order to quickly pass through the column, the carrier liquid must be pressured.

2. High speed: fast analysis speed, fast carrier liquid flow rate, much faster than classical liquid chromatography, usually analyze a sample in 15 30 minutes, some samples can even be completed in 5 minutes, generally less than 1 hour.

3. High efficiency: high separation efficiency. Stationary and mobile phases can be selected to achieve the best dissociation compared to industrial distillation columns and gas chromatography.

The separation efficiency is many times higher.

4. High sensitivity: ultraviolet detector can reach, and the injection amount is in the order of l.

5. Wide range of applications: more than 70% of organic compounds can be analyzed by high performance liquid chromatography, especially the separation and analysis of compounds with high boiling point, macromolecules, strong polarity and poor thermal stability, showing advantages.

HPLC separates mixtures based on a wide variety of chemical interactions. This interaction is usually a non-covalent property between the analyte and the analytical column. When using HPLC, the liquid analyte is injected into the column at different times, moved in the stationary phase by pressure, due to the different interactions between different substances in the analyte and the stationary phase, different substances leave the column in order, and different peak signals are obtained through the detector, each peak represents a different type of compound, and finally the analyte contains substances by analyzing and comparing these signals.

Chromatographic instruments designed with a liquid as the mobile phase are called liquid chromatographs. The liquid chromatograph with high-pressure infusion pump, high-efficiency stationary phase and high-pressure sensitive detector becomes a high-performance liquid chromatograph. There are many types of HPLCs, but no matter what type of HPLC, they are basically divided into 4 parts:

High-pressure infusion sets, injection systems, separation systems, and testing systems.

Simply put, the sample is dissolved in the mobile phase, enters the column with the mobile phase, and is adsorbed by the stationary phase (packing material). Different components in the sample are adsorbed differently, resulting in a different chronological order of the flow column.

In the case of reversed-phase chromatography, the stationary phase (silica alkane packing material) is less polar than the mobile phase (acetonitrile, methanol, etc.). For each component in the sample, the adsorption force of the stationary phase with small polarity is relatively large, and the column flows later, which is reflected in the spectrum and the peak is late.

Normal-phase chromatography is the opposite.

HPLC is a liquid chromatography method for high-pressure delivery of mobile phases for fast and efficient separation.