System composition and working principle of high performance liquid chromatography

The HPLC system consists of a reservoir, pump, injector, column, detector, and recorder. The mobile phase in the reservoir is pumped into the system by the high pressure pump. The sample solution enters the mobile phase through the sampler and is loaded into the column (stationary phase) by the mobile phase. Since the components in the sample solution have two phases, Different distribution coefficients, when used for relative motion in two phases, undergo repeated adsorption-desorption distribution processes, and each component produces a large difference in moving speed, and is separated into individual components and then flows out of the column. When passing through the detector, the sample concentration is converted into an electrical signal and sent to the recorder. The data is printed out as a map.

High performance liquid chromatography (HPLC) is also called high pressure liquid chromatography, high speed liquid chromatography, high resolution liquid chromatography, and the like. On the basis of classical liquid chromatography, the theory of gas chromatography was introduced in the late 1960s and developed rapidly. It differs from the classical liquid chromatography in that the filler particles are small and uniform, and small particles have high column efficiency but cause high resistance and require high pressure to transport the mobile phase. Therefore, it is also called high pressure liquid chromatography. Because of the fast analysis speed, it is called high-speed liquid chromatography.

High performance liquid chromatography is currently the most widely used chromatographic analysis method. The high performance liquid chromatography system consists of a mobile phase storage liquid bottle, an infusion pump, an injector, a column, a detector, and a recorder, and its overall composition is similar to gas chromatography. However, many adjustments have been made to the characteristics of the mobile phase as a liquid. The infusion pump of HPLC requires the infusion volume to be constant and steady; The injection system requires the injection to be convenient to switch tightly; Because the viscosity of the liquid mobile phase is much higher than that of the gas, the column to reduce the column pressure HPLC is generally thick and the length is much smaller Gas chromatography column. HPLC is very widely used, almost every field of quantitative qualitative analysis.

When using high-performance liquid chromatography, the liquid to be detected is injected into the column and moves through the stationary phase by pressure. Due to the different interactions between different species of the tested species and the stationary phase, different substances are separated from the column and passed through the detector. The peak signal is finally determined by comparing these signals by analysis to determine the substance contained in the side object. As an important analytical method, high performance liquid chromatography is widely used in chemical and biochemical analysis. There is no essential difference between high performance liquid chromatography and classical liquid chromatography. Its characteristic is that it uses a high-pressure infusion pump, a high-sensitivity detector, and a high-efficiency particulate stationary phase. It is suitable for analysis of high boiling point, low volatility, high molecular weight, and different Polar organic compounds.

Development History:

In the 1960s, due to the limitations of gas chromatography on the analysis of high-boiling organic compounds, the theory and methods of gas chromatography were reintroduced into classical liquid chromatography in order to separate proteins and nucleic acids that are difficult to gasify. In the late 1960s, Kirkland, Haber, Horvath, Hayes, Lipsky, and others developed the world's first high performance liquid chromatograph, which opened the era of high-performance liquid chromatography. High-performance liquid chromatography uses a finer particle size stationary phase to fill the column, increasing the number of columns in the column, and driving the mobile phase at high pressure, making it possible for classical liquid chromatography to take several days or months to complete the separation in a matter of hours. Even completed in dozens of minutes.

In 1971, Kirkland et al. published a book entitled "Modern Practice of Liquid Chromatography," marking the official establishment of high performance liquid chromatography (HPLC). In the following period, high-performance liquid chromatography has become the most commonly used separation and detection method. It is widely used in organic chemistry, biochemistry, medicine, drug development and detection, chemical engineering, food science, environmental monitoring, commodity inspection, and legal inspection. Applications. High performance liquid chromatography also greatly stimulates the development of stationary phase materials, detection techniques, data processing techniques, and chromatography theory.

Before 1960's, the use of filler particles greater than 100 μm, the efficiency of the increase faced with difficulties, later researchers will use a particulate stationary phase to overcome a bottleneck. Kochland and Hovas prepared a thin-shell stationary phase, which has a porous shell on the surface of the glass microspheres, achieving high-speed mass transfer and laying a solid foundation for the development of high-performance liquid chromatography. As the particle size of the filler decreases, higher efficiency can be achieved. In the 1960s, pneumatic amplifying pumps, syringe pumps, and low-flow reciprocating piston pumps were developed. However, the latter's pulse signals are large and it is difficult to meet the requirements of high-performance liquid chromatography. In the 1970s, a reciprocating double-plunger constant-flow pump solved this problem. After the 1970s, Kirkland produced a fully porous spherical silica gel with an average particle size of only 7 μm, excellent column efficiency, and gradually replaced the amorphous silica gel. Afterwards, the bonded stationary phase produced has greatly improved the stability of the column, making it possible to use it several times. After 1970, fillers suitable for the separation of biological macromolecules have become a research hotspot. After 1980, improving the selectivity of separation became a major problem for chromatographers. It is increasingly recognized that changing the composition of the mobile phase is the key to improving selectivity.

High-performance liquid chromatography features:

High pressure - pressure up to 150 ~ 300kg/cm2. The pressure drop per column is 75kg/cm2 or more.

High speed - The flow rate is 0.1 to 10.0 mL/min.

Efficient - the number of plates up to 5000/m. Simultaneous separation of up to 100 components in one column.

High sensitivity - UV detector sensitivity up to 0.01ng. At the same time consume less sample.

HPLC has the following advantages over classical liquid chromatography:

Fast - Usually one sample is analyzed for 15 to 30 minutes, and some samples can be completed within 5 minutes.

High resolution - the stationary phase and mobile phase can be selected for optimal separation.

High sensitivity - up to 0.01 ng for UV detectors and up to 0.1pg for fluorescence and electrochemical detectors.

Columns can be used repeatedly - using a single column to separate different compounds.

The sample size is small and it is easy to recover - the sample is not destroyed after passing through the column, and the single component can be collected or prepared.