Working principle of high performance liquid chromatograph; The high-pressure pump sends the mobile phase of the liquid storage tank into the chromatographic column through the sampler and then flows out from the outlet of the detector. At this point, the whole system is full of mobile phase. When the sample to be separated enters from the sampler, the mobile phase flowing through the sampler brings it into the chromatographic column for separation. After separation, different components enter the detector in turn, and the recorder records the signals entering the detector to obtain the liquid chromatogram.

High performance liquid chromatography refers to the theory of gas chromatography developed on the basis of classical chromatography. Technically, the mobile phase is transported by high pressure, and the chromatographic column is filled with small particle size filler in a special way, so that the column efficiency is much higher than that of classical liquid chromatography (the number of plates per meter can reach tens of thousands or hundreds of thousands), and a highly sensitive detector is connected behind the column to continuously detect the effluent.

Extended data

High-performance liquid chromatograph is equipped with high-pressure binary pump or low-pressure quaternary pump. The stroke volume of the pump and the volume of the mixer will affect the baseline noise level of chromatography, especially in gradient elution. Generally, the smaller the stroke volume of the pump, the larger the volume of the mixer, the smaller the pulse caused by infusion, the higher the response ability to gradient change and the smoother the baseline.

When using binary pump, it should be noted that when the proportion of a mobile phase in binary mixing is less than 5%, especially when normal phase elution is used for chiral resolution of some pharmaceutical intermediates and final products, it is best to use single pump premixing method. Avoid the peak related to the stroke on the chromatographic baseline, because the pumping accuracy of the pump is relatively poor at low ratio.

Reference source; Baidu encyclopedia-high performance liquid chromatograph