Effective Liquid Chromatography (HPLC for short) is an important branch of Chromatography. It can not only separate high-boiling polar and thermally unstable compounds, but also is suitable for ionic, macromolecular and biologically active compounds. The sample components after compound HPLC separation are easy to collect, so they can be used for preparative separation. HPLC has a variety of separation modes, and the Detectors mainly include UV-Vis, differential refractive index, fluorescence spectroscopy, electrochemistry and mass spectrometry. Here only Waters510HPLC instrument is taken as an example to introduce its structure and operation.
The structure diagram of Waters5lOHLC instrument is shown in Figure II-34. The flow of the instrument is as follows: the high-pressure pump is input into the system, the sample is introduced from the injection valve with a syringe, and the absorbance signal is recorded by the Recorder after being amplified.
operation method
1. Prepare Mobile Phase
Liquid chromatography requires that the mobile phase needs to be of high purity, and all solvents (including water) must be of HPLC grade. The Waters510 pump is a unit pump, so if the mobile phase is a mixed solvent, it needs to be mixed before use, and then filtered and degassed.
2. Pump operation
Waters5l0 pumps use the digital wheel to set the flow rate, and the knob to set the upper limit of the pressure. Operation is simple and intuitive.
The main issues to watch out for are:
(1) Before power on, the flow rate is set to 0.0mL·min-1. First use a 10mL special syringe to remove possible air bubbles in the pipeline before the pump from the drain valve, then power on, and then set the flow rate.
(2) The upper limit pressure should be set before analyzing the sample to protect the chromatographic column. Generally, the upper pressure limit of a chromatographic column with a length of 15cm can be set to 2x104kPa. When the pressure exceeds, the pump will automatically stop working. After troubleshooting, press the "Reset" button, the pump will work again.
3. Operation of the injector
(l) Put the injection valve handle and the sample holding handle in the "load" position and the open position respectively.
(2) Insert the sample holding plug into the hole on the handle of the injection valve, insert the syringe that has drawn the sample into the injection port (insert to the bottom), inject the required volume of sample into the sample ring of the injection valve, pull out syringe, put the sample retention plug back in place.
(3) Hold the sample back to the closed position, mark the button on the Detector, and record the chromatogram.
