Installation Procedures and Usage Method of HPLC Column
Procedures for Installation
1. Remove the column packing box and confirm the type, size, and solvent stored in the column.
2. Unscrew the sealing plug of the joint at both ends of the column and put it back to the packing box for reserve.
3. The inlet end of the column is connected to the outlet of the injection valve through the connecting pipe according to the flow direction indicated on the column tube. The exit is connected to the detector. Connect the pipe through the hollow screw, after crushing as hard as possible to insert to the end, and then clockwise tighten the hollow screw.
Usage Method of HPLC Column
1. Pretreatment of samples. It is best to use the mobile phase to dissolve the sample and remove particulate impurities with a filter membrane.
2. Formulation of the mobile phase. The viscosity of the mobile phase should be as small as possible so as to obtain good separation effect when using longer analytical columns, and simultaneously reduce the column pressure and prolong the service life of the liquid pump.
3. Choice of the flow velocity of the mobile phase. When the optimal flow rate is selected, the analysis time may be prolonged. The method of changing the washing strength of the mobile phase can be used to shorten the analysis time.
The function of the HPLC Column
High-performance liquid chromatography (HPLC) is a kind of chromatographic method, which uses liquid as the mobile phase to obtain a relatively high flow rate by means of a high-pressure infusion pump to improve separation speed. And it is also a chromatographic method for separation and analysis of HPLC columns made of highly efficient stationary phases with very fine particles. In high-performance liquid chromatography (HPLC), if the nonpolar stationary phase, such as the octadecyl bonding phase and polar mobile phase, is used, the reverse chromatographic separation system is formed. Otherwise, it is called a positive chromatographic separation system. The mobile phase used in reverse chromatography systems is less costly and more widely used.
Importance of Gradient Elution for HPLC Column
The importance of gradient elution for the HPLC column is the same as the importance of temperature-programmed for the HPLC column. Therefore, in the inquiry experiments, gradient elution must be continuously optimized to find the most suitable gradient elution procedure, so that the experiment can make the best results in the shortest time. And the higher the proportion of water in the mobile phase, the separation degree is better and the peak time is longer.
Determination of HPLC Column
Before the operation, it is critical to the configuration of the solvent to be tested. First, methanol was used as solvent to prepare the reserve solution, the steps are as followed: take some dimethyl phthalate (0.3880 g/L), diethyl phthalate (0.2770 g/L) and dibutyl phthalate (0.3776), and takes 1 mL reserve solution, dilute to 10 mL with water and methanol (20:80) each as the solution to be tested. Then start the equipment according to the operation rules and then set the operating conditions. After stabilization, it is better to start sampling. Absorb the mixture solution with a microsyringe, inject the instrument inlet, and pull the injection valve clockwise. Then, the retention time, peak area, and separation ratio of chromatographic peaks were recorded. After determination, cleaning the HPLC column with a configured ethanol solution.