About The HPLC Main Components And Its Column Maintenance And Use Notes
HPLC has been widely used in many fields such as food additives and pharmaceutical composition analysis due to its advantages of accurate quantitative analysis results, short analysis period, wide analysis range, and low detection limit.
Introduction of main components
The infusion system mainly includes an online degassing device, a high-pressure infusion pump, and a gradient elution device. The main function of the online degasser is to remove bubbles before the mobile phase enters the column. The packing particles of the liquid chromatography column are small, and the flow resistance through the 2 ~ 5 mm column is very large. Therefore, the mobile phase needs to be extracted by the high-pressure infusion pump and sent to the column.
High-pressure pumps can be divided into constant pressure and constant flow pumps according to infusion performance. Based on the mechanical structure, it can be divided into a hydraulic diaphragm pump, pneumatic amplification pump, screw injection pump, and reciprocating plunger pump.
The sampler is a device that sends analytical samples to HPLC chromatographic column. It is divided into manual and automatic injectors. The manual sampler is generally equipped with a 20-100 μL quantitative loop.
HPLC chromatography column
The column uses liquid as the mobile phase and the solid phase in the broad sense as the stationary phase to separate the analytical samples. Chromatography columns can be roughly divided into normal-phase chromatography columns and reverse-phase chromatography columns according to the separation mode.
The detector converts the physical or chemical characteristics of the sample separated by the chromatographic column into measurable electrical signals, records them through the chromatogram, determines the separation effect from the peak shape in the chromatogram, and performs qualitative and quantitative analysis on the sample according to the peak time and peak area of the chromatogram of the standard and analytical sample.
HPLC column maintenance and use notes
Adjust the pH value of all the analyzed samples to the pH range suitable for the column. Avoiding overloading during injection is one of the important methods to maintain long-lasting and good column performance. If the manual injector is used for injection, the action should be fast and accurate during the injection. When the analyte composition is complex, a chromatographic protection column should be added in front of the column to protect the column. At the beginning of each analytical work, the chromatographic column should be equilibrated. The aqueous solution containing 10% of the organic solvent in the mobile phase is used for exhausting and flushing the chromatographic column and pipeline. Replace, and then equilibrate the column with a mobile phase of at least 10 column volumes until the baseline is equilibrated before injecting.
After each analysis, the buffer salt solution in the HPLC chromatographic column must be thoroughly rinsed with a certain amount of aqueous solution, and then the column should be rinsed with a solvent that has a strong elution capacity for the test substance. The amount of desolvation is at least 20 times the column volume. If the chromatographic column is not rinsed in time, impurities will accumulate in it for a long time, which will easily block the chromatographic column and reduce the efficiency of the column. In the long run, this will accelerate the reduction of column life. If the chromatographic column is not used for more than 4 days, it should be removed from the instrument and sealed tightly with a head nut. When storing the column, be careful not to vibrate or collide it strongly.