Liquid chromatography of 5 large points need to remember!
# 01 Selection and treatment of liquid phase in liquid chromatography 1. Property requirements of mobile phase: low viscosity, good compatibility with detector, easy access to pure product and low toxicity characteristics.2. pH value selection: For the weak acid or weak base samples, increase the retention of components on the fixed phase and improve the peak shape by adjusting the pH value of the mobile phase.3. Selection of buffer: the pH value of the buffer is determined according to the pKa of the analyte, so that the compound exists in a form to obtain a good sharp peak.4. The degassing mobile phase: remove the dissolved gas in the mobile phase in advance to avoid the influence of bubbles on the system and analytical results.5. Filter mobile phase: All solvents should be filtered through a 0.45 μ m (or 0.22 μ m) filter membrane to remove impurity particles.6. Storage of mobile phase: it should be stored in glass, polytetrafluoroethylene or stainless steel containers, to avoid storage in plastic containers to prevent pollution and oxidation.7. Pay special attention to the problem of halogenated organic solvents: it may contain acidic impurities, easy to react with stainless steel, may produce corrosive products after mixing with ethers, and mixing with reactive organic solvents may produce crystallization.# 02 Selection of mobile phase solvent ?? Chemical stability: the selected mobile phase solvent should have a certain chemical stability, and do not react with the fixed phase and sample components.?? Do not interfere with the detector: the solvent shall not interfere with the operation of the detector and select the mobile phase that does not affect the normal operation of the detector.?? Easy to remove: the solvent should be easily removed without interfering with the recovery of the isolated components.?? Small viscosity: the viscosity of the solvent should be small, to ensure that the appropriate column pressure drop.?? Practicability: the solvent should be cheap, easy to buy, safe to use and high purity.# 03 Precautions for the use of mobile phase solvent ?? filtration and degassing: before the use of mobile phase, 0.45 μ m filter membrane to remove particles and dust.?? Use after returning to room temperature: use the mobile phase after returning to room temperature to avoid unstable baseline level.?? Selection of reagents: with HPLC grade reagent, like water, the water flow phase needs to be replaced frequently.?? Cleaning flow path: do HPLC mobile phase reagent application of HPLC grade reagent, mobile phase solvent must be often cleaning flow path.?? Gradient change: the gradient experiment should pay attention to the intersolubility of the mobile phase, to avoid the problem of buffer salt precipitation.# 04 Storage of liquid chromatography mobile phase ?? storage container: the mobile phase is generally stored in glass, polytetrafluoroethylene or stainless steel containers, can not be stored in plastic containers.?? Phosphate, acetate buffer: should be as fresh as possible, do not store.# 05 For each 10% reduction of the organic solvent (methanol or acetonitrile), the triple rule increases the retention factor by about 3 times. This is a clever and labor-saving way. In the process of adjustment, pay attention to observe the separation of each peak.