The function of the guard column is to filter out chemical “trash” from the mobile phase and sample, and also to effectively remove insoluble matter in the mobile phase and sample. Although current chromatographs are equipped with filters of different pore sizes such as 1 and 2μm at the mobile phase suction port, at the rear of the injection valve, and at both ends of the chromatographic column, the filters can only remove insoluble particles but not chemical contaminants. Therefore, , it is necessary to install a guard column, especially when analyzing complex mixtures and biological samples such as traditional Chinese medicines and proprietary Chinese medicines, it is an indispensable measure to protect the analytical column. The guard column packing should be consistent with the analytical column, and the particle size can be larger than that of the analytical column. The guard column is a consumable item and can be used after a certain number of times
After analyzing samples (50 to 100 times), if the column pressure increases significantly or the peak shape deteriorates or the baseline drifts, the guard column should be replaced.
The mobile phase is usually made of water or buffer solution mixed with an organic solvent. In principle, all mobile phases passing through the chromatographic column should be filtered, but the actual operation should be different depending on the specific situation: when the organic solvent is of chromatographic grade , when quartz sub-boiling water or other ultrapure water is used, filtration has no more practical significance when the quality of water and organic solvents can be ensured and there is no pollution.
When the mobile phase contains buffer salts, filtration is necessary. As mentioned above, when the buffer solution is left for a period of time (depending on the ambient temperature, generally no more than 48 h in summer and no more than a week in winter), It should also be re-filtered before use.
The prepared sample test solution must be filtered through a 0.45μm (0.22μm is better) pore size filter membrane before being injected into the flow system. Pay attention to distinguish between water system and oil system. The two cannot be mixed to prevent the filter membrane from dissolving and causing pollution. The container holding the mobile phase and the online filter in the chromatography system must be cleaned and replaced regularly to prevent the filter from being overloaded and unable to perform its filtration function.
When the sample contains chemical components that are harmful to the chromatographic column, such as permanently adsorbed proteins, sugars and other organic molecules and strongly adsorbent substances, as well as group ions that may cause damage to the column packing. Before injection, the sample should be separated and purified as much as possible to remove excess impurity components.
Although HPLC columns can withstand pressures up to 6000 psi (pounds per square inch), excessive pressure changes and excessive pressure changes will shorten the life of the column. The method to avoid this is
(1) When the column pressure is too high, use a smaller flow rate as much as possible, preferably no more than half of the maximum allowable pressure of the column.
(2) When the flow rate is large, the flow rate gradient method should be adopted to achieve the flow rate step by step.
(3) When using a manual injection valve, the injection valve must be switched as quickly as possible. otherwise
Pressure shocks exceeding 20% will quickly render the column useless. When using multiple columns, column switching should be avoided under high pressure.
Generally, the maximum operating temperature of chromatographic columns based on silica gel does not exceed 60°C, and is preferably lower than 40°C. Especially when the pH of the mobile phase is close to the operating limit, the operating temperature should be lowered. Excessive temperature will accelerate the hydrolysis of the bonded phase and the dissolution of silica gel, thereby changing the properties of the packing, collapse of the column bed, reducing column efficiency, and changing peak shape. The current marked pH range of bonded silica gel chromatography columns can reach 2 to 10, but extreme pH conditions should be avoided in actual use, especially when used under alkaline conditions (pH ≥ 8). If it must be used When used under extreme pH conditions, column damage can be avoided by:
(1) Install a pre-column (saturation column) between the pump and the injection valve to saturate the mobile phase;
(2) Reduce the use temperature;
(3) Immediately after inspection, clean thoroughly with a “mild solvent” that is miscible with the fluid used;
Before starting the experiment, you should know what solvent is currently in the column. For new chromatographic columns, unless otherwise specified, this is the mobile phase used in the evaluation report. The current solvent in the column must be miscible with the flow used to analyze the sample. If it is not miscible, it must be transitioned to a third solvent that is miscible with both. Isopropyl alcohol is the only reagent that is miscible with various solvents and can be used as an intermediary mobile phase. The suitable storage environment for reversed-phase bonded silica gel columns is pure methanol. If the mobile phase for analysis is buffer 2 organic solvent, the original pure methanol can be replaced with methanol 2 water with a lower methanol content (<20%), and then Add the mobile phase for analysis to prevent salt precipitation in the column; after the test is completed, flush the system with an appropriate solvent in a timely manner and finally transition to pure methanol flushing. For mobile phases containing buffer salts, the best way is to first Rinse with a solution that has the same composition as the mobile phase for analysis but does not contain buffer salts, and then gradually transition to pure methanol. Most reports indicate that the flushing fluid volume is about 15 to 20 times the column volume, but it is ultimately determined by whether the baseline is straight and whether the pressure is stable. Some manufacturers’ chromatographic columns indicate that the minimum concentration of organic phase in the mobile phase is ≥5%, so care should be taken when using them.