1. Removal of solid particles in the sample and mobile phase
The solid particulate matter contained in the sample and mobile phase will block the chromatographic sieve plate. Blocking the sieve plate will not only cause the column pressure to rise, but also cause the column efficiency to drop, because the blockage of the sieve plate will cause uneven flow. , causing the chromatographic peak shape to be tailed and widened, thereby reducing the efficiency of the column.
Second, the pH of the mobile phase should be within the range of use
Due to the presence of Si-C and Si-O bonds in the filler, the mobile phase exceeds its pH range, which leads to the loss of the silica matrix and the carbon chain breakage of the bonded phase, resulting in a decrease in column efficiency and a shortened service life. It is often difficult to recover the column due to damage to the column due to improper pH control of the mobile phase, so care must be taken to strictly control the pH of the mobile phase.
Third, use the guard column or online filter
After filtration of the sample and mobile phase, the solid particulate matter cannot be completely eliminated, as the wear of the pump, the aging of the seals and the tubing will also produce solid particulate matter, which is carried by the mobile phase into the column, blocking the sieve plate, resulting in The column pressure is increased and the column efficiency is lowered. Both the guard column and the in-line filter have a sieve plate with the same pore size as the column pore size, thus preventing solid particulate matter from reaching the column and preventing blockage of the column screen. Since the increase in column pressure is a large percentage of analytical failures, it is recommended that you add a guard column or inline filter to the column injection end, in addition to filtering the sample and mobile phase.
If you confirm that the column pressure rise is due to plugging of the injection end screen, you can choose to remedy the following:
1. Add a guard column or an in-line filter in front of the column, then back-flush the column with methanol and water = 20/80 ml/min for 180 min.
2. Add a guard column or inline filter to the column injection end and use it in reverse.
Fourth, the correct use of buffer salts
The buffer salt is usually soluble in water and is insoluble in organic solvents. Therefore, improper use of the buffer salt causes it to precipitate, blocking the pores between the pores and the pores on the filler matrix, causing the filler to be kneaded and the column pressure to rise, and hindering the substrate. The bonded carbon chain is free to stretch, which reduces the retention capacity of the column and reduces the column efficiency. After the buffer salt is precipitated, removal is very difficult, so proper use of buffer salts is very important to extend the life of the column.
The correct use of buffer salts is to prevent the precipitation of buffer salts, so the correct use of buffer salts can be attributed to one sentence: to filter before use, rinse after use. The specific method is as follows:
1. Isocratic conditions: Before using the buffered salt, the transitional mobile phase should be washed at a flow rate of 1.0 ml/min for 60 min. Another method of removing the buffered salt after use is to flush the column with a transient mobile phase at a flow rate of 0.2 ml/min. overnight.
2. Gradient conditions: Before running the gradient with the buffer phase containing mobile phase, rinse with the same mobile phase with the initial mobile phase at a flow rate of 1.0 ml/min for 60 min, and then rinse the column with 1.0 ml/min using the transition mobile phase. 120min. The gradient setting of the buffered salt mobile phase should be as gentle as possible to avoid buffer salt precipitation during the gradient process.
Note: The transition mobile phase means that the composition of the organic phase and the aqueous phase is the same as that of the analytical mobile phase, except that the transition mobile phase does not contain buffer salts.
3. Remedy for buffer salt precipitation:
1) Scheme 1: Backwash the column for 120 min with methanol/20/80 at a flow rate of 1.0 ml/min at 35 °C.
2) Scheme 2: The column was backflushed back and forth at a flow rate of 0.2 ml/min with methanol/water = 20/80 overnight.
5. Prevent strong retained substances from remaining on the column
Strongly retained substances and macromolecular compounds accumulate in the column, creating additional retention behavior for the compounds in the sample, which not only causes peak broadening, tailing, but also decreases column efficiency, and also causes retention time changes, accumulating To a certain extent, it will also cause the column pressure to rise. Since the influence of strongly retained substances and macromolecular compounds on chromatographic separation is a cumulative effect, it takes a certain amount of time to be reflected, but for many drugs, especially complex samples, it is difficult to judge whether they contain strong retained substances, so To prevent the accumulation of strongly retained materials, the column needs to be cleaned with pure methanol or acetonitrile in daily routine maintenance.
cleaning method:
1. Unused buffer salt: After the daily analysis is completed, the buffer salt is removed by the above method, and then the column is back-washed with methanol or acetonitrile for 60 min.
2. Using buffered salt: After the analysis is completed, the buffer salt is first removed by the above method.
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