Method for prolonging the service life of liquid chromatography column
The service life of the liquid chromatography column is not only related to the analyzed sample, mobile phase and frequency of use, but also the most important thing to do with the daily committee. In order to extend the life of the column and protect your interests, please read this section carefully.
The service life of a column is mainly measured by two indicators: local column efficiency and column pressure. If the efficiency of a column is too low or the column pressure is too high, it is usually considered that the column has ended. Therefore, the key to extending the life of the column is to eliminate the factors that cause a decrease in column efficiency and an increase in column pressure. The following is the daily maintenance method of the column:
1. The PH of the mobile phase should be within the range of use. The liquid chromatography column except the reverse phase cyano column PH1.5-9.0, the PH range of the reverse phase column is 1.5-10, due to the presence of Si-C in the packing Bonding with Si-O, the mobile phase exceeding its PH range will lead to the loss of silica gel matrix and the breakage of the bonded phase carbon chain, which will reduce the column efficiency and shorten the service life. Due to improper PH control of the mobile phase and damage to the column, it is usually difficult to recover the column. Therefore, it must be taken seriously and the PH value of the mobile phase should be strictly controlled.
2. Removal of solid particles in the sample and mobile phase The solid particulate matter contained in the sample and mobile phase will block the liquid chromatography column sieve plate. Blocking the sieve plate will not only cause an increase in column pressure, but also cause column efficiency. Decreased, because the clogging of the sieve plate will cause uneven liquid flow, resulting in tailing and broadening of the chromatographic peak shape, thereby reducing the efficiency of the column. Therefore, it is recommended to use ultrapure water and chromatographically pure reagents. Before analyzing the sample, perform a syringe filter on the sample, and the mobile phase passes through a 0.45 μm filter membrane.
Third, the use of guard columns or online filter samples and mobile phases can not completely eliminate solid particulate matter after filtration, because the wear of the pump, the aging of the seal ring and the pipeline will also produce solid particulate matter, these solid particles are carried by the mobile phase Into the liquid chromatography column, blocking the sieve plate, resulting in increased column pressure and decreased column efficiency. Both the guard column and the online filter have a sieve plate, and the pore size is the same as that of the chromatographic column, so it can prevent solid particulate matter from reaching the chromatographic column, and effectively prevent the clogging of the chromatographic column sieve plate. Since the increase in column pressure accounts for a large percentage of analysis failures, in addition to filtering the sample and mobile phase, it is recommended that you add a guard column or in-line filter to the sample inlet of the column.
If the increase in the column pressure of the depleted liquid chromatography column is caused by the clogging of the sieve plate at the injection end, Branch chooses the following methods to remedy:
1. First add a protective column or online filter in front of the liquid chromatography column, and then flush the column with methanol and water = 20 / 80ml / min for 180min.
2. First add a guard column or online filter to the inlet of the liquid chromatography column, and then use it in reverse.
4. Proper use of buffer salts Buffer salts are usually soluble in water and difficult to dissolve in organic solvents. Therefore, improper use of buffer salts will cause them to precipitate out, block the pores on the filler matrix and the gaps between the particles, and cause the packing to compact and increase the column pressure ; At the same time, it hinders the free expansion of the bonded carbon chain on the matrix, which reduces the retention capacity of the chromatography column and reduces the efficiency of the column. After the buffer salt is precipitated, it is very difficult to remove. Therefore, the correct use of buffer salt is very important to extend the life of the column.
The purpose of the correct use of buffer salts is to prevent the precipitation of buffer salts, so the correct use of buffer salts can be summed up in one sentence: filter before use and rinse after use. The specific method is as follows:
1. Isocratic conditions: Before and after using buffer salts, it is necessary to rinse with a transition mobile phase at a flow rate of 1.0ml / min for 60min; another method to remove buffer salts after use is to rinse the column with a transition mobile phase at a flow rate of 0.2ml / min overnight.
2. Gradient conditions: Before running the gradient with a mobile phase containing buffer salts, flush the column with the same mobile phase as the initial mobile phase at a flow rate of 1.0 ml / min for 60 min, and then use the transition mobile phase to flush the column at 1.0 ml / min 120min. The gradient setting of the mobile phase containing buffer salt should be as gentle as possible to avoid buffer salt precipitation during the gradient process.
Note: The transitional mobile phase means that the composition of the organic phase and the aqueous phase is the same as the analytical mobile phase, the difference is that the transitional mobile phase does not contain buffer salts.
3. Remedy for buffer salt precipitation:
1) Scheme 1: Backwash the liquid chromatography column with methanol / 20/80 at a flow rate of 1.0 ml / min at 35 degrees Celsius for 120 min
2) Scheme 2: Backwash the column with methanol / water = 20/80 at a flow rate of 0.2 ml / min overnight.
5. Prevent strong retention substances from accumulating on the column. Strong retention substances and macromolecular compounds accumulate in the chromatography column, causing additional retention behavior for the compounds in the sample, which not only causes peak shape broadening and tailing, and reduces column efficiency. At the same time, it will also cause changes in the retention time, which will also cause the column pressure to increase when accumulated to a certain extent. Since the effect of strongly retained substances and macromolecular compounds on chromatographic separation is a cumulative effect, it will take some time to manifest, but for many drugs, especially complex samples, it is difficult to judge whether they contain strongly retained substances, so To prevent the accumulation of strongly retained substances, the column needs to be cleaned with pure methanol or acetonitrile in daily routine maintenance.
the cleaning method:
1. Unused buffer salt: After the daily analysis is completed, the buffer salt is removed by the above method first, and then the column is backwashed with methanol or ethyl cyanide for 60 minutes.
2. Used buffer salt: After the analysis is completed, first remove the buffer salt using the above method, and then flush the column with pure methanol or ethyl cyanide for 60 min.
3. Remedy:
Water-ethyl cyanide-chloroform (or isopropanol)-ethyl cyanide-water Each step backwash the column at a flow rate of 1.0ml / min for 60min.
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