Five methods for prolonging the life of HPLC column

The service life of chromatographic column is closely related to routine maintenance besides the sample, mobile phase and usage frequency. In order to prolong the service life of chromatographic column. The service life of chromatographic column is mainly measured by local column efficiency and column pressure. If the column efficiency is too low or the column pressure is too high, it is generally considered that the chromatographic column has ended. Therefore, the key to prolong the service life of chromatographic column is to eliminate the factors causing the decrease of column efficiency and the increase of column pressure.
The following are the routine maintenance methods of chromatographic column:
1、 The pH of the mobile phase should be within the range of use. Except for the reversed cyano column with pH of 1.5-9.0, the pH range of the reversed-phase chromatographic column is 1.5-10. Due to the existence of Si-C and Si-O bonds in the packing, the loss of silica matrix and the fracture of carbon chain in the bonding phase will result in the decrease of column efficiency and the shortening of service life. Due to the improper pH control of mobile phase, it is usually difficult to recover the chromatographic column. Therefore, it is necessary to take it seriously and strictly control the pH value of mobile phase.
2、 Removing the solid particles in the sample and mobile phase will block the sieve plate of chromatographic column. Blocking the sieve plate will not only cause the increase of column pressure, but also cause the decrease of column efficiency, because the blockage of sieve plate will cause uneven liquid flow, leading to the tailing and broadening of chromatographic peak, thus reducing the column efficiency. Therefore, it is suggested to use ultrapure water and chromatographic pure reagent, filter the sample with syringe before analysis, and filter the mobile phase through 0.45 μ m membrane.
3、 The use of protective column or on-line filter sample and mobile phase after filtration can not completely eliminate solid particles, because the pump wear, seal ring and pipeline aging will also produce solid particles, these solid particles are carried into the chromatographic column by the mobile phase, blocking the sieve plate, resulting in the increase of column pressure and decrease of column efficiency. There are sieve plates on the protection column and on-line filter, and the pore size is the same as that of the chromatographic column, so it can prevent the solid particles from reaching the chromatographic column and effectively prevent the blockage of the sieve plate of the chromatographic column. Since the increase of column pressure accounts for a large proportion of analysis failures, in addition to filtering the sample and mobile phase, it is recommended that you add a protective column or on-line filter at the injection end of the chromatographic column.
If the increase of column pressure is caused by the blockage of the sieve plate at the injection end, the following methods can be selected for remedy: 1. Add a protective column or online filter in front of the chromatographic column, and then use methanol and water = 20 / 80ml / min to back flush the chromatographic column for 180min. 2. Add a protective column or online filter at the injection end of the chromatographic column, and then use it in reverse.
4、 The correct use of buffer salt buffer salt is usually soluble in water, but difficult to dissolve in organic solvent, so improper use of buffer salt will cause it to precipitate, block the micropores on the packing matrix and the space between particles, make the packing harden, and increase the column pressure; at the same time, it hinders the free expansion of the carbon chain bonded on the matrix, and reduces the retention capacity and column efficiency of the chromatographic column. It is very difficult to remove the buffer salt after it is separated out. Therefore, the proper use of buffer salt is very important to prolong the service life of chromatographic column. The purpose of proper use of buffer salt is to prevent buffer salt out, so the correct use of buffer salt can be summed up as a sentence: filter before use, rinse after use. The specific methods are as follows: 1. Isocratic conditions: before and after the use of buffer salt, the transitional mobile phase should be used to flush the chromatographic column at the flow rate of 1.0ml/min for 60min; another method to remove the buffer salt after use is to flush the chromatographic column with the flow rate of 0.2ml/min with the transitional mobile phase overnight. 2. Gradient conditions: before running the gradient with the mobile phase containing buffer salt, the transitional mobile phase with the same composition as the initial mobile phase was used to flush the chromatographic column at the flow rate of 1.0 ml / min for 60 min, and then the transitional mobile phase was used to flush the chromatographic column at 1.0 ml / min for 120 min. the gradient setting of the mobile phase containing buffer salt should be as gentle as possible to avoid the buffer salting out during the gradient process. Note: the transitional mobile phase refers to the organic phase and water phase, which are the same as the analytical mobile phase, except that the transitional mobile phase does not contain buffer salts. 3. The remedy of buffer salting out: 1) scheme 1: reverse flush the chromatographic column with methanol / 20 / 80 at a flow rate of 1.0ml/min at 35 ℃ for 120min; 2) scheme 2: reverse flush the chromatographic column with methanol / water = 20 / 80 at a flow rate of 0.2ml/min overnight.
5、 To prevent the strong retention substances from remaining on the chromatographic column, the accumulation of strong retention substances and macromolecular compounds in the chromatographic column will produce extra retention behavior for the compounds in the sample, which will not only cause the broadening and tailing of the peak, but also cause the change of the retention time, and the column pressure will increase when the accumulation reaches a certain degree. Since the influence of strong retention substances and macromolecular compounds on chromatographic separation is a cumulative effect, it takes a certain time to reflect. However, for many drugs, especially for complex samples, it is difficult to judge whether they contain strong retention substances. Therefore, to prevent the accumulation of strong retention substances, it is necessary to clean the chromatographic column with pure methanol or acetonitrile in daily maintenance. Cleaning method: 1. No buffer salt: after the analysis is completed every day, first remove the buffer salt by the above method, and then wash the chromatographic column with methanol or cyanide for 60min. 2. Use the buffer salt: after the analysis, remove the buffer salt with the above method, and then wash the chromatographic column with pure methanol or acetonitrile for 60min 3. Remedy: water - acetonitrile - chloroform (or isopropanol) - acetonitrile - water, and wash the chromatographic column at the flow rate of 1.0ml/min for 60min at each step.
For more related technologies, please consult MS technologies technicians