The clothes have to be worn for a long time, and the column has been used for a long time. However, washing the column is not as simple as washing clothes. How to wash it, there are still some doorways:
The first question: what is used?
1.1: What is flushing a column? Usually, column flushing is to flush out the dirty matter on the column and the damage to the column, and store it in a suitable solvent. In another case, it can be counted as The scope of rinsing is the process of re-balancing the column between gradient methods.
1.2: Commonly used solvents are used for reversed-phase columns. Usually, pure water and organic solvents are used. Water is used as a salt and other additives in the column, while organic solvents are used to rinse off some of the chromatograms. There are strong contaminants on the column. In addition to the methanol and acetonitrile commonly used in the analysis, there are some organic solvents that can remove more pollutants, such as isopropyl alcohol.
The second question: How long does it take to rush the column? This is a problem that often plagues everyone, 20 minutes, 30 minutes, or an hour?
2.1.1: The calculation principle of column rinsing In fact, rinsing the column is not a time concept, but a volume problem: usually it is necessary to completely replace the mobile phase in the column, which requires at least ten column volumes, that is, A solvent with at least ten times the column volume should flow through the column to completely replace the original solvent.
2.1.2: Column volume calculation The column volume of the column is the cylindrical volume of the column multiplied by the porosity of the column packing (generally about 0.6), so calculate a common 4.6 × 150mm chromatogram The column volume is approximately 1.5 mL, and if it is 10 column volumes, it is 15 mL, which is 15 min according to the usual flow rate of 1 mL/min.
2.2.1: Flushing time for gradient rebalance. However, this is only the time to replace all solvents. It is only suitable for short-term flushing of the column, such as the equilibration time between gradient methods, but it is also affected by the delay volume of the instrument. It is already easy to re-achieve the chemical equilibrium of the mobile phase. Generally, the larger the delay volume of the instrument, the longer this time. If the mobile phase used is not easy to reach the chemical equilibrium, it will take a long time, such as using some buffer salts. Case. Of course, in many cases, the balance time between gradient methods does not need to be so long.
2.2.2: Cleaning the column wash time If it is necessary to completely rinse the column, it takes a longer time. It is usually recommended to be 20 times the column volume. That is, for a 4.6×150mm column, it takes 30 minutes to ensure Flush completely. Columns of other sizes can be easily calculated based on column specifications and flow rates.
The third question: how to flush the column, there are no two cases: normal flushing and abnormal flushing
3.1: Flushing step of the column under normal conditions
3.1.1: Flushing with a mobile phase without additives Normally, only the mobile phase is replaced and purified. For the general method, only a high proportion of organics such as methanol or acetonitrile is used after the end of the process. Rinse the column approximately 20 times the column volume.
3.1.2: Flushing with salt or additives If the mobile phase of the method used contains buffer salts or other additives, the pure water phase with the same initial ratio of mobile phase should be used instead of the buffer salt. After one phase, rinse 20 times the column volume, then replace with a high proportion of organic phase to wash 20 column volumes.
3.1.3: Preservation after column washing For the preservation of the column, it is generally stored in pure organic phase, short-term storage, methanol acetonitrile can be stored for a long time, it is recommended to store in methanol.
3.2: Flushing under abnormal conditions If it is an abnormal condition, consider some washing methods that do not take the usual path:
What is abnormal? There are two common types, the efficiency of the column is reduced and blocked:
3.2.1: Column efficiency drop column flushing For column efficiency, we first need to determine the column problem rather than the instrument or analytical method, excluding the two, if not the column damage, such as collapse (physical impact Cause), bonded phase hydrolysis (long-term use at high temperature and low pH), electrolyte degradation caused by irreversible damage such as silica matrix hydrolysis (used at high pH), because most of the above problems are excluded because the column is not too Normally “dirtyâ€, you can first rinse the column with pure methanol or acetonitrile for a long time. If it doesn't work, you can consider using pure isopropanol to wash the column. The elution ability of isopropanol is stronger than methanol acetonitrile. It can elute many pollutants that cannot be washed by these two solvents from the column. If the effect is still not obvious, refer to the “Column Regeneration†section of the column instructions for a more powerful solvent wash, but these solvents are usually not the usual reversed-phase LC solvents.
3.2.2: Flushing the column. If it is a blockage, first determine what cause the blockage is caused. If it is caused by the residual buffer salt in the column, this is the most fortunate blockage. Try flushing the column at a higher temperature with a higher proportion of water phase (95% recommended).
If the column is clogged due to the sample, another method should be considered. If the sample contains too much highly adsorbable substance (sometimes it is a certain impurity in a complex sample, it cannot be removed by filtration, etc.) Adsorption on the column head leads to blockage, and you can try to wash it slowly with a different solvent (which has better solubility for this type of material).
If some of the particulate impurities are clogged up due to improper sample filtration or precipitation of the sample on the column, in addition to trying to find a suitable solvent rinse, only backflushing can be considered. Generally, backflushing columns are not recommended because it is possible It causes great damage to the column, so recoil is a must-have method for dead horses to be a live horse doctor. It must be used with caution. Preparing the sample and using a pre-column or in-line filter is a good way to protect the column!
3.3: The idea of ​​quickly rinsing the column looks very good and powerful. It's actually very simple. When rinsing the column, you can shorten the rinsing time by increasing the flow rate. We already know that the completeness of column rinsing is measured by volume. So increasing the flow rate can save time. However, it is important to note that for blocked columns, increasing the flow rate may not be a viable option...
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