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Daily use and maintenance of liquid chromatography column
The correct use and maintenance of the column is very important, and a slight carelessness will reduce the efficiency, shorten the service life and even damage.9mm LC-MS chromatography vials During the chromatographic operation, the following issues need to be noted to maintain the column.
1 Avoid sudden changes in pressure and temperature and any mechanical shock. Sudden changes in temperature or dropping the column from a height will affect the filling condition in the column; a sudden increase or decrease in column pressure will also impede the packing in the column, so it should be slow when adjusting the flow rate, when the valve is injected. The rotation of the valve cannot be too slow (as described above).
2 The composition of the solvent should be gradually changed, especially in reversed-phase chromatography, and should not be changed directly from organic solvent to all, and vice versa.
3 Generally speaking, the column cannot be backflushed. 9mm LC-MS chromatography vialsOnly when the producer indicates that the column can be backflushed, the impurities remaining in the column head can be backflushed. Otherwise, the recoil will quickly reduce the efficiency of the column.
4 Choose to use a suitable mobile phase (especially pH) to avoid damage to the stationary phase. Sometimes a pre-column can be connected in front of the injector. When the analytical column is bonded to silica gel, the pre-column is silica gel, so that the mobile phase can be “saturated” by the silica gel before entering the analytical column to avoid dissolution of the silica matrix in the analytical column. .
5 Avoid injecting samples with complex matrices, especially biological samples, directly into the column. Pretreatment of the sample is required or a guard column is connected between the injector and the column. The guard column is typically a short column filled with a similar stationary phase. The guard column can and should be replaced frequently.
6 Rinse the column with a strong solvent often to remove impurities that remain in the column. When cleaning, the displacement of the mobile phase in the flow path system should be gradually transitioned with a solvent that is miscible. The volume of each mobile phase should be about 20 times the volume of the column, that is, 50 to 75 ml for routine analysis.
Here are some cleaning solvents and sequences for the column. For reference: the silica gel column is washed sequentially with 9mm LC-MS chromatography vialsn-hexane (or heptane), dichloromethane and methanol, and then washed sequentially in reverse order. All solvents must be strictly dehydrated. Methanol can wash away residual strong polar impurities, and hexanes reactivate the surface of the silica gel. The reverse phase column was washed successively with water, methanol, acetonitrile, methyl chloride (or chloroform), and washed sequentially in the reverse order. If the mobile phase for the next analysis does not contain buffer, then you can omit the step after rinsing with water. Methyl chloride can wash away residual non-polar impurities, and repeated injections of 100-200 μl of tetrahydrofuran several times in methanol (acetonitrile) can help remove strong hydrophobic impurities. A mixed solution of tetrahydrofuran with acetonitrile or methanol can remove lipids. Sometimes dimethyl sulfoxide is injected several times. In addition, protein contamination was removed by gradient elution with acetonitrile, acetone and trifluoroacetic acid (0.1%).
The cation exchange column can be washed with a dilute acid buffer, and the anion exchange column can be washed with a dilute alkali buffer to remove the salt with strong exchangeability, and then water, methanol, dichloromethane (removing the organic matter adsorbed on the surface of the stationary phase), methanol, and water in turn. rinse.
7 When storing the column, fill the column with acetonitrile or methanol. The column joint should be tightened to prevent the solvent from evaporating and drying. It is absolutely forbidden to leave the buffer solution in the column for a night or more.
8 During the use of the column, if the pressure rises, one may be that the sintered filter is clogged. At this time, the filter should be replaced or taken out for cleaning. Another possibility is that the macromolecule enters the column and the stigma is contaminated 9mm LC-MS chromatography vials. If the efficiency of the column is reduced or the peak of the chromatogram is deformed, the stigma may collapse and the dead volume may increase.
In the latter two cases, carefully unscrew the column joints, remove the column head packing with clean small steel to a height of 1~2mm (note that the contaminated packing is removed) and then level the packing in the column. The column is then filled with the appropriate solvent-wet phase (same as in the column), flattened, and the column joints are tightened. After this treatment, the efficiency of the column is improved, but it is difficult to restore to the level of the new column.
Column failure is usually the end of the column. 9mm LC-MS chromatography vials A short column (5~30mm) with the same stationary phase as the analytical column in front of the analytical column can protect and extend the life of the column. It is worthwhile to use a guard column to lose some efficiency.
Usually the column life is up to 2 years when used correctly. Silica gel-based fillers can only be used in the pH range of 2~9. After the column has been used for a period of time, some substances with strong adsorption may remain on the top of the column. In particular, some colored substances are more likely to see the filler adsorbed on the top of the column. After the new column is used for a period of time, the top packing may collapse and the efficiency of the column may decrease. At this time, the filler may be added to restore the efficiency of the column.
After each work, the Zui is rinsed with a highly eluting eluent. For example, the ODS column should be flushed to baseline with methanol. When using a salt buffer solution as the mobile phase, rinse with a salt-free mobile phase after use. Compounds containing halogen elements (fluorine, chlorine, bromine) may corrode stainless steel pipes and should not be in contact with them for long periods of time. If the column is not used frequently on the HPLC instrument, it should be turned on for 15 minutes every 4~5 days 9mm LC-MS chromatography vials.
This is the end of the introduction of LC-MS column daily use and maintenance. I hope it can help you.