Balance, regeneration and maintenance of chromatographic col

Before using a new column, you should conduct a performance test on your own liquid chromatograph, that is, to determine the column efficiency by using the test conditions and samples in the inspection report attached to the column. In addition, the chromatographic column should be tested frequently in the future.
Installation of ferrule column (without pre column)
1. Put the ferrule frame into the column core;
2. Insert two jacket sheets into the groove of the column core to make the column core higher than the jacket;
3. Push the ferrule frame which has been sheathed on the column core upward until it is higher than the jacket piece;
4. Screw the ferrule cap and ferrule frame together, and then tighten them by hand;
5. Then connect the other end of the column in the same order;
6. Connect to the liquid chromatograph and screw the peek connector by hand; if it is a stainless steel joint, use a special wrench.
Note: when using the ferrule column, the ferrule at both ends should always be connected to the column core. No matter you are balancing the chromatographic column or cleaning, you can not take off the card sleeve at any time, otherwise it will cause the loss of packing.
Installation of ferrule column (with pre column)
1. Put the ferrule frame into the column core;
2. Insert two jacket sheets into the groove of the column core to make the jacket higher than the column core;
3. Push the ferrule frame which has been sheathed on the column core upward until it is higher than the jacket piece;
4. Put the "bullet head" precolumn into the ferrule piece;
5. Screw the ferrule cap and ferrule frame together, and then tighten them by hand;
6. Then connect the other end of the column in the same order;
7. Connect to the liquid chromatograph and screw the peek connector by hand; if it is a stainless steel joint, use a special wrench.
Replace the filter screen of chromatographic column and glass wool filter (the chromatographic column can be repaired at the same time)
Note: before taking out the filter screen and glass sheet of the reversed phase column core, the chromatographic column should be washed with water and methanol / acetonitrile, and the head of the repair tool should also be dipped with a small amount of methanol / acetonitrile, so as to avoid taking out the filler in the column when taking out the filter screen and glass wool filter sheet.
1. Set 2 of the repair tools into the top of the column core;
2. Screw 3 of the repair tool into the core of the column covered with 2, and turn it clockwise until it is tightened;
3. Hold the column core with one hand and pull it out gently with the other hand. 3. Take out the filter screen at the top of the column core;
4. Gently take out the glass wool and the contaminated packing under the filter screen with a small shovel;
5. Wet the new filler with methanol, then fill the excavated part and flatten it;
6. Install a new glass wool filter screen and press the glass wool into the top of the column core with 4 of the repair tools;
7. Put 2 on the top of column core, and then put the filter screen in;
8. Press down, then take down 2, and then use 4 to flatten the edge of the filter screen.
Equilibrium column
The reversed phase chromatographic column was stored in acetonitrile / water after factory test. Make sure that the mobile phase you use is miscible with acetonitrile / water. Since the chromatographic column may dry out during storage or transportation, a methanol or acetonitrile equilibrium column with 10-20 times the volume of the column should be used before analyzing the sample with the mobile phase; if the mobile phase you use contains buffer salt, you should pay attention to "transition" with pure water.
Silica gel column or polar column is stored in n-heptane after factory test. If an aqueous mobile phase is required for the column, equilibrate with ethanol or isopropanol before using the mobile phase.
How to balance the column?
In the process of equilibrium, the flow rate is increased slowly.
The column was equilibrated with the mobile phase until a stable baseline was obtained (if the buffer salt or ion pair reagent level is low, it will take a long time to equilibrate).
Regeneration of chromatographic column
A modest pump should be used for column regeneration. We recommend that the pump on your HPLC should not be used.
Table 1 recommended solvent volumes for flushing
Column size column volume volume volume of solvent used
125-4 1.6ml 30ml
250-4 3.2ml 60ml
250-10 -20ml 400ml
Please select your regeneration method according to the following table:
The regeneration of polar stationary phases (such as Si, NH2, diol based chromophore fillers) was studied
Heptane chloroform ethyl acetate acetone ethanol water
The regeneration of nonpolar stationary phases (such as RP-18, RP-8, CN, etc.) was studied
Water & acetonitrile chloroform (or isopropanol) acetonitrile water
0.05m dilute sulfuric acid can be used to clean the contaminated column.
be careful:
In the regeneration of NH2 modified chromatographic column, NH2 may exist in the form of ammonium ion, so it should be washed with 0.1M ammonia water after water washing, and then washed with water until the alkali solution completely flows out.
If the simple organic solvent / water treatment can not completely remove the impurities adsorbed on the silica gel surface, washing with 0.05m dilute sulfuric acid is very effective.
Maintenance of chromatographic column
1. Using pre column to protect the analytical column (silica gel has a certain solubility in polar / ionic mobile phase);
2. The pH stability range of most RP-HPLC columns is 2-7.5, which should not exceed the pH range of the column as far as possible;
3. Avoid drastic changes of mobile phase composition and polarity;
4. The mobile phase must be degassed and filtered before use;
5. If polar or ionic buffer solution is used as mobile phase, the column should be washed clean after the experiment and stored in acetonitrile;
6. The pressure rise is a signal that the precolumn needs to be replaced.
For more related technologies, please consult MS technologies technicians