Manual for Normal Phase, Reversed Phase Flash Column ( Part 2)

6. Guard column

Be sure to use a guard column, because sample and eluent contamination may increase column pressure and affect selectivity. Generally, the selection of guard column is based on the principle of the same model, so that the packing material of the column is similar to the material of the chromatographic column used for analysis. The guard column needs to be replaced when the column pressure increases or the column efficiency is observed to decrease.

7. Injection volume and concentration

The maximum sample load of the column depends on the type of column, the conditions used, and the type of sample. It is difficult to give general instruction. It is easier to suggest the injection volume (see typical values ​​in the table).

Injecting a sample that is too large or too concentrated will cause peaks to broaden or merge.
empty flash column chromatography
8. Temperature

Flash columns are best used in column ovens. Repeatability depends on temperature control. The optimal temperature is related to the specific application. Temperature affects the linear velocity of eluent flow. When using ChromSep glass columns, be sure to adjust the flow rate to keep the pressure below 3000psi.

9. Store

Be sure not to store the column when the column is filled with buffer or other salt-containing eluents. The storage solvent should contain at least 20% organic solvent to prevent the growth of bacteria.

10. Possible causes of column efficiency loss

a. Additional peak broadening. When using small diameter or short length columns, peak broadening may be more obvious. Make sure that the length and the inner diameter of the pipeline are kept to a minimum. Check the injection volume and detector check whether the cell volume is suitable for the column volume.
b. The equilibration time for elution is insufficient.
c. Incorrect column temperature.
d. Incorrect correction density.
e. Bed compression. An excessive elution flow rate was used. Reverse the column and use a low flow rate.

11. Loss of column efficiency and/or high back pressure

a. Particles accumulate on the sinter or resin bed (both of which increase the backpressure). If the column pressure increases, disconnect the column from the injector and run the pump to verify that the source of the backpressure is indeed the particle contamination from the column (sample, eluent, and system). Invert the column and flush the column with reverse flow. If this does not solve the problem, replace the inlet filter or sieve plate.
b. Microorganisms grow in the eluate. Turn the column upside down and try to flush the contaminants out of the column with a reverse flow. Replace the inlet filter or sieve plate.
c. There are protein fat, grease pollution, or polar compound pollution. Regenerate the column (see section 12).

12. Regeneration

(1). Regenerated reversed-phase column

a. First turn the column upside down.
b. Rinse the column at a flow rate of about 40% of the optimal flow rate for about 45-60 minutes.
The order of the solvents used should be water-methanol-isopropanol-dichloromethane-isopropanol-methanol-water.
c. Turn the column back to the normal direction and equilibrate with the eluent used for the analysis.

Note:
When using another elution method, be sure to start rinsing with water to remove the buffer to ensure that the subsequent eluent is miscible.

(2). Regenerated silica gel column

a. First turn the column upside down.
b. Flush the column at a flow rate of about 40% of the optimal flow rate for about 45-60 minutes. The order of the solvents used should be isooctane (or hexane)-ethyl acetate-dry isooctane (or hexane).
c. Turn the column back to the normal direction and equilibrate with the eluent used for the analysis.

(3). Regenerated polar bonding column

Depending on the conditions used, either reversed phase conditions or silica gel column conditions can be used.

Note: Regeneration will reduce the efficiency of the column. In addition, never use ketones or aldehydes to wash the amino column because it may react with the stationary phase. Tetrahydrofuran can be used as an alternative.