Manual for Normal Phase, Reversed-Phase Flash Column

Flash column is packed with modified silica material. Introducing alkaline solvents (pH>7.0) or acidic solvents (pH<2.0) into the column will damage the flash column.

1. Introduction

Flash column is filled with silica gel which is a reversed-phase or polar gel-linked silica matrix material. Flash columns in the form of silica gel are used for normal phase (non-aqueous) conditions.
Reverse phase (C8, C18, ODS, RP-8, and RP-18) is usually used for reverse-phase conditions (phase).

Polar glue-linked types (APS, diol, CN, and NH2) can be used in normal phase and reverse phase conditions according to the application purpose. It is recommended not to use the same column under very different conditions. This is because the properties of the stationary phase will change under certain conditions, so under other conditions, column efficiency will be affected. It is also not recommended to use silica gel flash columns under reversed-phase conditions or reverse-phase flash columns under normal-phase conditions. This is because the process will have poor repeatability (comparison between each injection and between columns ).

2. Flash column aging

Before starting the analytical work, the column must be properly aged. An improperly conditioned column may cause problems, such as poor column efficiency or changes in separation conditions.

A. Aging under reversed conditions
To age, this type of column, first rinse with acetonitrile or methanol, and then equilibrate with your chosen eluent.

Before shipment, each flash column has been tested and aged. Therefore, it is not necessary to rinse with water during the first use).

If additives (such as buffers or ion-pair reagents) are used in the mobile phase, it is recommended to use a mobile phase with the correct ratio but without these additives for buffer washing. Buffer flushing should be performed at a low flow rate first, and then the normal flow rate will be used last.

B. Aging under normal phase conditions
Flash column efficiency may be severely affected by the bonding of water to the stationary phase. Drying (activation) or wetting (inactivation) of the column may be required. The solvent used may be water-saturated or anhydrous. Dry the column can use anhydrous methylene chloride.

C. Special instructions for polar bonding columns
Since these columns can be used in reversed-phase or normal phase conditions, before aging, you must first check whether the eluent or eluent you want to use is miscible with the solvent packaged in the flash column. If these solvents are not miscible, you must first use a suitable buffer solvent to rinse.

3. Eluent

Pay attention to section 1 and section 2. Be sure not to use buffers with a pH lower than 2 or higher than 7, because they will change the nature of the stationary phase. It is best to use polar bonding columns between 3 and 5. Before use, the eluent should be degassed and filtered with a 0.5-micron filter membrane to avoid detection and pumping problems.

Be sure to check for microbial growth in the aqueous solution before you start using the system, otherwise, your column will become clogged and the column pressure will rise to unacceptable levels.

4. Flow and pressure

Note: Maximum pressure: stainless steel column: 4500psi; glass column: 3000psi
To increase or decrease the flow rate, small interval changes are required to prevent disturbance of the packed bed.

If you want to replace the column, reduce the flow rate to 0 and wait for the eluent to completely flow out of the column (2 minutes).
Removing the column without waiting for a decrease in pressure will damage the column.
The high column pressure is generally the result of incorrect use of the column.

The use of guard columns (see section 6) will prevent contaminants from depositing on the analytical column.

5. Sample preparation

The key to maintaining the longevity of the column is proper sample handling before injection. You must prevent the pumping of compounds that are highly hydrophobic/polar from the mobile phase into the column, whether from the mobile phase or the sample. In particular, it is forbidden to introduce particulate impurities. These will eventually increase the operating pressure and are very difficult or impossible to remove.

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.

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.