What are the steps to clean or regenerate columns?

Steps To Clean Or Regenerate Columns

Depositions on the inlet frit, in the column, or on the surface of the stationary phase can cause increased back-pressure, changes in retention times, or loss of column performance. A correctly applied washing procedure can resolve these problems most of the time.

You have more chances to recover your column’s original performance if you use the column regeneration procedures as soon as possible.

When washing the column with a reversed flow, the solvent-inlet end is collected with strongly adsorbed species, making the reversed flow beneficial.

According to popular belief, reversing the flow of a well-packed column will have no detrimental effects. The direction in which the flow was normally going is ‌the‌ ‌direction‌ ‌in‌ ‌which‌ ‌the‌ ‌column‌ ‌was‌ ‌originally‌ ‌packed.

It is important to note, however, that any irregular/amorphous original silicas can nonetheless pose problems when reversing the flow, since this could result in the restructure of the packed bed.

Reversing the flow on these irregular silicas must always be done with extreme caution.

Basically, check the regeneration procedures for different phases (RP, NP, Ion Exchange, etc.). If the original pressure is restored, then you should perform the CoA column test to determine the column’s peak symmetry and therefore its performance.

Let’s understand the steps to clean or regenerate columns.

Procedures for cleaning and regenerating SiliaChrom

Maintaining column efficiency and reliability for a long time is possible if proper care is taken. Information is provided on different methods to extend the lifespan of HPLC columns in this section.

First of all important to understand what is difference between cleaning and regeneration.

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What is the difference‌ ‌between‌ ‌cleaning‌ ‌and‌ ‌regeneration

Most of the time, we assume that the material eluted from a column or cartridge has all been removed. After a run, It is simply necessary to perform a second separation on the column after it has been run once or twice with the initial solvent mixture.

Even if the mobile phase is sufficiently strong to elute them during regular runs, some impurities that are strongly retained on the column can accumulate at the inlet. There can be a variety of problems resulting from such a circumstance, such as performance loss, back-pressure building up, peak tailing, retention time shift,‌ ‌or‌ ‌baseline‌ ‌drift.

The best way to prevent these symptoms is to have the column regularly cleaned before any of the above symptoms occur.

The procedure is simple, and chromatography system configuration does not have to be changed. If cleaning is not adequate, a more thorough treatment, i.e. regeneration, may be required to prevent the need for a column replacement.

Procedures for Cleaning and Regeneration

Cleaning procedures become more efficient as you use them more often. After running a sample that is deemed “dirty”, cleaning should be performed.

In order to prevent column clogging, users should perform regeneration before storing columns.In general, the flow rate is set lower in separation than during separation itself (20%‌ ‌to‌ ‌50%‌).

The column volume (packing volume included) in mL is equal to‌ ‌‌π ‌x‌ ‌[Column‌ ‌Radius‌ ‌in‌ ‌cm]‌2‌‌ ‌x‌ ‌[Column‌ ‌Length‌ ‌in‌ ‌cm]

ColumnSuggested Cleaning proceduresSuggested Regeneration Procedures
Reverse Phase Column (C8,C18,C1, C4, Amine,
Cyano, Phenyl, PFP.)
1. Water:ACN (95:5)- To remove buffer
2. Water:ACN (5:95)
3. Mobile phase used during phase separation
1. Water:ACN (95:5)
2. THF
3. Water:ACN (5:95)
4. Mobile phase used during phase separation
Normal Phase Columns
(Cyano, Diol, Amine)
Note: Not to Use Water
1. Methanol:Methylene chloride (50:50)
2. Ethyl Acetate
3. Mobile phase used during phase separation
1. Methanol:Methylene chloride (50:50)
2. IPA/Ethyl Acetate
3. Mobile phase used during phase separation
Unbonded Silica Columns (Silica)1. Hexane, IPA, Dichloromethane
2. Mobile phase used during phase separation
1. Hexane, IPA, Dichloromethane
2. Mobile phase used during phase separation
Ion Exchange Column
( SCX, SAX)
1. 5 mM phosphate buffer pH 7.0
2. Acetic acid:Water (10:90)
3. Water, Methanol & water
1. 5 mM phosphate buffer pH 7.0
2. Acetic acid:Water (10:90)
3. Water, Methanol & water
Steps to Clean Or Regenerate Columns

It is necessary to recommend that after restoration procedure in order to evaluate the column performance verify the column performance efficiency after running the column.

These are some of the Steps to Clean or Regenerate columns in pharmaceutical industry.

Steps To Clean Or Regenerate Columns
HPLC Columns

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