Silica columns (normal or straight phase chromatography) 
 
Regeneration: 

1. Flush with 30 ml hexane
2. Flush with 30 ml dichloromethane
3. Flush with 30 ml isopropanol
4. Flush with 30 ml dichloromethane
5. Put the column in the original position
6. Equilibrate with mobile phase

Polar bonded columns (cyano-, amino-, diol-) 

Regeneration for use with apolar solvents

1. Reverse the flow direction of the column
2. Flush with 30 ml hexane
3. Flush with 30 ml dichloromethane
4. Flush with 30 ml isopropanol
5. Flush with 30 ml dichloromethane
6. Put the column in the original position
7. Equilibrate with mobile phase  
   

Regeneration for use with aqueous solvents:

1. Reverse the column
2. Flush with 30 ml water
3. Flush with 30 ml methanol
4. Flush with 30 ml isopropanol
5. Flush with 30 ml methanol
6. Put the column in the original position
7. Equilibrate with mobile phase

Reversed phase columns are normally used with aqueous eluents. Apolar pollutants are easily adsorbed on this type of phases. When acidic compounds are separated it is very important that the pH of the eluent is low (ion suppression). Reproducibility of retention for a large extent depends on reproducibility of the eluent pH.

To condition this type of columns:

1. Rinse with water (20 ml),
2. Rinse with acetonitrile or methanol (20 ml).
3. Then equilibrate with the eluent of choice.  

Regeneration:

1. (Preferably) wash the column in back flush
2. Flush with 30 ml methanol – water = eluent ratio (v/v)
3. Flush with 30 ml methanol
4. Flush with 30 ml isopropanol for strongly retained impurities only.
5. Flush with 30 ml methanol
6. Put the column in the original position
7. Equilibrate with methanol – water followed by the mobile phase 
   

Silica based ion exchange columns usually contain quaternary amine groups as functional moieties for anion exchange (AX) or sulphonic acid groups for cation exchange (CX) chromatography. These functional groups are often connected to the silica back bone by apolar spacer arms.

Sample pollutants which should be avoided are strong anions which are adsorbed by the quaternary amines or strong cations which are adsorbed by the sulphonates. Strongly apolar compounds interact with the spacer arms and do not come off because of the low organic modifier content usually applied. To condition this ion exchange columns first rinse with methanol (20 ml), followed by HPLC water (20 ml). Then equilibrate with the eluent of choice.  

Regeneration:

1. Rinse with 30 ml 1 M ammonium nitrate at 0.4 ml/min
2. Rinse with 30 ml water at 0.4 ml/min
3. Rinse with 30 ml methanol at 0.4 ml/min Check your column manual if this modifier is allowed
4. Rinse with 30 water at 0.4 ml/min
5. Rinse with 30 ml of eluent at 0.4 ml/min
6. Invert the column to the original position
7. Equilibrate with the eluent

Most polymer based columns for HPLC are based on polystyrene/divinylbenzene. Polymer based columns cannot withstand high pressures to the same extent as silica columns. Normally one should not work above 150 bars. The pH resistance of polymer columns is higher than those of silica columns (usually from pH 1 to pH 14).

Organic modifiers however can only be used as indicated by the manufacturer.
Polymer columns must always be stored in such a way that they are prevented from drying out. When a polymer dries out it shrinks, resulting in total disturbance of the packing bed.
In all cases refer to the column manual for proper use and regeneration!

Eluents commonly used are tetrahydrofuran (THF), toluene, chloroform and 1,2-dichlorobenzene. Less common apolar solvents such as methylene chloride can also be used with most columns.
For separating many modern polymers more polar or stronger eluents are required, such as dimethylformamide (DMF), N-methylpyrollidone (NMP) and o-chlorophenol (o-CP). Not all polymer packings can handle these solvents, and guide lines of the manufacturer must be followed!

Switching from apolar to stronger solvents and vice versa can be critical, so an intermediate polarity solvent must be chosen. Again refer to the column manual for proper use and regeneration procedure. Unstabilized THF (for example, HPLC grade) is not always recommended as eluent or storage solvent. Peroxide formation may change the packing surface which results in tailing peaks and adsorption of polar polymer. When this process has already taken place, it is in some cases possible to regenerate the packing by pumping through 10 column volumes of 0.1 w/v/ % triphenylphosphine in THF.
 

For reversed phase chromatography usually aqueous mobile phases with polar organic modifiers in the concentration range of 0 to 100% can be used. It is not recommended to use 100% aqueous eluents because this might cause loss of efficiency. For improving the peak symmetry and efficiency when using methanol/water eluents, addition of 10% of tetrahydrofuran or acetonitrile might help. 

Column regeneration:

1. When used in the reversed phase mode pump consequently at a flow of 0.5 ml/min for 30 min:
   10% in THF in deionized water
2. THF
3. isopropanol
4. THF
5. water / modifier (mobile phase)

Anion Exchange Columns (polymer based stationary phases)

Just like silica based AX columns, these columns in most cases contain quaternary amines as functional groups, bonded to a polymer based porous particle.
 
Column regeneration:

1. Reverse the column, if allowed (see manual)
2. Pump 30 ml deionized water through the column
3. Pump 30 ml 0.1 M NaOH through the column
4. Pump 30 ml deionized water through the column
5. Pump 30 ml 0.1 M acetic acid or HCl through the column
6. Pump 30 ml deionized water through the column
7. Pump 30 ml isopropanol with 0.1% trifluoroacetic acid through the column
8. Pump 30 ml deionized water through the column
9. (Put the column back in the original position)
10. Equilibrate with the eluent  
    

Sulphonated polystyrene/divinylbenzene columns can serve different purposes. One of the purposes is the use for cation chromatography. 

Column regeneration:

1. Invert the column, if allowed (see manual)
2. Pump 30 ml deionized water through the column
3. Pump 30 ml 0.01 NaOH through the column
4. Pump 30 ml de'ionized water through the column
5. If contamination by fats, oils, or similar organics is suspected, pump 60 ml acetonitrile/water 80 : 20 (v/v), followed by 30 ml deionized water. Adjust flow rate, if necessary, to prevent column pressure from exceeding 150 bar.
6. Prepare a special flushing solution by dissolving 2.1 g distilled ethylenediamine (EDA) plus 19.2 g reagent citric acid and diluting to 1.0 liters with distilled water. Filter through 0.45 μm membrane and degas. This solution is 35 mM EDA plus 100 mM citric acid. Pump 40 ml of this solution through the column.
7. Put the column back in the original position
8. Equilibrate the column in whatever eluent you normally use at flow rates you normally use until baseline stabilizes (usually 30 - 60 min). If the column does not return to normal performance, it may be permanently damaged and requires replacement.
    
    

As mentioned before, for cleaning and regeneration procedures it is best to follow the column manual carefully or contact your column supplier.

Reversing the column flow will increase the effect of the cleaning process, but it is not always recommended for certain types of columns.

A relatively old column, which does not produce double peaks, should not be reversed. On the other hand, columns with double peaks in the chromatograms can be restored very often just be reversing the flow of the mobile phase in the column. In those cases it is best to order a new column during the time that the “old one” is still working well.