HPLC Column Cleaning Procedures (silica-based columns)

 The use of guard cartridges is recommended for protection of HPLC columns from both frit blockage and irreversible sample adsorption. However, over a period of time, columns may become contaminated by strongly adsorbed sample components. This may be indicated by a deterioration in column performance and an increase in back pressure.

It is recommended that the column efficiency is measured before and after the clean-up procedure. Please note that in cases of irreversible compound adsorption or column voiding, it may not be possible to regenerate the column.

 The following general procedures are recommended for regeneration of column performance.

1. Disconnect and reverse the column.
2. Connect the column to the pump, but not the detector.
3. Follow the appropriate flushing procedure, using 10 – 20 column volumes of each solvent. Always make sure that the last solvent used will be compatible with the mobile phase. Do not use a flow rate higher than that specified on the QC chromatogram.

Various Manufacturers may have different recommendation. Please find below the recommendation for Hichrom packed columns

Reversed-phase columns

(C18, C8, C4, Phenyl, CN, ‘AQ’ type)

1. a)    Mobile phase without buffer
2. b)    Methanol
3. c)    Acetonitrile
4. d)    Acetonitrile/IPA (75:25)
5. e)    IPA
6. f)     Dichloromethane
7. g)    Hexane

 It many cases, the sequence a) to e) may be sufficient. If step f) or g) is necessary, flush with IPA before returning to mobile phase.

 If metal ions are thought to be causing contamination, flush with aqueous 0.05M EDTA followed by water.Columns which have been used with ion-pairing reagents are best kept for this purpose.

Do not use  100% water for flushing conventional reversed phase as "dewetting" may  occur. The water forms beads on the surface of the packing which can trap  contaminants in the pores of the stationary phase. Use a mobile phase with at least 5% organic.

Reversed-phase protein/peptide columns

1. a)    Mobile phase without buffer
2. b)    Gradient of 10 – 90% B where       A = 0.1% TFA in water     B = 0.1% TFA in acetonitrile

Unbonded silica columns (SIL)

1. a)    IPA
2. b)    Methanol
3. c)    Ethyl acetate

Bonded normal-phase columns

(CN, NH2, Diol)

1. a)    Chloroform
2. b)    IPA
3. c)    Methylene chloride
4. d)    Hexane

Anion-exchange columns

(SAX, WAX)

1. a)    Water
2. b)    Methanol
3. c)    Chloroform
4. d)    Methanol
5. e)    Water

Cation-exchange columns

(SCX, WCX)

1. a)    Water (inject 4x 200ml DMSO during flush)
2. b)    Tetrahydrofuran

Size exclusion columns for proteins

For weakly retained proteins

1. a)     0.1M phosphate buffer, pH 3

For strongly retained proteins

1. a)     Gradient of 100% water to 100% acetonitrile over 60 minutes 

Column Volumes (ml)

I.D.	Length 50 mm	Length 150 mm	Length 250 mm
2.1 mm	0.17	0.52	0.87
3.2 mm	0.4	1.2	2.0
4.6 mm	0.83	2.5	4.2
10.0 mm	3.9	11.8	19.6
21.2 mm	19.3	57.8	96.3