Strategies for Column Care
Column flushing is a simple strategy for Column Care that can extend column lifetime by washing strongly retained compounds from the column. At the end of each day’s use of the column remove any buffer and flush the column with 100% of a strong solvent.
Below is a list of general flushing and storage procedures for various types of columns and the removal of retained compounds.
Applying these procedures will restore column performance. Please always check for manufacturers’ recommendations before applying the flushing procedures below.
The method includes
Manual mechanical procedures:
Procedures for Silica based materials
Procedure for Silica columns
Procedures for bonded Silica columns
Procedure for Reversed Phase Columns based on silica ( C18, C8, C4, C1, Phenyl, CN and AQ types)
Procedure for Reversed Phase Columns based on wide pore silica for Protein and Peptide separation( C18, C8, C4, columns)
Procedure for silica based Anion Exchange Columns (SAX, WAX)
Procedure for silica based Cation Exchange Columns (SCX, WCX)
Procedure for Silica based Size Exclusion Columns for Proteins
Procedures for Silica hydride based materials
Procedures for Silica hybride based materials
Procedures for Zirconia/Titania based materials
Procedure for Reversed Phase Columns based on polymer coated zirconia
Procedures for Polymer based materials
Procedure for Reversed Phase Columns based on polymers ( C18, C8, C4, columns)
Column Volumn
Manual mechanical procedures:
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.
Disconnect and reverse the column
Connect the column to the pump but not to the detector
Flush with 10 to 20 column volumes of solvent at a high flow rate no higher than that used for the QC chromatogram
If altering the procedures below be sure to use miscible solvents for each successive step
General consideration for Silica based materials
Silica is used and basis for the manufacture of many different chromatography and solid phase extraction materials
Silica gel based materials have free silanol groups on the surface. The silanol groups have different acidities. Being weakly acidic in character the silanol groups can interact with certain analytes and matrix components. As the pKa of the silanol groups is roughly 4.5, ionisation can occur at intermediate pH values and thus the possibility of electrostatic interaction with cationic species exist. The older type A silica and some prep grade silica contain high concentration of metal ions (sometime more than 100 ppm). Metal ions impart even greater acidity to the silica surface and can also interact with chelating or scavenging compounds.
Residual silanol groups are more bothersome on non end-capped bonded silicas and on short chain bonded phases such as C2 or C4 phases.
Very hydrophobic compounds have a strong influence such as corn oils, highly aromatic materials and waxes can stick to the reverse phase materials and change their characteristics.
Biological fluids contain proteinaceous materials that can adsorb on the surface. Despite the chromatographers attempts to protect HPLC columns from foreign substances, eventually certain analytes-matrix combinations can affect stationary phases detrimentally. After a column is contaminated it will perform differently from a uncontaminated one. The can exhibit back pressure problems
Contaminated columns must be cleaned and regenerated to return to its original operating conditions. Below we will discuss cleaning procedures for various column materials
Procedure for straight Silica columns
Flushing:
IPA
MeOH
Ethyl Acetate
Column Storage :
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer.
Cap the column securely to prevent mobile phase evaporation
Procedure for silica based bonded Normal Phase columns (CN, NH2, Diol)
Flushing:
Chloroform
IPA
Methylene Chloride
Hexane
Column Storage:
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer
Cap the column securely to prevent mobile phase evaporation
Procedure for Reversed Phase Columns based on silica ( C18, C8, C4, C1, Phenyl, CN and AQ types)
Flushing :
a) Mobile phase without buffer
b) Methanol
c) Acetonitrile
d) Acetonitrile/IPA (75:25)
e) IPA
f) Dichloromethane
g) Hexane
In 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.
Do not flush with 100% water except for that carry polar embedded – groups so called AQ type Phases!
Flush with 10 to 20 Column Volume of solvent such as MeOH or MeCN but without buffer
Metal ions cause contamination! if you suspect metal ion contamination, flush with aqueous 0.05M EDTA, then water followed by above sequence.
Columns which use ion-pairing reagents should be dedicated to ion-pairing applications
Column Storage :
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer
Cap the column securely to prevent mobile phase evaporation
Procedure for Reversed Phase Columns based on wide pore silica for Protein and Peptide separation ( C18, C8, C4, columns)
Flushing
a) Mobile phase without buffer
b) Gradient of 10 – 90% B where
A = 0.1% TFA in water
B = 0.1% TFA in acetonitrile
Column Storage :
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer
Cap the column securely to prevent mobile phase evaporation
Procedure for silica based Anion Exchange Columns (SAX, WAX)
Flushing
Water
Methano
Chloroform
Methano
Water
Column Storage :
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer
Cap the column securely to prevent mobile phase evaporation
Procedure for silica based Cation Exchange Columns (SCX, WCX)
Flushing
Water (inject 4 x 200 ul DMSO during flush
THF
Column Storage :
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer
Cap the column securely to prevent mobile phase evaporation
Procedure for Silica based Size Exclusion Columns for Proteins
Flushing
For weakly retained proteins
0.1 M phosphate buffer pH 3
For strongly retained proteins
Gradient of 100% water to 100% MeCN in 60 min.
Column Storage :
Flush with 10 to 20 Column Volumes of solvent recommended by manufacturer
Cap the column securely to prevent mobile phase evaporation
Procedure for Silica hydride, Cogent Type C silica based columns
Flushing:
Overnight Storage
Rinse Column with 8 – 10 column volume of 40% DI Water containing 0.1% v/v formic acid (or 0.05% Phosphoric acid if you are using UV detector) and 60% Acetonitrile. If desired 100 % Acetonitrile can be used instead
Cap the column securely with the plugs provided to prevent mobile phase evaporation
Storage Long term
Rinse Column with 15 – 20 column volume of 40% DI Water containing 0.1% v/v formic acid (or 0.05% Phosphoric acid if you are using UV detector) and 60% Acetonitrile. If desired 100 % Acetonitrile can be used instead
Cap the column securely with the plugs provided to prevent mobile phase evaporation
Return column to the box
Store column at room temperature
Procedures for Silica hybride based materials