An increased back-pressure, altered retention times and loss of column performance are all symptoms of deposits in the column or on the surface of the stationary phase. Most of the times, these problems can be overcome by the use of a correctly applied washing procedure. What should also be kept in mind is that in most cases the sooner washing (regeneration) of the column is performed, the better.
Strongly adsorbed species are collected at the solvent-inlet end of the column and in many cases it is a benefit to use a reversed flow during washing. Worth mentioning is that a well packed column should not loose performance as a result of reversed flow.
Deposits are most commonly present as surface adsorbed species or precipitations.
|Suspected Deposition/Impurity||Recommended solvents||Examples|
|Lipohilic||Strongly Lipophilic solvents||Al, Tol|
|Polar (Small peptides)||Versatile solvents||DCM, THF, DMF|
|Strongly Polar / Ionic (water sol.)||Aqueous solvents||50:50 DMF/w, THF/w|
|Ion-exchange supressing mixtures||DMF/AA (1%), DMF/TFA (0.1%)|
|Macromolecular Depositions (Protein/ Large Peptide precipitations)||Strongly interaction breaking mixtures||DMF/1% SDS(aq), ACN/1% SDS(aq), Alc/AA, Alc/NEt3 (0.1%) Alc/10-100mM aqueous NaOH*|
|Highly aqueous mixtures||Alc (10%)/w|
Al = Alkanes,
Tol = Toluene,
DCM = Dichloromethane,
THF = Tetrahydrofuran,
DMF = Dimethylformamide,
AA = Acetic acid,
TFA = Trifluoroacetic acid,
ACN = Acetonitrile,
Alc = Alcohol (MeOH, EtOH),
w = water,
NEt3 = Triethylamine,
SDS = Sodiumdecylsulphate
* Use as last measure for not more than 10 column volumes, also end by acidifying the phase sing Alc/1% aqueous AA 50:50.
For polar phases, especially bare silica, very strong adsorption of polar residues is common. While under normal phase conditions highly polar impurities are not eluted, however the solution to this is the use of high levels of protic solvents, possibly in combination with acid.
If there are problems with altered retention times, first check the phase - water (moisture) equilibrium. The use of dry solvents could gradually "dry" the stationary phase, resulting in altered retention times. This is especially rue for bare silica.
?: While using highly polar solvents, especially for bare silica, the stabilization of the system after washing could take a significant amount of time. This is due to the silica surface - water equilibrium being distorted.
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