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Frequently Asked Questions

How can the columns be cleaned / regenerated?

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.

RP (Hydrophobic Phases)

Deposits are most commonly present as surface adsorbed species or precipitations.

Examples of recommended solvents
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
Polar, positively
charged (amines)		 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*
Salt/Buffer
precipitations		 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.
Regeneration protocol
  1. Choose solvent/solvent mixture based on impurity. If not known, assume strongly polar / ionic species in order to avoid further precipitation.
  2. Use a low flow rate (10% of normal flow rate), possibly in reversed flow mode and at slightly elevated temperatures (< 40°C), for up to ten column volumes.
  3. If problems with back-pressure, occasionally check progress at normal flow.
  4. If the problem remains apply conditions for polar, positively charged and/or macromolecular depositions. If SDS is used, wash thoroughly with pure THF, DMF or ACN afterwards.
  5. If problems persist please consult our technical support for further advice.

NP (Polar Phases)

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.

Regeneration protocol?
  1. Eqilibrate the column using an aqueous-lipophilic mediating solvent such as THF.
  2. Use a low flow rate (10% normal flow rate) of pure Alcohol (MeOH), possibly in combination with acid (Acetic acid or Formic acid, 1-5%), for up to ten column volumes.
  3. If the problem persists, use a mixture of Alcohol and water, with added acid, under the same conditions as in 2.
  4. As a final measure: 0.5-1.5% NH3(aq), followed by 0.5-1.5% aqueous acid (HCl, AA etc.), water, alcohol, THF and finally the actual eluent.
  5. If problems persist please consult our technical support for further advice.

?: 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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