UltiMate 3000 RSLCnano – Standard Applications
34
2.9.3 Installation Tips
• Follow the General Recommendations for Applications (→ page 6).
• The design of the nano trap column provides the easiest connections, but must only be
used in forward flush operation. Please check the indicated flow direction when
installing a nano trap column.
• If the loss of hydrophilic peptides is observed, the concentration of acetonitrile in the
loading solvent can be decreased down to 99/1 water/ACN + 0.025% TFA.
• If too much hydrophobic secondary interaction is observed on the IEX column, the
amount of ACN can be increased up to 5% or 10%. This will be at the expense of the
loading efficiency for hydrophilic peptides on the trap column.
• The loading time and desalting time are highly dependent on the sample quantity and
purity. They can be adjusted to meet customer needs. However the desalting step must
be kept long enough to avoid the formation of adducts between salt and sample.
• To limit the breakthrough on the SCX column, the loading solvent must contain as little
TFA as possible (for example, max. 0.025%) or FA should be used.
• The salt plugs listed here have been chosen to check the system and work fine for the
separation of the protein mix digest. The best sequence of plugs will highly depend on
the affinity of the peptides present in the sample with the IEX column.
• It is useful to inject several times the last salt plug or to prepare one with a higher salt
concentration in order to make sure that the column is clean before repeating an
experiment.
• After each series of injection, it is useful to wash the column with consecutive 2M salt
injections. When the salt is washed out from the column, a 60/40 water/ACN solution
can also be used to wash out peptides which might be bound to the column due to
hydrophobic interactions.
• SCX column regeneration can be performed by flushing the column overnight with a
10 mM phosphate buffer pH 3, 20% ACN and 600 mM NaCl salt solution. Please
ensure that the column is conditioned with loading solvent before using it in the 2D salt
plug application.
Tips: The combination of salts, buffers and organic solvents can result in precipitation.
When preparing the solution described above, carefully evaluate if salts precipitate.
Dilute or remove precipitation before using the wash solvent, to prevent system
damage.
To Next Page
Loading...
