Manual

Fig. 1. Schematic gradient for separation on UNO S-1 or UNO Q-1 column.
Use of Detergents
Cationic or non-ionic detergents may be used with the UNO Q support. Anionic or non-
ionic detergents may be used with the UNO S support. We recommend the use of the reduced
form of Triton X-100 to minimize UV absorption artifacts. It is essential to thoroughly equi-
librate the column with the detergent-containing buffer prior to sample application. Pay par-
ticular attention to pH, which influences the solubility of the various classes of detergents.
Problems may arise when using salt gradient elution if the starting conditions include deter-
gent below its critical micelle concentration (CMC). As the salt concentration (i.e. the counter-
ion concentration) increases, the CMC drops and eventually micelles will form. This may
cause a sudden increase in the UV baseline as the micelles themselves scatter light. We rec-
ommend using a concentration of detergent above the CMC during gradient elution. If sub-
sequent chromatographic steps (e.g. hydrophobic interaction) or other biochemical
manipulations will be affected by the presence of the detergent, then the initial choice of deter-
gent may be influenced by the availability of a protocol for its removal.
See Reference 2 for a more detailed explanation of the characteristics and use of detergents
in biology and biochemistry and their removal from biological samples.
Chromatography Pre-Runs
Prior to loading the sample, it is recommended running a blank gradient to check that the
column is clean. In addition, detergents and other buffer components will themselves elute as
sharp, UV-absorbing peaks during the gradient, complicating subsequent analysis of the chro-
matogram.
Column Hygiene
Careful preparation (especially filtration) of the sample and the buffers will maintain the
column performance and lifetime. Normally, washing with 1.0 M NaCl or KCl will remove
most bound components. However, if there is a significant decrease in column performance
(i.e. increasing backpressures or a significant drop in resolution) then a more extensive clean-
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Load Sample
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[Buffer] M
Volume (ml)
Load Sample
Scale-up
Recommended gradient
volumes:
S-1 or Q-1 10 to 30 ml
S-6 or Q-6 60 to 120 ml
S-12 or Q-12 120 to 240 ml