Manual
Elution Conditions
Separations by ion-exchange chromatography are typically accomplished by increasing
the salt concentration of the eluent either as a “step” or as a “continuous” gradient. For many
separations, varying the pH of the elution buffer in addition to its salt concentration may be
advantageous. Generally, it is best to choose initial pH and ionic strength conditions such that
the protein of interest elutes early in the gradient. This is especially true for labile proteins or
where a higher salt concentration is undesirable.
Choice of Elution Salt
Sodium or potassium chloride are the most common elution salts and are recommended
for use with UNO columns. Other ions may be used and may show different selectivities
based on their relative elution strengths and chaotropic nature.
The following ions are shown below in order of elution strengths:
Cations for UNO S Columns
Barium > Calcium > Magnesium > Potassium > Sodium > Lithium
Anions for UNO Q Columns
Citrate>Sulfate>Iodide>Chloride>Formate>Acetate
See Reference. 1 for a more detailed explanation of ion selectivity in chromatographic sep-
arations.
Gradient Volumes & Salt Concentrations
As a starting point for developing a separation, we recommend using the UNO S or UNO Q
columns with a simple gradient profile over 10 column volumes.
Protocol: Use a flow-rate within the specified range in Table 1. Following sample appli-
cation, wash unbound proteins from the column with 4 column volumes of Start buffer A. For
elution, use a gradient volume of 10 column volumes to a NaCl concentration of 0.5 M
(50% B). Follow this segment of the gradient by raising the salt concentration to 1.0 M
(100% B) over 4 column volumes and then hold at 1.0 M for 4 column volumes before re-
equilibrating the column with 5 column volumes of start buffer A. This gradient is shown
schematically in Figure 1. Once an initial separation has been performed and the elution posi-
tion of the protein of interest determined, the gradient composition and volume is adjusted to
achieve maximum resolution. Normally, a gradient volume of 10 to 20 ml per ml of column bed
volume is sufficient. The slope of the gradient will affect resolution. A steep gradient will result
in relatively small peak volumes but short peak-to-peak distances. A shallower gradient nor-
mally gives greater resolution but peak volumes are larger.
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