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6.1.2 Predefined methods
The predefined methods that are available are described in the table below.
Description Method
After equilibration and sample application, the protein of
interest is adsorbed to the column ligand. After a wash to
remove unbound sample, elution is performed either by using
a buffer containing a competitor to displace the protein of
interest, or by changing the pH or ionic strength. Finally, the
column is re-equilibrated with start buffer. Affinity
chromatography
(AC)
After equilibration and sample application, negatively charged
proteins are adsorbed to the column ligand. After a wash, to
remove unbound sample, elution is performed using a gradient
of increasing salt concentration (of e.g., NaCl). Finally, the
column is washed and re-equilibrated with start buffer. Anion Exchange
Chromatography
(AIEX)
After equilibration and sample application, positively charged
proteins are adsorbed to the column ligand. After a wash, to
remove unbound sample, elution is performed using a gradient
of increasing salt concentration (of e.g., NaCl). Finally, the
column is washed and re-equilibrated with start buffer. Cation Exchange
Chromatography
(CIEX)
After equilibration and sample application, elution is performed
using a pH gradient. The proteins separate and elute according
to their isoelectric points. Finally, the column is re-equilibrated. Chromatofocusing
(CF)
The column is filled with a cleaning solution. Select inlet
positions. Enter the solution identity, volume, flow rate and
incubation time. By adding steps, several cleaning solutions
can be used. Suggestions for cleaning steps are available for
a number of column types. Column CIP
After equilibration of the column, sample is injected via a
capillary loop and eluted isocratically. A non-adsorbing sample
like acetone or salt should be used. After the run, calculate
column performance in the Evaluation module. The efficiency
of the column is determined in terms of height equivalent to
a theoretical plate (HETP), and the peak asymmetry factor (A
s
).
The result is logged in the column logbook.
Column
Performance Test
The column is filled with buffer solution. Select inlet positions.
Enter the solution identity, volume, flow rate and incubation
time. By adding steps, several preparation solutions can be
used. Column
Preparation
Getting Started with ÄKTAavant and UNICORN 6 28-9440-69 AB 65
6 Create a method
6.1 Guide to method creation
6.1.2 Predefined methods