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5 Create a method
5.1 Guide to method creation
5.1.2 Predefined methods
5.1.2
Predefined methods
The predefined methods that are available are described in the table below.
Method
Description
Affinity Chromatography (AC)
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 reequilibrated with start buffer.
Anion Exchange
Chromatography
(AIEX)
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.
Cation Exchange
Chromatography
(CIEX)
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.
Chromatofocusing (CF)
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.
Only available for ÄKTA avant 25.
Column CIP
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 Performance Test
After equilibration of the column, sample is injected 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 (As). The result is logged in the column
logbook.
Column Preparation
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.
Getting Started with ÄKTA avant 29-0351-83 AA
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