Bio-Rad Macro-Prep 25 S Media - Manual (Page 6)

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Section 4
Preparation for Use
Macro-Prep 25 supports are supplied fully hydrated in 20% (v/v) ethanol. Wash the
ethanol storage solution from the support with deionized water before packing the
column. Small volumes of the Macro-Prep 25 supports are easily washed in a
Büchner funnel with 4–5 volumes of deionized water. For large volumes (for
example, >1 liter), it is more convenient to pour the desired amount of support into
a suitable container, allow the support to settle, and decant the ethanol solution.
Add one volume of deionized water, resuspend the support, allow it to settle, and
decant the supernatant. Repeat this procedure with deionized water (approximately
4–5 times, or until the ethanol is removed) and then pack the column (see Section
5 Column Packing). The support may be prepared by decanting the ethanol
solution and resuspending it in the application buffer prior to column packing.
Section 5
Column Packing
The Macro-Prep 25 S and Macro-Prep 25 Q ion exchange supports have a
nominal particle size of 25 µm, thus affording high resolution separations at
medium pressures. In order to obtain the most resolution from these supports, a
well packed column is essential. Empty columns designed for use at medium
pressures (up to 100 PSI/7 bar) such as the Bio-Scale
™ MT columns (see Section
9 Ordering Information) from Bio-Rad Laboratories are recommended. These
columns must be used with bed supports with 10 µm or smaller pore sizes to
prevent high backpressure due to bed support plugging.
Note: NaCl solution or buffers containing 0.5–1.0 M NaCl is recommended for
optimum column packing.
1. Slurry, approximately 1:1 (v/v), the prepared matrix in 0.5–1.0 M NaCl and
degas.
2. Close the outlet of column. Fill 10% of the column with degassed 0.5–1.0 M
NaCl. Remove any air bubbles trapped in the bed support or the column end
piece.
3. Add an appropriate amount of the support in a homogeneous slurry to the
column.
4. Fill the remainder of the column with 0.5–1.0 M NaCl.
5. Connect the flow adapter to the pump, fill it with 0.5–1.0 M NaCl, and make
sure it is free from air bubbles. Attach the flow adapter to the column.
Inserting it at a slight angle makes it easier to avoid trapping air bubbles.
6. Open the column outlet and pump 4–5 column volumes (CV) of 0.5–1.0 M
NaCl through the column at the highest flow rate your pump/column system
permits. A dense, well-packed bed gives better chromatography.
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