Bio-Rad Media Sampler Pack - Manual (Page 7)

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2.3 CFT pH Change and Equilibration
The most rapid method for changing the pH of CFT is with 2.5–5 CV of >200 mM
sodium phosphate at the desired pH.
If reequilibrating from the sanitizing-in-place (SIP) solution or storage in strong base,
use 400 mM sodium phosphate at the desired pH. Once the desired pH is
achieved, wash with equilibration buffer, e.g., 10 mM sodium phosphate, at the
same desired pH.
2.4 Column Qualification
The packing efficiency of CFT can be evaluated by calculating HETP (height
equivalent to theoretical plate) and peak symmetry. Use 2–5% (v/v) acetone,
0.1% (w/v)
DL -tryptophan, or salt. Due to the dynamics of counterion exchange, a
concentration of at least 0.15 M NaCl should be included in the equilibration buffer
if salt is being used. For calculation of HETP, up to 1.75 M NaCl may be used.
Section 3. Chromatography
CFT chromatography is performed similarly to CHT chromatography; phosphate
buffer systems with increasing concentrations of phosphate elute bound
molecules. The following is the recommended protocol for a typical separation:
1. Samples containing high concentrations of phosphates should be diluted to
the concentration of the starting buffer (typically 10 mM phosphate, pH 6.8).
2. Buffer solutions and samples should be filtered through a 0.2–0.45 µm filter
before use.
3. Apply the sample and allow unbound material to pass through the column.
4. Elute with a gradient of increasing concentration of phosphate buffer
(10–400 mM). If performing chromatography at
≤12°C, use potassium
phosphate because sodium phosphate exhibits lowered solubility at these
lower temperatures.
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