Bio-Rad Bio-Scale™ CHT™ Type I Columns - Manual (Page 5)

or diluted to the start buffer’s concentration. Buffer exchange can be accomplished using
Bio-Spin
® 6 or Bio-Spin 30 columns, Econo-Pac ® 10DG desalting columns, Bio-Gel ® P-
6DG size exclusion gel or the Econo-Pac P6 cartridge. The choice of product depends on
sample volume. Always centrifuge or filter the sample (0.2-0.45 mm filter) to remove par-
ticulates. Application of turbid or lipid-containing samples may reduce the column lifetime.
See Ref. 1 for further information about sample preparation.
Sample Load
The recommended sample load for each column is shown in Table 1. This amount may
vary somewhat depending on the actual sample composition. We do not recommend over-
loading the column as both resolution and column lifetime will decrease. For larger loads,
either change to a larger column or perform several chromatographic runs with a reduced
loading. Ideally, samples should be bound in a concentrated zone at the top of the column.
Higher sample loads produce a broad application zone in which components with less affin-
ity for the support are displaced by more tightly bound components. This may result in a shift
of certain peaks to an earlier elution position in the gradient.
2.3 Elution Conditions
Separations using hydroxyapatite are typically accomplished by increasing the phosphate
concentration of the eluent either as a “step” or as a “continuous” gradient. For certain sepa-
rations, varying the pH of the elution buffer in addition to its phosphate 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 phosphate concentration is undesirable. If Tris buffer must be used, do not
use at a concentration
≥
10 millimolar.
Gradient Volumes & Phosphate Concentrations
As a starting point for developing a separation, we recommend using the Bio-Scale CHT2-I
column with a simple gradient profile over 48 ml.
Protocol: Use a flow-rate of 2.0 ml/min. Following sample application, wash unbound pro-
teins from the column with 6 ml (3 bed volumes) of Start buffer A. For elution, use a gradi-
ent volume of 24 ml (12 bed volumes) to a phosphate concentration of 0.5 M (100% B). Hold
at 0.5 M for 6 ml before re-equilibrating the column with 12 ml of start buffer A. This gradi-
ent is shown schematically in Fig. 1. Once an initial separation has been performed and the elu-
tion position 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 shal-
lower gradient normally gives greater resolution but peak volumes are larger.
Where binding and elution conditions are well established for a particular biomolecule,
the sample concentration effect seen with hydroxyapatite means that this form of chro-
matography is ideal for the recovery of low abundance proteins from a large sample volume.
References 2 and 3 provides useful background information on the theory and applications
of hydroxyapatite chromatography.
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