The effect of flow rate on the dynamic binding capacity of the Macro-Prep DEAE support. A sample containing 3 mg/ml BSA was separated on a 1 x 10 cm (3 ml) Macro-Prep DEAE column. Sample was loaded onto the column in 10 mM Tris-HCl, pH 7.6 (buffer A) and was eluted in 1 M NaCl in buffer A. Sample was applied until the eluting buffer had an A280 value 50% of the incoming sample.
The effect of flow rate on separation and peak symmetry. A 5 ml sample containing 3.65 mg/ml each myoglobin, conalbumin, BSA, and soybean trypsin inhibitor was loaded onto a column containing 6.56 ml Macro-Prep DEAE support. The sample was loaded in 50 mM Tris-HCl, pH 7.6 (buffer A) and eluted in a gradient of 0–1 M NaCl in buffer A as follows: buffer A for 15 ml, 0–0.35 M NaCl in 85 ml, 0.35–0.65 M NaCl in 15 ml, 0.65–1 M NaCl in 15 ml.
Comparing Purifications of Different DEAE Supports
Sample separation with the Macro-Prep DEAE support and two other commercially available DEAE supports. A 100 µl sample containing 9 mg/ml each of myoglobin, conalbumin, ovalbumin, BSA, and soybean trypsin inhibitor was loaded onto a 0.5 cm ID column containing 2 ml of support. Sample was loaded onto the column in 50 mM Tris-HCl, pH 7.6 (buffer A) and was eluted with 1 M NaCl in buffer A. The flow rate was 153 cm/hr (0.5 ml/min). Scales offset for comparison.
Macro-Prep DEAE Support: Scaling Up Purification Process
Scalability of separations on Macro-Prep DEAE support. Separation of a yeast enzyme concentrate (Sigma) under various conditions of scale. A, a 50 mg sample was separated on a 7.4 ml column (10 mm ID) using a flow rate of 78 cm/hr (1 ml/min) in an analytical flow cell; B, a 500 mg sample was separated on a 64 ml column (25 mm ID) using a flow rate of 78 cm/hr (6.4 ml/min) in an analytical flow cell; C, a 1 g sample was separated on a 64 ml column (25 mm ID) using a flow rate of 305 cm/hr (25 ml/min) in a preparative flow cell. For all separations, sample was loaded in 50 mM Tris-HCl, pH 8.3, washed with 1 column volume of the same buffer, and eluted in a linear gradient of 0–0.35 M NaCl in the same buffer for 22.5 column volumes.
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