Bio-Rad ENrich™ High-Resolution Size Exclusion Columns - Manual (Page 8)

4 ENrich SEC Instruction Manual
2.3 Sample considerations
1. Always filter (0.22–0.45 μm filter) and/or centrifuge your
sample to remove any particulates.
2. In size exclusion chromatography, mass loading is less critical
than the volume of the sample. As a general rule, to obtain
maximum performance, the sample volume should not
exceed 1% of the bed volume (that is, ~0.25 ml for a 24 ml
CV) and the sample concentration should not exceed 20 mg
protein/ml. Larger volumes can be applied, but resolution may
decrease.
2.4 Sample Elution
All species within the sample should elute within 1 full CV. The flow
rate for optimal resolution depends on sample composition and
purity. In general, lower flow rates provide greater performance.
Optimal flow rates for both ENrich SEC 70 and ENrich SEC 650
columns are 0.5–1.25 ml/min (see Table 1).
2.5 Column Calibration
Use the Bio-Rad size exclusion standards (catalog #151-1901).
One vial contains 18 mg of a lyophilized mixture of thyroglobulin
(Mr 670,000), bovine
γ-globulin (Mr 158,000), chicken ovalbumin
(Mr 44,000), equine myoglobin (Mr 17,000), and vitamin B12
(Mr 1,350).