Separation Options with Superdex

Extracted from Size Exclusion Chromatography Principles and Methods, (PDF)
GE Healthcare, 2014

Superdex is produced with different average particle sizes and different selectivities (see Table 2.2).

  • Use the prepacked columns with Superdex Peptide, Superdex 75, and Superdex 200 Increase for small-scale preparative purification and analytical runs.
  • Use columns prepacked with Superdex 30 prep grade, Superdex 75 prep grade, or Superdex 200 prep grade media for preparative applications.
  • Use bulk media of Superdex 30 prep grade, Superdex 75 prep grade, and Superdex 200 prep grade for preparative purification with larger sample volumes of up to 13 ml.
  • The maximum pressure drop over the packed bed is a typical value. Note that the actual value is individual for each column and needs to be determined (see Setting column pressure limits, Chapter 1).
  • Start with Superdex 200 Increase when the molecular weight of the protein of interest is unknown. Superdex 200 Increase or Superdex 200 prep grade are especially suitable for the separation of monoclonal antibodies from dimers and from contaminants of lower molecular weight, for example albumin and transferrin.
  • Start with Superdex Peptide or Superdex 30 prep grade for separations of peptides, oligonucleotides and small proteins below Mr 10 000.
  • Exposure to temperatures outside the range 4°C to 40°C will negatively affect the efficiency of the packed bed and the column will need to be repacked.

Table 2.2. Separation options with Superdex media
 

Product* Fractionation range, Mr (globular proteins) Sample Loading capacity† Maximum pressure drop over the packed bed Recommended Operational Flow rate‡
Superdex 200 Increase 3.2/300 1 × 104 to 6 × 105 4 to 50 μl 3.0 MPa, 30 bar, 435 psi** 0.075 ml/min
Superdex 200 Increase 5/150 GL 1 × 104 to 6 × 105 4 to 50 μl 3.0 MPa, 30 bar, 435 psi** 0.45 ml/min
Superdex 200 Increase 10/300 GL 1 × 104 to 6 × 105 25 to 500 μl 3.0 MPa, 30 bar, 435 psi** 0.075 ml/min
Superdex Peptide 3.2/300 1 × 102 to 7 × 103 < 50 μl 2.0 MPa, 20 bar, 290 psi 0.05 ml/min
Superdex Peptide 10/300 GL 1 × 102 to 7 × 103 25 to 500 μl 2.0 MPa, 20 bar, 290 psi < 1.0 ml/min
HiLoad 16/600 Superdex 30 pg§ < 1 × 104 ≤ 5 ml 3 MPa, 3 bar, 42 psi 1.0 ml/min
HiLoad 26/600 Superdex 30 pg§ < 1 × 104 ≤ 13 ml 3 MPa, 3 bar, 42 psi 2.6 ml/min
Superdex 30 pg§ (Bulk medium) < 1 × 104 0.5% to 4% of total column volume Column-dependent 10 to 50 cm/h
Superdex 75 3.2/300 3 × 103 to 7 × 104 < 50 μl 2.4 MPa, 24 bar, 350 psi 0.05 ml/min
Superdex 75 5/150 GL 3 × 103 to 7 × 104 4 to 50 μl 1.8 MPa, 18 bar, 260 psi 0.3 ml/min
Superdex 75 10/300 GL 3 × 103 to 7 × 104 25 to 500 μl 1.8 MPa, 18 bar, 260 psi 0.5 to 0.75 ml/min
HiLoad 16/600 Superdex 75 pg§ 3 × 103 to 7 × 104 ≤ 5 ml 0.3 MPa, 3 bar, 42 psi 1.0 ml/min
HiLoad 26/600 Superdex 75 pg§ 3 × 103 to 7 × 104 ≤ 13 ml 0.3 MPa, 3 bar, 42 psi 2.6 ml/min
Superdex 75 pg§ (Bulk medium) 3 × 103 to 7 × 104 0.5% to 4% of total column volume Column-dependent 10 to 50 cm/h
Superdex 200 3.2/300 1 × 104 to 6 × 105 < 50 μl 1.5 MPa, 15 bar, 220 psi 0.05 ml/min
Superdex 200 5/150 GL 1 × 104 to 6 × 105 4 to 50 μl 1.5 MPa, 15 bar, 220 psi 0.3 ml/min
Superdex 200 10/300 GL 1 × 104 to 6 × 105 25 to 500 μl 1.5 MPa, 15 bar, 220 psi 0.5 to 0.75 ml/min
HiLoad 16/600 Superdex 200 pg§ 1 × 104 to 6 × 105 ≤ 5 ml 0.3 MPa, 3 bar, 42 psi 1.0 ml/min
HiLoad 26/600 Superdex 200 pg§ 1 × 104 to 6 × 105 ≤ 13 ml 0.3 MPa, 3 bar, 42 psi 2.6 ml/min
Superdex 200 pg§ (Bulk medium) 1 × 104 to 6 × 105 0.5% to 4% of total column volume Column-dependent 10 to 50 cm/h

* HiLoad is packed with Superdex prep grade.

† For maximum resolution, apply as small sample volume as possible. Note that sample volumes less than 0.5% normally do not improve resolution.

‡ See Appendix 5 to convert linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa. § prep grade.

** Typical pressure drop over the packed bed.

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