Separation Options with Superdex^®

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

* 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.

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