Separation Options with Sephadex^®

For group separations the medium should be selected so that the high molecular weight molecules are eluted at the void volume with minimum peak broadening or dilution and minimum time on the column. The lowest molecular weight substances should appear by the time one column volume of buffer has passed through the column. Sephadex^® media are available in prepacked columns (also spin columns), microplates, and in bulk media packs (Table 5.2).

Table 5.2. Group Separation options with Sephadex^® media

Chromatography Medium
Columns and 96-well plates	Loaded volume (mL)	Eluted volume (mL)	Dilution factor	Operation
Sephadex® G-25 Medium
PD SpinTrap™ G-25	0.07 to 0.13	0.07 to 0.13*	No dilution	Centrifuge
PD MultiTrap™ G-25	0.07 to 0.13	0.07 to 0.13*	No dilution	Centrifuge
PD MiniTrap™ G-25	0.2 to 0.5 0.1 to 0.5	0.1 to 0.5 1.0	No dilution 2 to 10	Centrifuge Gravity flow
PD MidiTrap™ G-25	0.75 to 1.0 0.5 to 1.0	0.5 to 1.0 1.5	No dilution 1.5 to 3	Centrifuge Gravity flow
PD-10 Desalting columns	1.75 to 2.5 1.0 to 2.5	1.0 to 2.5 3.5	No dilution 1.5 to 3.5	Centrifuge Gravity flow
Sephadex® G-10 Medium
PD MiniTrap™ G-10	0.1 to 0.3	0.5	1.7 to 5	Gravity flow
PD MidiTrap™ G-10	0.4 to 1.0	1.2	1.2 to 3.0	Gravity flow
Sephadex® G-25 Superfine
HiTrap® Desalting	0.25 0.5 1.0 1.5 (max.)	1.0 1.5 2.0 2.0	4 (approx.) 3 (approx.) 2 (approx.) 1.3 (approx.)	Syringe/pump/system Syringe/pump/system Syringe/pump/system Syringe/pump/system
2× HiTrap® Desalting	3.0 (max.)	4.0 to 5.0	1.3 to 1.7	Syringe/pump/system
3× HiTrap® Desalting	4.5 (max.)	6.0 to 7.0	1.3 to 1.7	Syringe/pump/system
Sephadex® G-25 Fine
HiPrep™ 26/10 Desalting	10 15 (max.)	10 to 15 15 to 20	1.0 to 1.5 1.0 to 1.3	Pump/system Pump/system
2× HiPrep™ 26/10 Desalting	30 (max.)	30 to 40	1.0 to 1.3	Pump/system
3× HiPrep™ 26/10 Desalting	45 (max.)	45 to 55	1.0 to 1.2	Pump/system
4× HiPrep™ 26/10 Desalting	60 (max.)	60 to 70	1.0 to 1.2	Pump/system

* Applied volume = eluted volume; For sample volumes less than 100 µL it is recommended to apply a stacker volume of 30 µL equilibration buffer after the sample has fully absorbed.

For convenience and reliable performance, use prepacked Sephadex columns such as HiTrap^® Desalting 5 ml and HiPrep™ 26/10 Desalting.

Always use disposable columns if there is a risk of biological or radioactive contamination or when any possibility of carryover between samples is unacceptable.