Separation Options with Sephadex®

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

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.

Materials

     
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