Seplife® 4AG Bu/90 Hydrophobic Interaction Chromatography Agarose Resin

Hydrophobic Interaction Chromatography - Agarose

Appearance: White spherical beads
Type: Hydrophobic chromatography media
Matrix: 4% cross-linked agarose
Ligand: Butyl
Particle size range (μm): 45-165
pH stability: 3-13 (operational), 2-14 (CIP)
Chemical Stability: Stable in all common aqueous buffers, 1 mM HCl, 1.0 M NaOH, 30% isopropanol, 70% ethanol, 6 M guanidine hydrochloride
Flow rate* (cm/h): Max 420 cm/h
Shipped as: 20% ethanol slurry

*Testing conditions: Chromatography column 16mm×400mm; column bed height 25cm; temperature 25°C; mobile phase water.

Column packing
Column loading should be performed in accordance with standard operating procedures. It is important to ensure that each material is at its working temperature, and when possible, the media should be degassed before column packing.

Equilibration
Equilibrate the column with an equilibration buffer solution of 2 to 5 times the volume of the column bed until the conductivity and pH of the effluent are consistent with those of the sample feeding buffer. The equilibration buffer is generally a buffer with high salt concentration, such as 0.02-0.05mol/L PBS plus 1-2.5mol/L (NH 4 ) 2 SO 4 , etc.

Sample feeding
1. Samples are prepared with an equilibration solution, and cloudy samples need to be centrifuged and filtered before loading.
2. Typically, the resin is used in bind-elute mode: let the target product bind to the column, wash off the impurities with the equilibration solution, and then choose an eluent to wash off the target product.
3. The extent to which the media adsorbs sample components depends on the hydrophobic nature of the sample, the ionic strength of the mobile phase, and the temperature. When the salt concentration is high, or the temperature is high, or the sample components are Highly hydrophobic, the media will adsorb the components firmly.

Elution
The adsorbed biomolecules can be eluted by reducing the salt concentration. The elution can be enhanced by adding surfactants or organic solvents, the most commonly used is a low-salt concentration buffer, such as 0.02-0.05 mol/L PBS.

Regeneration
Generally, first wash with more than 10 BV of a buffer solution of low salt concentration. Then, wash with the equilibration solution previously used for binding proteins until the equilibrium is reached. If there are inactivated proteins or lipids that cannot be washed off, they can be removed by cleaning-in-place.

Cleaning-in-place (CIP)
1. For proteins bound by ionic bonds, 0.5~1 BV of 2M NaCl can be used to remove them.
2. For precipitated proteins, hydrophobically bound proteins or lipids, first wash with 1 BV of 0.1M NaOH. Then wash with the equilibration buffer until pH is reached.
3. For proteins and lipids with strong hydrophobic binding, wash with 4-10 BV of 70% ethanol or 30% isopropanol.
It is important to note that the concentration of the organic solvent should increase gradually, otherwise bubbles may be created.

Storage
Sealed and stored at 4~30°C (preservation solution is 20% ethanol) in a ventilated, dry and clean place, do not freeze.