Seplife® 6AG CM/90 Weak Cation Exchanger Agarose Resin

Ion Exchange Chromatography - Agarose

Appearance: White spherical beads
Type: Weak acid cation - Carboxy methyl
Matrix: 6% cross-linked agarose
Ion exchange capacity (mmol/ml): 0.09-0.13 (H + )
pH ligand fully charged: Negatively charged at pH>5
Particle size range (μm): 45-165
pH stability: 4-13 (operational), 2-14 (CIP)
Chemical Stability: Stable in all common aqueous buffers; 1M sodium hydroxide; 8M urea; 6M guanidine hydrochloride; 70% ethanol.
Flow rate* (cm/h): Max 750 cm/h, 0.3MPa
10% dynamic binding capacity (mg/ml)**: ≥35
Shipped as: Slurry in 20% ethanol solution

*Testing conditions: Chromatography column 16mm×200mm; column bed height 20cm; temperature 25°C; mobile phase water.
** Testing conditions: Binding buffer: 20mM PB, pH 6.7; Elution buffer: 20mM PB+1M NaCl, pH 6.7 Sample: Lysozyme. Column 8mm*100mm, room temperature, Retention time 2 minutes.

Column packing
Column packing should be done according to standard operating procedures. It is important to ensure that each material is at its working temperature, and when possible, the chromatography media may be degassed before column packing.

Equilibration
Equilibrate the column with an appropriate 2-5 column volume buffer. Ensure the conductivity and pH of the effluent are exactly the same as the buffer. The equilibration solution should be a low concentration (20-50mM) buffer such as Tris or PBS.

Sample feeding
1. The sample is prepared in the equilibration buffer; turbid sample should be centrifuged and filtered before loading. Samples with high conductivity (too high salt concentration) should be processed before loading.
2. Generally, the target product is bound to the media, the impurities are washed away with the equilibration buffer solution, and then an eluent is selected and used to wash off the target product.
3. The extent to which the media adsorbs sample components depends on the charged nature of the sample, the ionic strength and pH of the mobile phase. The lower the salt concentration, the stronger the adsorption of the sample components by the media. When using CM chromatography media, the recommended pH value is 1 unit less than the isoelectric point of the target product.

Elution
Elution can be carried out by increasing the salt concentration or changing the pH value.

Regeneration
Generally, use high salt concentration buffer (containing 1-2mol/L NaCl) or lower the pH to wash more than 10 times the volume of the column. Then wash with the equilibration solution that was used for binding proteins until the equilibrium is reached.
If there are inactivated proteins or lipids that cannot be washed away during regeneration, they can be removed by cleaning in place (CIP).

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, and then wash with equilibrium buffer solution until the pH is neutral.
3. For proteins and lipids with strong hydrophobic binding, wash with 4-10 BV of 70% ethanol or 30% isopropanol. It should be noted that the concentration of the organic solvent should gradually increase to avoid bubbles.

Storage
Sealed and stored at 4-30°C (preservation solution is 20% ethanol in 0.2M sodium acetate) in a ventilated, dry and clean place, do not freeze.