This product is an affinity chromatography medium formed by coupling heparin to agarose gel base beads. The mechanism of heparin affinity is complex and generally involves multiple driving forces such as ionic interactions, hydrogen bonding, and van der Waals forces. Heparin-based resins are primarily used for the separation and purification of antithrombin III, coagulation factors, lipoproteins, esterases, hormones, interferons, and more. Some literature also reports the use of heparin resins for exosome purification. This product retains the excellent hydrophilicity and large-pore structure of agarose, ensuring good compatibility with bioactive macromolecules. It features high binding capacity, low non-specific adsorption, and fast flow rates, making it suitable for various heparin affinity chromatography applications.
Aladdin Heparin Agarose Resin is stored in 20% (v/v) ethanol (containing 0.05 M sodium acetate), with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Value / Description
| Matrix
| 6% cross-linked agarose
| Ligand
| Porcine-derived heparin
| Particle Size Range ①
| 45~165 μm
| Average Particle Size
| ~90 μm
| Binding Capacity ②
| ≥6 mg aFGF/mL
| Recommended Operating Flow Rate
| 60~300 cm/h
| Maximum Flow Rate & Pressure ③
| 1200 cm/h，0.3 MPa
| pH Operating pH Range
| 4–12 (long-term), 4–13 (short-term, e.g., CIP)
| Chemical Stability
| Stable in common aqueous buffers, 0.5 M NaOH, 8 M urea, 30% isopropanol, 70% ethanol, etc.
| Storage Conditions
| 20% ethanol (with 0.05 M sodium acetate), 2–30°C
| Shelf Life
| 3 years |
|
Notes:
① Over 90% of the beads fall within this size range.
② Binding capacity measured as pure recombinant human acidic fibroblast growth factor (rh-aFGF) yield per mL resin.
③ Maximum tested flow rate at 10 cm column height.
Protocol
1. Column Packing
The following procedure describes column packing when connected to a chromatography system:
(1) Equilibrate all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation:
Settled resin volume = Column volume × Compression factor (~1.15 for this resin).
(3) Resin Preparation: Resuspend the resin slurry thoroughly. Measure the required volume, remove storage solution by filtration, and wash 3 times with ~3 resin volumes of purified water.
(4) Packing Slurry Preparation: Transfer resin to a suitable container and add packing solution to achieve a 50–75% (v/v) slurry. Mix well before use.
(5) Pre-packing Setup: Fill the bottom of the clean column with packing solution to remove air from the bottom frit and outlet. Retain a small amount of liquid, tighten the bottom end fitting, and ensure the column is vertical.
(6) Packing: Pour the well-mixed slurry into the column in one slow, continuous motion (use a packing reservoir if needed). After adding all resin, fill the reservoir with packing solution, attach the top lid, and connect the column to the system.
(7) Compression: Allow the resin to settle naturally (or use a low flow rate of 50–150 cm/h). Apply a high flow rate (600 cm/h; ensure pressure ≤0.3 MPa) until the interface is stable. Mark the stable height, stop the pump, and lower the adapter to the position corresponding to the compression factor (~1.15). Tighten the adapter and equilibrate the column at a high flow rate.
Packing Condition
| Heparin Agarose Resin
| Compression Factor
| 1.15
| Packing Flow Rate
| 600 cm/h |
|
2. Column Efficiency Testing
After packing, assess column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor) with acetone or NaCl as tracers:
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
| Sample Volume
| 1.0% CV
| 1.0% CV
| Mobile Phase
| Pure Water
| 0.4 M NaCl
| Flow Rate
| 30 cm/h
| 30 cm/h
| Detector
| UV-280 nm
| Conductivity |
|
Calculate HETP, N, and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
HETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.
3. Equilibration and Loading
Equilibrate the column with initial Buffer A (e.g., 10–20 mM phosphate, citrate, or Tris-HCl buffer, pH 7–8) at the operating flow rate until baseline (conductivity/pH) stabilizes.
Dissolve or dialyze the sample into Buffer A. Load at 50–80% of DBC₁₀% (e.g., 5–8 mg rh-aFGF/mL resin for a DBC₁₀% of ~10 mg/mL). Alternatively, determine the load by monitoring target protein breakthrough in the flow-through.
4. Washing
5. Elution
6. Regeneration and Cleaning-in-Place (CIP)
Regenerate with 5 CV of elution buffer. For stubborn contaminants (e.g., denatured proteins, lipids), use CIP agents such as 0.1 M NaOH or non-ionic surfactants. Reverse flow may enhance cleaning efficiency.
7. Storage
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