This product is an affinity chromatography resin prepared by direct sulfate esterification of cellulose microspheres. Like agarose matrix microspheres, it belongs to natural polymer-derived "soft gels". Compared with agarose, cellulose microspheres use raw materials with standard grades, and the cellulose backbone contains no anionic groups. The non-specific adsorption of cellulose microspheres is 1/10 to 1/3 of that of agarose microspheres. For example, the non-specific adsorption of cytochrome C on the cellulose base matrix of this product can be as low as 2 μg/mL.
After sulfate esterification, the resin exhibits affinity for a variety of live viruses, inactivated or split viruses, viral or microbial antigens, and heparin-binding proteins.
Meanwhile, sulfate ester is an excellent salt-tolerant cation exchange ligand. It can also be used as a cation exchange medium, with a binding capacity for lysozyme up to 180 mg/mL, and remains well-bound at NaCl concentrations of 50–200 mM.
Aladdin Sulfate-modified Cellulose Affinity Resin is stored in 20% ethanol with a gel-to-storage solution volume ratio of 1:1. The product specification indicates the actual gel volume.
Product Parameters
Parameter | Specification | Matrix | Highly cross-linked cellulose | Ligand | Sulfate ester, 160–200 μmol H + /mL | Particle size range ① | 45–165 μm | Mean particle size | ~90 μm | Binding capacity ② | ≥120 mg/mL | Recommended operating flow rate | 60–150 cm/h | Maximum flow rate and pressure ③ | 300 cm/h, 0.3 MPa | Working pH | 4–8.5 (recommended); 3–12 (long-term stable); 2–14 (short-term stable) | Chemical stability | Stable in common aqueous buffers, 0.5 mol/L NaOH, etc. | Storage | 20% ethanol, 2–8 °C | Shelf life | 5 years |
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Notes：
①More than 90% of microspheres by volume fall within this range;
②Test method: 2-hour incubation; result is close to DBC₁₀%;
③ Maximum tested flow rate at 10 cm bed height.
Instructions for Use
1. Column Packing
The following describes column packing for connection to a chromatography system.
(1)Equilibrate all materials to chromatography temperature; degas liquids if possible.
(2)Calculate medium volume: Settled medium volume = Column volume × Compression factor. Settled volume is the stable volume after full sedimentation in 20% ethanol storage solution. Compression factor of sulfate-modified cellulose affinity chromatography medium: ~1.15. Achieve compression by lowering the adapter or high-flow packing.
(3)Wash medium: Mix medium suspension thoroughly, measure required volume, remove liquid, and wash with 3× medium volume of purified water (repeat 3 times) to remove storage solution.
(4)Prepare packing suspension: Transfer medium to a suitable container, add packing buffer to make 50–75 v% suspension; mix well before use.
(5)Pre-pack preparation: Add packing buffer to the bottom of clean column to eliminate air under the bottom frit; leave a small volume of distilled water, tighten bottom plug, and set column vertically.
(6)Packing: Pour mixed suspension slowly and in one batch into the column (use a packing reservoir if needed), running down the inner wall to avoid bubbles. Fill reservoir with packing buffer, seal top, and connect to system.
(7)Compaction: Let medium settle naturally (or at 50–150 cm/h). After complete settling (clear gel–liquid interface), remove reservoir, install top adapter, and lower adapter to interface. Compact at high flow (see table below; do not exceed 0.3 MPa) until interface is stable; mark bed height. Stop pump, open top vent/plug, close bottom valve/plug, lower adapter to marked position corresponding to compression factor, tighten adapter. Equilibrate column at high flow after packing.
Packing condition | Sulfate-modified Cellulose Affinity Resin | Compression factor | 1.15 | Packing flow rate | 150–300 cm/h |
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2. Column Efficiency Test & Evaluation
Column efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).
Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.
Item | 1.0% Acetone | 0.8–1.0 M NaCl | Sample volume | 1.0% column volume | 1.0% column volume | Mobile phase | Pure water | 0.4 M NaCl | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
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Calculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:
- HETP = L / N
- N = 5.54 × (Vr / Wh)2
- As = a / b
Where:L = column height; Vr = retention volume; Wh = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.Generally, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.
3. Equilibration & Loading
Equilibrate column with starting buffer A at operating flow rate until conductivity, pH, etc., are stable.Common buffers: phosphate, citrate, Tris–HCl, pH 4–9 (e.g., 10 mM PB + 50–150 mM NaCl, pH 7.5).
4. Elution
Elute target protein by increasing salt concentration in buffer A, typically with 0.5–2 M NaCl.
Process screening: linear gradient elution to determine optimal salt concentration.
Process optimization: step gradient elution to save buffer and improve efficiency.
5. Regeneration & Cleaning-in-Place (CIP)
Standard regeneration cleans medium thoroughly. For elevated backpressure or cross-contamination prevention, use:1–2 mol/L NaCl, 0.5 mol/L NaOH, 70% ethanol, or 30% isopropanol. CIP removes impurities effectively; reverse flow is preferred.Wash with 3–5 column volumes (CV) of pure water after cleaning.
CIP procedure | Purpose | 1–2 mol/L NaCl, 0.5–2 CV; water, 3–5 CV | Remove ionically bound impurities | 0.5 mol/L NaOH, 1–2 h contact time (longer for endotoxin); buffer A, 5–10 CV; water, 3–5 CV | Remove protein precipitates, hydrophobic proteins, or pyrogens | 30% isopropanol or 70% ethanol, 5–10 CV; water, 3–5 CV | Remove tightly bound lipoproteins and liposomes |
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6. Resin Storage
The initial storage solution for the resin is 20% ethanol. After use, it can still be stored in 20% ethanol. The suitable storage temperature is 2–8°C; do not freeze.
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