DEAE HR Agarose (High Resolution) is a weak anion exchange chromatography medium that combines high resolution with high flow rates. It is produced by coupling diethylaminoethyl (DEAE) groups to highly rigid agarose gel microspheres. The product has a mean particle size of 35 μm and can still achieve flow rates exceeding 900 cm/h. Thanks to the optimized ion exchange capacity (ligand density), the dynamic binding capacity for lysozyme is greater than 60 mg/mL at a residence time of 2 minutes. It is mainly used for intermediate purification and polishing. When the purification scale is relatively small, or when the starting sample is relatively clean (e.g., supernatants with high expression levels), this product can also achieve high purity through a single-step purification.
Aladdin DEAE HR Agarose (High Resolution) is supplied in 20% ethanol, with a gel-to-preservative solution volume ratio of 1:1. The product specification provided by our company refers to the actual volume of the gel.
| Parameter | Specification |
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| Matrix | High-resolution, high-rigidity agarose microspheres | | Functional group | Diethylaminoethyl, –CH₂CH₂NH(CH₂CH₃)₂⁺ / mL | | Mean particle size | 35 μm | | Ion exchange capacity | 60–100 μmol OH⁻/mL | | Dynamic binding capacity① | ≥60 mg/mL | | Recommended operating flow rate | 50–150 cm/h | | Maximum flow rate② | 900 cm/h | | pH stability | 6–10 (operating pH); 3–12 (long-term stability); 2–14 (short-term stability) | | Chemical stability | Stable in the following solutions: common aqueous buffers, 1 M sodium hydroxide, 8 M urea, 6 M guanidine hydrochloride, 70% ethanol, 30% isopropanol, etc. | | Storage | 20% ethanol, 2–8 °C | | Shelf life | 5 years |
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Notes:
① DBC<sub>10%</sub> test conditions: 10% breakthrough, BSA, 2 min residence time.
② Maximum test flow rate at a column height of 10 cm.
Ion exchange chromatography is a separation and purification process in which charged target molecules undergo ion exchange with an ion exchange chromatography medium. It primarily relies on electrostatic interactions to separate charged molecules based on their differences in charge. Since all biomolecules are polar, they can carry charges under certain conditions. Taking proteins as an example, when the solution pH is below the protein's isoelectric point (pI), the protein molecules carry a positive charge and can bind to cation exchange media via ion exchange. Different molecules have different net charges and thus different binding affinities to the chromatography medium. By adjusting the elution conditions, different molecules can be eluted sequentially, thereby achieving separation and purification.
Figure 1 is a schematic diagram of a typical anion exchange chromatography process. Anion exchange chromatography refers to the ability of the chromatographic medium to exchange ions with anions in the solution. The DEAE group has a low degree of ionization and is a weak-base ion exchanger; hence, this product is referred to as a weak anion exchange medium. When the medium is in an equilibrium state, the counterions surrounding the DEAE anions are negatively charged, typically Cl⁻. During the binding process via ion exchange with the target molecules, negatively charged molecules are bound, while uncharged or positively charged molecules are not, achieving an initial separation effect. Anion exchange chromatography can elute target molecules by increasing the ionic strength of the mobile phase or by lowering the pH. Increasing the ionic strength of the mobile phase results in a competitive elution process (Figure 1a): as the ionic strength increases, negative ions compete with target molecules to become the counterions of the DEAE groups. Depending on the net charge and binding affinity, different molecules are eluted sequentially, thereby achieving purification.
On the other hand, the charge carried by target molecules is pH-dependent. Lowering the pH reduces the net negative charge on the target molecules (Figure 1b), weakening their binding to the medium. Therefore, lowering the pH is also an elution method in anion exchange chromatography.
Figure 1: Schematic diagram of the working principle of a cation exchange medium.
图中文字翻译
微球及内部骨架的平衡状态
Equilibrium state of the microspheres and their internal matrix
上样结合目标分子
Sample loading and binding of target molecules
提高离子强度洗脱
Elution by increasing ionic strength
升高 pH 洗脱
Elution by increasing pH
Instructions for Use
1. Column Packing
The following describes the column packing method for the resin when connected to a chromatography system.
(1) All materials used should be at the same temperature as the chromatography operation. Liquids should preferably be degassed.
(2) Calculation of medium volume: The settled gel volume is used to quantify the medium. Required settled gel volume = column volume × compression ratio (also called compression factor). The settled gel volume refers to the stable volume read after the medium has settled completely in the 20% ethanol storage solution. The compression ratio of CM HR Agarose (High Resolution) is ~1.10. To achieve this compression ratio, either the column adapter can be lowered, or high flow rates can be used.
(3) Resin Washing: Thoroughly resuspend the resin slurry, measure the required volume, and vacuum filter to remove the liquid. Wash the resin three times with approximately 3 volumes of purified water each time to remove the storage solution.
(4) Preparation of the packing suspension: Transfer the medium to a suitable container, add an appropriate amount of packing solution to prepare a 50–75 v% packing suspension, and mix well before use.
(5) Pre-packing preparation: Add packing solution to the bottom of the cleaned chromatography column to remove air from the bottom frit and the lower part of the column, leaving a small amount of distilled water in the column. Tighten the bottom cap and adjust the column so that it is perpendicular to the ground.
(6) Packing: Pour the mixed medium suspension slowly into the chromatography column in one go (using a packing reservoir if necessary). To avoid introducing air bubbles, allow it to flow down naturally along the inner wall of the column. After all the medium has been added, fill the packing reservoir with packing solution, tighten the top cap of the reservoir, and connect the column to the chromatography system.
(7) Compression: Allow the resin in the column to settle naturally (or at a low flow rate of 50-150 cm/h). Once the resin is completely settled (a clear interface between the resin and the liquid is visible), remove the packing reservoir, attach the top adaptor, and lower the adaptor to the interface. Continue to compress the resin using a high flow rate (see recommended flow rates below, ensuring column backpressure does not exceed 0.3 MPa) until the interface is clear and stable. Mark the column height at the stable interface. Stop the pump. Open the valve/plug on the top adaptor, close the valve/plug at the column bottom, lower the adaptor to the position corresponding to the compression ratio below the marked position, and tighten the adaptor. Packing is complete. After packing, equilibrate the resin in the column using a high flow rate.
Packing Condition | CM HR Agarose (High Resolution) | Compression ratio | 1.10 | Packing flow rate | 600–1500 cm/h (varies by column type; the compression factor shall prevail) |
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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
When using anion exchange media, commonly employed buffer systems include Tris-HCl, ethanolamine, histidine, and similar buffers, at a concentration of approximately 20 mM. These buffers without any additional salts are referred to as equilibration buffer, designated as Buffer A. Buffer A is characterized by low ionic strength and a pH at least one unit above the isoelectric point (pI) of the target protein.
Before loading, equilibrate the column with Buffer A at the operating flow rate. This process typically requires 5–10 column volumes of Buffer A, until the conductivity, pH, and other detector signals remain constant and correspond to those of Buffer A.
The most commonly used purification mode for ion exchange media is the "binding mode," where the target protein binds to the medium via ion exchange while impurities flow through. In some cases, the "flowthrough mode" is also used, where impurities bind to the medium and the target protein flows through. This mode is more often employed in intermediate or polishing purification stages.
The sample is typically dissolved in Buffer A, or the sample solution is buffer-exchanged into the Buffer A system. The loading amount can be set as "mg target protein / mL medium," usually at 50–80% of the DBC10%. For example, DEAE HR Agarose (High Resolution) has a DBC10% of ~60 mg/mL for BSA, so the loading amount for purification could be 30–48 mg/mL. In addition to DBC10%, the loading amount can also be determined by testing the target protein in the flowthrough fraction.
After loading, re-equilibrate the column with 3–10 column volumes of Buffer A.
4. Wash
Wash the loaded column with 3–10 column volumes of equilibration buffer (Buffer A). Monitor the detector signals until the conductivity, pH, and other parameters remain constant, indicating that unexchanged components have been washed out.
5. Elution
The most commonly used elution method is to increase the NaCl concentration in the elution buffer (Buffer A with NaCl added). A step elution or linear gradient elution within the range of 0–500 mM NaCl is recommended. In some cases, elution can also be achieved by changing the pH or by simultaneously altering both ionic strength and pH. Optimization of the elution conditions can improve the chromatography performance.
6. Regeneration
Use a higher ionic strength NaCl solution (1–2 M NaCl) to elute strongly bound proteins that were not eluted, thereby regenerating the medium. This product can be reused multiple times after regeneration. Alternatively, regeneration can also be achieved by changing the pH or simultaneously changing both ionic strength and pH. After regeneration, equilibrate with Buffer A.
7. Clean-in-Place (CIP)
If protein precipitates, lipoproteins, or hydrophobic proteins bind to the medium and cannot be removed during the above steps, CIP is required for removal. The following options can be used for CIP: 1 M NaOH, 70% ethanol, 30% isopropanol, etc. Use approximately 2.5 CV of the CIP solution with a contact time of about 30 min. After CIP, re-equilibrate the column with Buffer A.
8. Resin Storage
The initial storage solution for the medium is 20% ethanol. After use, it can continue to be stored in 20% ethanol. The recommended storage temperature is 2-8°C. Do not freeze.
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