BioReagent, 70% v/v; 40 μm BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Store at 2-8°C,Do not freeze Ships Wet ice,Do not freeze Check lot-specific COA for exact specifications.
SDS, COA, datasheet, and spec sheet available for download. Lot-specific COA accessible via lot number lookup.
Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Ion Exchange Chromatography (IEC) is a chromatographic method that uses ion exchange ligands as the stationary phase. Separation is achieved based on the difference in binding strength during the reversible exchange between component ions in the mobile phase and counter ions on the stationary phase. Featuring high resolution, large binding capacity and easy operation, IEC has become one of the most widely used purification techniques for the separation and purification of biochemical products, proteins, peptides and other biomolecules.Ion exchange media mainly consist of three parts: a cross-linked network framework (i.e. base matrix), functional groups immobilized on the framework, and mobile counter ions with opposite charges to the functional groups that participate in ion exchange.
Q Agarose (High Resolution) is prepared by chemical modification of highly rigid agarose beads with an average particle size of 40 μm. It delivers improved mechanical properties and excellent separation performance at elevated flow rates.
With a small particle size and superior resolution, this product also combines high flow rate, high pressure resistance and low back pressure, making it suitable for intermediate purification and polishing of biomolecules.
Aladdin Q Agarose (High Resolution) is stored in 20% ethanol, with a volume ratio of gel to preservative solution of 7:3. The product specification of our company refers to the actual volume of the gel.
Table 1 Product Specifications of Q Agarose (High Resolution)
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Notes:
① Working pH range: the pH range within which the separation and purification function can be achieved under the premise of stable chromatography medium.
② Long-term pH range: the pH range within which the medium remains stable for a long time without adverse effects on its subsequent performance. Short-term pH range: the empirically determined pH range for medium regeneration, cleaning in place and sanitization.
Instructions for Use
1. Column Packing
1.1 Preparation of Packing Buffer
1.2 Preparation of Chromatography Medium
1.3 Preparation of Chromatography Column
1.4 Column Packing (taking a chromatography column with 16 mm inner diameter and 10 cm bed height as an example)
2. Column Efficiency Test
Table 2 Solution Preparation for Column Efficiency Test
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Calculate the Height Equivalent to a Theoretical Plate (HETP), number of theoretical plates (N) and Asymmetry factor (As) according to the UV or conductivity curve, using the following formulas:
HETP = L / N
N = 5.54 × (VR / Wh )2
As = a / bWhere: L is the column height;
VR is the retention volume; Wh is the peak width at half height; a is the first half peak width at 10% of the peak height; b is the second half peak width at 10% of the peak height.In general, the HETP value should be less than three times the average particle size of the packing material (i.e., HETP/D50 < 3, where D50 is the average particle size of the packing material), and As should be between 0.8 and 1.5.
3. Separation and Purification
3.1 Column Equilibration
Connect the chromatography column to the purification equipment, and rinse with binding buffer for more than 3-5 column volumes (CV) until the pH and conductivity of the column effluent are consistent with those of the binding buffer. Zero the UV detector after equilibration is completed.
3.2 Sample Loading
Load the sample solution filtered through 0.22/0.45 μm membrane onto the ion exchange chromatography column. The loading volume depends on the binding capacity of the chromatography medium, the concentration of the molecules bound to the medium in the sample solution, and other chromatographic conditions.
3.3 Column Washing
Rinse with binding buffer for 3-5 CV to remove impurities that do not bind to the medium under this condition (until the UV 280 detection value returns to near the baseline). Alternatively, perform column washing according to the impurity elution conditions determined in previous experiments.
3.4 Elution
Rinse the chromatography column with elution buffer, and start collecting the eluted sample when the target UV peak rises.
3.5 Washing and Storage
Wash with 1-2 M NaCl for 5 CV to remove reversibly bound impurities, then rinse with purified water until the conductivity of the effluent is 0, and finally rinse with 20% ethanol for 2-3 CV. Store the chromatography column in an environment at 2-8℃.Do not freeze.
4. Cleaning In Place (CIP)
Cleaning In Place (CIP) is the process of removing strongly bound, precipitated or denatured substances from the chromatography medium. Residual impurities will affect the chromatographic performance of the column. Severe aggregation will clog the chromatography column, increase back pressure and reduce flow rate. Therefore, regular CIP can prevent the accumulation of contaminants in the column bed and help maintain the binding capacity and flow rate of the medium.CIP should be performed after the chromatography column has been used for 3-5 cycles to restore the medium performance. Inorganic salts, acids, bases or organic solvents are usually used for cleaning. The recommended cleaning conditions for different types of impurities and contaminants are as follows:
4.1 Removal of impurities adsorbed by ion exchange
Wash the chromatography column with 2-3 CV of 2 M NaCl solution, then rinse the column with 3-5 CV of purified water.
4.2 Removal of protein precipitates and hydrophobic impurities
Soak the chromatography column in 1 M NaOH for more than 1 hour, then rinse the column with 5-10 CV of purified water.
4.3 Removal of strongly bound hydrophobic impurities
Rinse the chromatography column with 3-5 CV of 70% ethanol or 30% isopropanol for 15-20 minutes, then rinse the column with 3-5 CV of purified water.
4.4 Removal of nucleic acids
After rinsing with 1-2 CV of neutral buffer, wash with 2-5 CV of 0.1 M acetic acid at pH 3.0, then perform reverse flow washing with 1 M NaOH for 15-30 minutes. If the column is to be used immediately after cleaning, simply rinse with binding buffer for 3-10 CV.
5. Sanitization and Sterilization
Sanitization and sterilization are performed to minimize microbial contamination in the chromatography column. NaOH solution can be used as a disinfectant for this product. NaOH solution can effectively remove viruses, bacteria, yeast and endotoxins with extremely low operating costs. The sanitization steps are as follows:
5.1 Rinse the chromatography column with binding buffer for 3-5 CV.
5.2 Rinse the chromatography column with 0.5 M NaOH solution for 2-5 CV.
5.3 Soak the chromatography column in 0.5-1 M NaOH solution for 1 hour.
5.4 Rinse the chromatography column with binding buffer with pH between 7 and 8 for 5-10 CV to complete the sanitization process.
Precautions: When the chromatography column is severely contaminated, use 0.5 M NaOH mixed with 30-40% propanol for cleaning. High concentration of NaOH or prolonged NaOH treatment will reduce the binding capacity of the medium. Please pay attention to the cleaning concentration and time during sanitization.
6. Storage
Unused medium should be stored in a dry, ventilated and clean environment at 2-8 ℃, ensure the container mouth is completely sealed, and never freeze. Pre-packed chromatography columns should be soaked and sealed in 20% ethanol or 2% benzyl alcohol for storage to prevent microbial contamination.
7. Linear Scale-up
The optimized purification process at laboratory scale can be linearly scaled up to pilot or production scale. The following points should be noted during the scale-up process:
Keep the residence time unchanged to ensure the stability of the dynamic binding capacity.
Select the column volume according to the required binding capacity. If the column height is changed, pay attention to whether it will affect the purification steps.
Determine the column diameter according to the flow rate requirement, and determine the bed height according to the known residence time. The recommended bed height is generally 10-25 cm.
Ensure the sample concentration is uniform and consistent, and the elution conditions are the same.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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