Agarose 4FF - BioReagent, 70% v/v; 90 μm

Cat. No.: A1524346

GRADE & PURITY BioReagent, 70% v/v; 90 μm

Storage

Store at 2-8°C,Do not freeze

Shipped In

Wet ice

Application

Protein purification

Overview

 Agarose 4FF is a gel filtration chromatography medium, also known as a size exclusion chromatography medium. This separation technique primarily separates and purifies substances based on differences in the molecular size, shape, and molecular weight of sample molecules. In the industry, it is commonly used for sample desalting and is suitable for the separation and purification of various samples including proteins, peptides, polysaccharides, nucleic acids, antibiotics, and natural products.Gel filtration chromatography media have specific separation ranges: small molecules require a medium with a low separation range, while larger molecules (e.g., antibodies) require a medium with a higher separation range. In terms of separation principle, this medium relies on its specific pore size to achieve separation and operates in two modes: when the target molecules can enter the pores of the medium, a semi-exclusion mode is used, with a recommended sample loading volume of 0.5%–5% of the column bed volume; if the molecules cannot enter the pores, a total exclusion mode is used, with a sample loading volume of up to 30% of the bed volume.

The product is manufactured using a high-density crosslinking process. Compared with traditional chromatography media, it features a higher degree of crosslinking and superior physicochemical properties. It not only supports high-flow-rate operation to improve separation efficiency but also exhibits tolerance to organic solvents, making it directly applicable for separating samples containing organic reagents. This product has strong applicability: it can be used directly for the purification and detection of biomacromolecules such as polysaccharides, nucleic acids, and viruses; it can also be modified through various chemical derivatizations to produce multifunctional separation media that serve as matrices for ion exchange, hydrophobic interaction, affinity, mixed-mode, and other chromatography methods.

The product offers excellent overall performance. The medium particles have uniform and consistent size, providing outstanding separation stability and experimental reproducibility. It supports high-flow-rate operation, effectively shortening the chromatography separation time. The process adaptability is strong, allowing easy scale-up from laboratory scale to industrial production scale. The medium exhibits excellent hydrophilicity and extremely low nonspecific adsorption to samples, maximizing sample integrity and improving sample recovery. Moreover, the product has broad applicability, covering a wide molecular weight separation range and meeting the chromatographic separation needs of various industries and diverse biological sample types.

Aladdin Agarose 4FF is stored in 20% ethanol, with a gel-to-preservation-solution volume ratio of 7:3. Our product specifications refer to the actual gel volume.

Table 1 Product Specifications of Agarose 4FF

Parameter	Specification
Matrix	4% highly crosslinked agarose
Mean particle size	90 μm
Separation range for globular proteins (Mr)	6×10⁴ – 2×10⁷
Separation range for dextrans (Mp)	3×10⁴ – 5×10⁶
Separation range for nucleic acids (bp)	45–872
Recommended flow rate	<250 cm/h
Recommended pressure	<0.3 MPa (3 bar)
pH stability	2–12 (long-term), 2–14 (short-term)①
Chemical stability	Stable in common aqueous buffers, e.g., 6 M guanidine hydrochloride, 8 M urea, 2 M NaOH, 1 M acetic acid, 70% ethanol, 30% isopropanol, 1% SDS, etc.
Storage	20% ethanol, 2–8°C
Shelf life	5 years

Notes: 

① Long-term refers to the pH range in which the medium remains stable for an extended period without adverse effects on subsequent performance. Short-term refers to the pH range empirically recommended for regeneration, cleaning-in-place, and disinfection.

Instructions for Use

1. Column Packing

1.1 Preparation of packing buffer

Use purified water and degas by ultrasonication for 15 minutes.

1.2 Preparation of chromatography medium

Calculate the required medium volume (compression factor: approx. 1.15) and weigh the medium. Replace the storage solution with packing buffer via vacuum filtration, then add an appropriate amount of packing buffer to prepare a 50% gel slurry.

1.3 Preparation of chromatography column

Inspect the column to ensure all components are intact and clean. Install the bottom adapter and tighten the O-ring. Fix the column vertically on a stand and calibrate its verticality with a level gauge. Draw packing buffer with a syringe, connect it to the column outlet and slowly push the liquid to remove air bubbles from the bottom mesh. Replace the syringe with a stopper, and add packing buffer to a height of about 2 cm inside the column.

1.4 Column packing (Example: 16 mm inner diameter, 10 cm bed height)

Mix the gel slurry thoroughly and pour it slowly into the column along a glass rod. Top up with packing buffer if necessary. Connect the column regulator to the chromatography system, start the pump to remove air bubbles in pipelines and top mesh, then stop the system. Insert the regulator into the column at a 45° angle, fix it and tighten the sealing ring to avoid air entrapment.

Remove the bottom stopper and place the outlet tube into a waste container. Set the flow rate to 60 cm/h until the medium bed interface stabilizes. Adjust the flow rate to 600 cm/h, then gradually reduce it to 300 cm/h and maintain for 45 minutes. Mark the bed interface with a marker and stop the system. Replace the bottom stopper, disconnect the column top from the pump, slightly loosen the sealing ring of the regulator, lower the adapter to 3 mm below the gel surface, then retighten the ring. Connect both ends of the column to the system for column efficiency testing.

2. Column Efficiency Test

After column packing is completed and before use, the packing quality of the chromatography column can be confirmed through column efficiency measurement and evaluation. Column efficiency is usually evaluated by the Height Equivalent to a Theoretical Plate (HETP) and the Asymmetry factor (As). Acetone or NaCl can be used as the sample for column efficiency measurement, and the sample solution and mobile phase are prepared according to the following table.

Table 2 Solution Preparation for Column Efficiency Test

Test Sample	Sample Solution Preparation	Mobile Phase Preparation	Detection Method
Acetone	0.1% (v/v) acetone in purified water	Purified water	UV detection at 280 nm
NaCl	1 M NaCl in purified water	Purified water	Conductivity detection

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 × (V_R / W_h )² As = a / bWhere: L is the column height; V_R is the retention volume; W_h 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 column to the purification system. Wash with at least 2–3 column volumes (CV) of binding buffer until the pH and conductivity of the column effluent match those of the binding buffer. After equilibration, zero the UV detector.

3.2 Sample loading

Load the sample solution, which has been concentrated and filtered through a 0.22/0.45 μm filter, onto the gel filtration column. The loading volume depends on the specific application. For semi-exclusion separation, load 0.5–5% CV; for total exclusion separation, load ≤30% CV. (Note: Smaller loading volumes yield better separation.)

3.3 Elution

At a constant flow rate, wash the column with equilibration buffer. Collect UV absorbance peaks corresponding to the target molecule size.

3.4 Cleaning and storage

Wash with 2–3 CV of equilibration buffer, then rinse with purified water until the effluent conductivity reaches zero. Finally, wash with 2–3 CV of 20% ethanol. Store the column at 2-8°C. Note: It is recommended to add 0.15 M NaCl to the equilibration buffer to avoid any ionic interaction between solutes and the medium. The buffer should be filtered through a 0.2 μm filter.

Note: Column equilibration is not required between consecutive sample-loading–elution cycles.

4. Cleaning-In-Place (CIP)

Cleaning-in-place (CIP) removes strongly bound, precipitated, or denatured substances from the chromatography medium. Residual impurities can affect column performance. If accumulation is severe, it can clog the column, increase backpressure, and reduce flow rate. Regular CIP helps prevent contaminant build-up in the bed and maintains flow rate and separation performance.

If you observe increased backpressure, pigment attachment, or reduced resolution, perform CIP promptly. (Note: When backpressure increases, first check valves, tubing, etc., for blockages before starting CIP.) Typically, cleaning is recommended after 3–5 cycles to restore good medium performance. Common cleaning agents include inorganic salts, acids, bases, or organic solvents. Recommended cleaning conditions for different types of impurities and contaminants are as follows:

4.1 Removal of impurities bound via ion exchange: Wash the column with 2–3 CV of 2 M NaCl solution, then rinse with 3–5 CV of purified water.

4.2 Removal of protein precipitates and hydrophobic impurities: Soak the column in 1 M NaOH for at least 1 hour, then rinse with 5–10 CV of purified water.

4.3 Removal of strongly bound hydrophobic impurities: Wash the column with 3–5 CV of 70% ethanol or 30% isopropanol (15–20 min), then rinse with 3–5 CV of purified water.

4.4 Removal of nucleic acids: After washing with 1–2 CV of neutral buffer, wash with 2–5 CV of 0.1 M acetic acid at pH 3.0, then backwash with 1 M NaOH for 15–30 min. If used immediately after cleaning, rinse with 3–10 CV of equilibration buffer.

5. Sanitization and Sterilization

Disinfection/sterilization minimizes microbial contamination in the column. This product can be disinfected using NaOH solution. NaOH solution is effective against viruses, bacteria, yeast, and endotoxins, and has very low operating cost. Disinfection procedure:

5.1 Rinse the chromatography column with binding buffer for 2-3 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 containing 0.2 M sodium acetate or 2% benzyl alcohol for storage to prevent microbial contamination.

Replace the preservation solution every 3 months to avoid microbial growth due to ethanol evaporation.

Note: Perform CIP and equilibrate with binding buffer for at least 5 CV before using stored medium.

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.

Specifications

Specifications & Purity

BioReagent, 70% v/v; 90 μm

Stability And Storage

Store at 2-8℃ long term (60 months). Do not freeze.

Storage

Store at 2-8°C, Do not freeze

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent