SP Agarose HP (High Performance) - BioReagent, 70% v/v; 34 μm

Cat. No.: S1524352

GRADE & PURITY BioReagent, 70% v/v; 34 μm

Storage

Store at 2-8°C,Do not freeze

Shipped In

Wet ice

Application

Protein purification

Descripción general

Ion Exchange Chromatography (IEC) is a chromatographic method that uses ion exchange ligands as the stationary phase and separates components based on differences in binding affinity during reversible exchange between solute ions in the mobile phase and counter-ions on the stationary phase. IEC offers high resolution, high working capacity, and ease of operation, making it one of the most frequently used purification techniques for biochemical products, proteins, peptides, and similar substances. An ion exchange chromatography medium consists of three main parts: a cross-linked network skeleton (the matrix), functional groups fixed onto the skeleton, and mobile counter-ions that carry opposite charge to the functional groups and can be exchanged. Featuring high resolution, large processing capacity, and simple operation, IEC is widely used for the separation and purification of biomacromolecules, proteins, peptides, and other biochemical products.

SP Agarose HP (High Performance) is based on a 6% highly cross-linked agarose microsphere matrix with an average particle size of 34 μm. Specific functional groups are immobilized onto the surface of the agarose microspheres via stable chemical bonds. It is a strong cation exchange chromatography medium. This product is specifically developed for large-scale separation and purification of biomacromolecules, is compatible with downstream processing of biological products, and is perfectly suited for intermediate purification and polishing steps – making it a highly practical and specialized medium in the field of biopurification.

SP Agarose HP (High Performance)  exhibits excellent overall performance with a broad range of core advantages: high sample loading capacity to meet the demands of large-scale batch purification; uniform particle size for improved separation resolution, ensuring exceptionally high resolution; low material loss throughout the purification process, enabling high product recovery; outstanding physicochemical stability, tolerant of various buffer systems and operating conditions, providing excellent experimental and production reproducibility; highly hydrophilic matrix resulting in extremely low non‑specific adsorption of target biomacromolecules, reducing ineffective material loss; and low operating difficulty with strong process adaptability, allowing easy scale‑up from laboratory to industrial production. Its versatility makes it suitable for the separation and purification of many types of biomacromolecules.

Aladdin  SP Agarose HP (High Performance) is stored in 20% ethanol containing 0.2 M sodium acetate, with a gel-to-preservative volume ratio of 7:3. The product specification refers to the actual gel volume.

Table 1 Product Specifications of SP Agarose HP (High Performance)

Parameters	Specifications
Matrix	6% highly cross-linked agarose
Ligand	Sulfopropyl
Average particle size	34 μm
Ion capacity	150–200 μmol H⁺/mL medium
Recommended flow rate	< 150 cm/h
Recommended pressure	< 0.3 MPa (3 bar)
Working pH range①	4–13
pH stability	4–13 (longterm), 3–14 (shortterm)②
Chemical stability	Stable in common buffers, e.g., 1 M NaCl, 1 M NaOH, 6 M guanidine hydrochloride, 8 M urea, 70% ethanol, 30% isopropanol, etc.
Storage	20% ethanol containing 0.2 M sodium acetate, 2–8 °C
Shelf life	5 years

Notes:

① Working pH range refers to the pH range within which the medium can achieve separation and purification while remaining stable.

② Long‑term refers to the pH range in which the medium remains stable over an extended period without adverse effects on subsequent performance. Short‑term refers to the pH range recommended for regeneration, cleaning‑in‑place, and sanitization based on empirical data.

Instructions for Use

1. Column Packing

1.1 Packing buffer preparation

Purified water, degassed by sonication for 15 min.

1.2 Medium preparation

Calculate the required amount of medium (compression factor approx. 1.25) and weigh it out. Exchange into packing buffer using a vacuum suction flask. Add packing buffer to the exchanged medium to prepare a slurry of approx. 50% concentration.

1.3 Column preparation

Inspect the column to ensure all parts are intact and clean. Install the bottom adaptor, tighten the O‑ring, then fix the column vertically on a stand. Use a level to check and adjust the column to be vertical. Attach a syringe filled with packing buffer to the outlet at the column bottom, slowly push the buffer to remove air bubbles from the bottom frit, then remove the syringe and cap the outlet. Add about 2 cm height of packing buffer into the column.

1.4 Column packing (using a 16 mm diameter column with 10 cm bed height as an example)

Mix the slurry well and slowly pour it into the column using a glass rod. If the liquid level is below the column top, add packing buffer to fill. Connect the adaptor to the chromatography system, start the pump at a certain flow rate to remove bubbles from the tubing and the top frit, then stop. Insert the adaptor at a 45° angle into the column, fix it and tighten the seal ring, taking care to avoid introducing air bubbles.

Unscrew the bottom cap of the column and place the bottom tubing into a waste container. Set the flow rate to 60 cm/h until the medium interface stops moving. Then set the flow rate to 720 cm/h and gradually reduce to 360 cm/h. Maintain this flow rate for 45 min. Mark the interface with a marker and pause the system. Screw on the bottom cap, disconnect the column top from the pump, slightly loosen the seal ring on the adaptor, press the adaptor down to 3 mm below the gel surface, and tighten the seal ring. Connect the column top and bottom tubing to the chromatography system and perform 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 3–5 CV of binding buffer until the pH and conductivity of the column effluent match those of the binding buffer. Zero the UV after equilibration.

3.2 Sample loading

Load the sample solution (filtered through 0.22/0.45 μm) onto the ion exchange column. Loading volume depends on medium capacity, the concentration of binding molecules in the sample, and other chromatographic conditions.

3.3 Column wash

Wash with 3–5 CV of binding buffer to remove impurities that do not bind under these conditions (until the UV 280 signal returns near baseline). Alternatively, wash according to impurity removal conditions determined in preliminary experiments.

3.4 Elution

Elute with elution buffer. Begin collecting the eluted sample when the target UV peak rises.

3.5 Cleaning and storage

Wash with 5 CV of 1–2 M NaCl to remove reversibly bound impurities, then wash with purified water until the effluent conductivity reaches zero. Finally, wash with 2–3 CV of 20% ethanol containing 0.2 M sodium acetate. Store the column at 2–8 °C.

4. Cleaning-In-Place (CIP)

CIP removes tightly bound, precipitated, or denatured substances from the chromatography medium. Residual impurities can affect column performance. Severe aggregation may block the column, increase backpressure, and reduce flow rate. Regular CIP helps prevent contaminant accumulation in the bed, maintaining medium capacity and flow rate.

The column should be cleaned after 3–5 cycles to restore good performance. Inorganic salts, acids, bases, or organic solvents are commonly used. Recommended cleaning conditions for different types of impurities and contaminants:

4.1 Removal of impurities bound by ion exchange: Wash the column with 2–3 CV of 2 M NaCl, then wash 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 wash with 5–10 CV of purified water.

4.3 Removal of tightly bound hydrophobic impurities: Wash the column with 3–5 CV of 70% ethanol or 30% isopropanol (15–20 min), then wash 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 back‑wash with 1 M NaOH for 15–30 min.

If the column is to be used immediately after cleaning, wash with 3–10 CV of equilibration buffer.

5. Sanitization and Sterilization

Sanitization minimizes microbial contamination in the column. NaOH solution can be used as a sanitizing agent. NaOH solution effectively removes viruses, bacteria, yeast, and endotoxins with very low operating cost.

Sanitization steps:

5.1 Wash the column with 3–5 CV of binding buffer.

5.2 Wash the column with 2–5 CV of 0.5 M NaOH.

5.3 Soak the column in 0.5–1 M NaOH for 1 hour.

5.4 Wash the column with 5–10 CV of binding buffer at pH 7–8 to complete sanitization.

Note: For heavily contaminated columns, use 0.5 M NaOH mixed with 30–40% isopropanol for cleaning. High NaOH concentrations or prolonged NaOH exposure may reduce medium capacity. Pay attention to cleaning concentration and duration.

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

Especificaciones y pureza

BioReagent, 70% v/v; 34 μm

Estabilidad y almacenamiento

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

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C, Do not freeze

Enviado en

Wet ice

Este producto requiere envío en cadena de frío. Los servicios terrestres y otros servicios económicos no están disponibles.

Grado

BioReagent