Phenyl Agarose 6FF（Low Sub） - BioReagent, 70% v/v; 90 μm

Cat. No.: P1524350

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

Storage

Store at 2-8°C,Do not freeze

Shipped In

Wet ice,Do not freeze

Application

Protein purification

Overview

Hydrophobic Interaction Chromatography (HIC) is a chromatographic method that uses ligands( e.g. Butyl, Octyl, and Phenyl, ordered from weakest to strongest hydrophobicity ) with moderate hydrophobicity as the stationary phase and a saline aqueous solution as the mobile phase. Separation is achieved based on differences in the strength of hydrophobic interactions between solute molecules and the stationary phase. The characteristic feature of hydrophobic chromatography media is that their surface contains hydrophobic ligands, which are alkyl chains of varying lengths or aromatic compounds.

Phenyl  Agarose 6FF（Low Sub）is based on a highly cross-linked agarose microsphere matrix with an average particle size of 90 μm. Functional groups are immobilized onto the agarose microspheres via stable chemical bonds. The ligands contain no charged groups, enabling true hydrophobic interaction chromatography without interference from ionic effects. It offers high flow rates and wide applicability, making it a commonly used medium for large-scale separation and purification of biomacromolecules.

Phenyl  Agarose 6FF（Low Sub）exhibits excellent overall performance. It supports high-flow-rate loading, effectively improving purification efficiency. The uniform particle size ensures consistent separation results in each chromatographic run. Its excellent physicochemical stability allows it to withstand repeated cleaning and regeneration, providing a long service life. Good reproducibility means minimal performance variation between batches, ensuring high data consistency. The mature production process enables easy linear scale-up from laboratory trials to industrial mass production. The highly hydrophilic matrix results in extremely low non-specific adsorption of biological samples, effectively reducing sample loss. The product is highly versatile and applicable to a wide range of scenarios, meeting the hydrophobic chromatography purification needs of various biomacromolecules. It is particularly suitable for the initial capture and intermediate purification stages.

Aladdin  Phenyl  Agarose 6FF（Low Sub）is stored in 20% ethanol, with a gel-to-preservative volume ratio of 7:3. The product specification refers to the actual gel volume.

Table 1 Product Specifications of Phenyl  Agarose 6FF（Low Sub）

Parameters	Specifications
Matrix	6% highly cross-linked agarose
Average particle size	90 μm
Ligand	Phenyl
Ligand density	25 μmol/mL
Recommended flow rate	< 400 cm/h
Recommended pressure	< 0.3 MPa (3 bar)
Chemical stability	Stable in common aqueous buffers, e.g., 1 mM HCl, 1 M NaOH (for cleaning only), 30% isopropanol, 6 M guanidine hydrochloride, 8 M urea, 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 Packing buffer preparation

Purified water, degassed by sonication for 15 min.

1.2 Medium preparation

Calculate the required amount of medium (compression factor approx. 1.15) 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. 50–70% 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 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 600 cm/h and gradually reduce to 300 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 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.

Note: A binding buffer containing 1.7 M (NH₄)₂SO₄ is recommended, filtered through 0.22 μm. The type of salt, specific concentration of (NH₄)₂SO₄, and pH should be selected based on actual conditions.

3.2 Sample loading

Load the sample solution (filtered through 0.2/0.45 μm) onto the hydrophobic column. Recommended loading flow rate: 75–150 cm/h. Loading volume depends on medium capacity, the concentration of binding molecules in the sample, and other chromatographic conditions.

Note: Protein binding to hydrophobic media is influenced by ligand type, ligand density, buffer ionic strength, salting-out effect, temperature, etc. Appropriate chromatographic conditions must be selected.

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 signal returns near baseline). Alternatively, wash according to impurity removal conditions determined in preliminary experiments.

3.4 Elution

Elute with elution buffer at a recommended flow rate of 75–150 cm/h. Collect the target elution peak.

Note: Elution buffer without (NH₄)₂SO₄ is recommended, filtered through 0.22 μm. The type of salt, specific concentration of (NH₄)₂SO₄, and pH should be selected based on actual conditions.

3.5 Cleaning and storage

Wash with 5 CV of purified water, then with 5 CV of 20% ethanol. Store at 2-8 °C.

Note: Salting-out effect of anions (strong to weak): PO₄³⁻ > SO₄²⁻ > CH₃COO⁻ > Cl⁻ > Br⁻ > NO₃⁻ > ClO₄⁻ > I⁻ > SCN⁻; salting-out effect of cations (strong to weak): NH₄⁺ > Rb⁺ > K⁺ > Na⁺ > Cs⁺ > Li⁺ > Mg²⁺ > Ba²⁺. Stronger salting-out effect leads to stronger hydrophobic interactions. Additional notes: Hydrophobic interaction chromatography is often used after ammonium sulfate precipitation and ion exchange chromatography. Common HIC buffers are neutral sodium or potassium phosphate buffers, e.g., 20 mM PB, 1.7 M (NH₄)₂SO₄, pH 7.0. Typically, (NH₄)₂SO₄ concentration is below 2 M, and NaCl concentration below 3 M. Gradient elution is recommended during process development, i.e., gradually increasing the proportion of elution buffer until salt concentration reaches a minimum. Collect fractions during elution and analyze each fraction. Gradient elution results can serve as a basis for subsequent optimization. If the target protein binds too strongly to the medium, organic solvents such as ethanol, isopropanol, or ethylene glycol (add below 30%) or surfactants such as Tween 20 or Triton X-100 can be added to the elution buffer.

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. During CIP, the column can be back-flushed.

The column should be cleaned after 3–5 cycles to restore good performance. Recommended cleaning conditions for different types of impurities and contaminants:

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

4.2 Removal of tightly bound hydrophobic proteins, lipoproteins, lipids, and other hydrophobic contaminants: Wash the column with 4–10 CV of 70% ethanol or 30% isopropanol (15–20 min), then wash with 3–5 CV of purified water.

4.3 Removal of other contaminants: Wash the column with 4 CV of 0.5–1 M NaOH at a flow rate of 40 cm/h, then wash with 5–10 CV of purified water.

Note: When using organic solvents for cleaning, introduce them gradually to avoid bubble formation at high organic solvent concentrations.

If the column is to be used immediately after cleaning, wash with 3–10 CV of binding 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. Disinfection procedure:

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

5.2 Wash the column with 0.5–1 M NaOH solution at 40 cm/h for 0.5–1 hour.

5.3 Wash the column forward with 5–10 CV of buffer at pH 7–8 at 40 cm/h to complete sanitization. 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.

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 3-15 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, Do not freeze

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

Grade

BioReagent