疏水层析法 (Hydrophobic Interaction Chromatography, HIC) 是采用具有适度疏水性的配基作为固定相 （如Butyl、Octyl 、Phenyl， 疏水性从弱到强排序 ），以含盐的水溶液作为流动相，利用溶质分子与固定相间疏水相互作用的强弱不同而实现分离的层析方法。疏水层析介质特征是其表面含有疏水性配基，是不同长度的烷烃或芳香族化合物。
苯基琼脂糖凝胶6FF (高取代) 是以高度交联的琼脂糖微球为基质，平均粒径 90 μm，用稳定的化学键把功能基团固定到琼脂糖微球上，配基不含带电荷基团，使得真正的疏水层析成为可能，不会干扰离子效应，流速快、应用广，是生物大分子规模分离纯化常用的介质之一。 相较于 Phenyl 琼脂糖凝胶6FF（低取代） 流速快、配基密度高。
苯基琼脂糖凝胶6FF (高取代) 综合使用性能优异，支持高流速上样，能够有效提升纯化作业效率；微球粒径均匀统一，保障每一次层析实验的分离效果；理化稳定性优异，耐受反复清洗与再生，使用寿命更长；实验重复性佳，批次之间性能偏差小，数据稳定性高；生产工艺成熟，可轻松实现从实验室小试到工业化大规模生产的线性放大；基质亲水性优良，对生物样品非特异性吸附极低，有效减少样品损耗；产品适配场景广泛，通用性强，可满足多种生物大分子的疏水层析纯化需求, 特别适用于分离的初始捕获和中间纯化阶段。
阿拉丁的苯基琼脂糖凝胶6FF (高取代) 储存在20%乙醇 ，凝胶和保护液的体积比为7:3，我司产品规格为实际凝胶的体积。
| 参数 | 指标 |
| 基质 | 6%高度交联琼脂糖 |
| 平均粒径 | 90 μm |
| 配基 | 苯基 |
| 配基密度 | 45 μmol/mL |
| 建议流速 | ＜400 cm/h |
| 建议压力 | ＜0.3 MPa（3 bar） |
| pH稳定性① | 3-13（长期）；2-14（短期） |
| 化学稳定性 | 在常用水溶性缓冲液中稳定，如 1 mM HCl、1 M NaOH(仅供清洗使用）、30% 异丙醇、6 M 盐酸胍、8 M 尿素等 |
| 储存 | 20%乙醇，2~8℃ |
| 保质期 | 5年 |
注：
① 长期，指介质在很长时间内保持稳定而不对其后续性能产生不利影响的 pH 范围。短期，指根据经验给出的介质再生、在位清洗和消毒的 pH 范围。
使用说明
1、装柱
1.1 装柱缓冲液准备
纯化水，超声脱气15min。
1.2 层析介质准备
计算所需层析介质的量（压缩系数约为1.15），并称取备用。用真空抽滤瓶置换至装柱缓冲液中。置换的介质加入一定量装柱缓冲液，制成约 50-70%浓度的胶悬液。
1.3 层析柱准备
层析柱准备检查层析柱，确保各部件完整、干净。安装下柱头，拧紧O型圈后，将层析柱垂直固定在铁架台上，用水平仪检查并调整使层析柱保持竖直。用注射器吸取装柱缓冲液，接于柱底的出口处，缓慢推出注射器内液体，排除底部筛网的气泡，取下注射器后拧上堵头。层析柱中加入约2cm 高的装柱缓冲液。
1.4 装柱（以直径 16mm，床高 10cm 的层析柱为例）
混匀胶悬液，用玻璃棒引流缓慢倒入层析柱中，若液面离柱顶还有空隙，加装柱缓冲液充满。将调节器连接至层析系统，启动泵以一定流速排除管路及顶部筛网中的气泡后暂停。将调节器沿 45°放入柱中，固定好调节器并拧紧密封圈，注意避免气泡进入。
拧开层析柱底部的堵头，将底部管路放入到废液缸中，流速设为60cm/h，至介质界面无变化后，设置流速为600cm/h， 并逐步降低流速到 300 cm/h ，维持该流速 45min，用记号笔在界面处做好标记，暂停系统。拧上底部堵头，层析柱上端与泵断开，稍拧松调节器上的密封圈，将柱头压到胶面以下3mm 处，拧紧密封圈。将层析柱顶部和底部管路连接到层析系统上，进行柱效测定。
2、柱效检测
完成装柱后、使用前可通过柱效测定和评价可以确认层析柱装填质量。柱效通常用理论塔板高度（HETP）和非对称因子（As）来评价。柱效测定可以采用丙酮或者NaCl 作为样品进行，按照下表配制样品溶液和流动相。
样品 | 1.0%丙酮 | 0.8 M NaCl | 样品体积 | 1.0%柱体积 | 1.0%柱体积 | 流动相 | 纯水 | 0.4 M NaCl | 流速 | 30 cm/h | 30 cm/h | 检测器 | UV-280 nm | 电导 |
|
根据UV或者电导率曲线计算理论塔板高度（HETP）、理论塔板数（N）和非对称因子（As），公式如下：
HETP = L / N
N = 5.54 × (V<sub>R</sub> / W<sub>h</sub> )<sup>2</sup>
As = a / b
其中：L为柱高； V<sub>R</sub>为保留体积；W<sub>h</sub>为半高峰宽；a为在10%峰高处的第一个半峰宽；b为在10%峰高处的第二个半峰宽。
一般来说，HETP的数值应小于填料平均粒径的三倍（即HETP/D50<3，D50为填料的平均粒径），As应在0.8~1.5之间。
3、分离纯化
3.1 柱平衡
将层析柱连接至纯化设备，使用结合缓冲液冲洗柱 5 CV 以上，至层析柱流出液pH、电导同结合缓冲液的 pH、电导一致。平衡结束后将紫外归零。
备注：结合缓冲液推荐含 1.7 M (NH₄)₂SO₄的缓冲液，经过 0.22μm 过滤，缓冲液中盐种类或 (NH₄)₂SO₄具体浓度和 pH 等根据实际情况选择。
3.2 加载样品
经 0.2 / 0.45 μm 过滤后的样品溶液加载在疏水层析柱上，上样流速推荐 75-150 cm/h，上样体积取决于层析介质载量、样本溶液中与介质结合分子的浓度和其他 层析条件。 备注：蛋白与疏水层析介质的结合受配基种类、配基密度、缓冲液的离子强度、 盐析效应和温度等因素影响，在使用疏水层析分离方式时，需要选择合适的层析 条件。
3.3 柱冲洗
使用结合缓冲液冲洗 3-5 CV，将该条件下不与介质结合的杂质洗去（至紫外检测值回归基线附近）。或根据前期实验确定的杂质淋洗条件进行柱冲洗。
3.4 洗脱
使用洗脱缓冲液冲洗层析柱，洗脱流速推荐 75-150 cm/h，收集目标洗脱峰。备注：洗脱缓冲液推荐不含((NH₄)₂SO₄的缓冲液，经过 0.22μm 过滤，缓冲液中盐种类或(NH₄)₂SO₄具体浓度和 pH 等根据实际情况选择。
3.5 清洗和保存
使用纯化水冲洗 5 CV，然后用 20% 乙醇冲洗 5 CV，置于 2-8 ℃环境中保存。 备注：阴离子盐析效应由强到弱顺序：PO₄³⁻、SO₄²⁻、CH₃COO⁻、Cl⁻、Br⁻、NO₃⁻、ClO₄⁻、I⁻、SCN⁻。 阳离子盐析效应由强到弱顺序：NH₄⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Mg²⁺、Ba²⁺。盐析效应越强则疏水相互作用越强。 备注：疏水层析通常用于硫酸铵沉淀和离子交换层析之后；疏水层析缓冲液常选用中性磷酸钠或磷酸钾缓冲液，如 20 mM PB, 1.7 M (NH₄)₂SO₄, pH7.0，通常 (NH₄)₂SO₄浓度在 2 M 以下，NaCl 浓度在 3 M 以下；工艺开发阶段宜采用梯度洗脱模式，即逐渐增加洗脱缓冲液的比例，直到盐浓度达到最低，洗脱过程中分步收集，分别鉴定不同洗脱位置样品，梯度洗脱的结果可作为后续优化分离的基础；当目的蛋白同介质结合较强时，可在洗脱液中加入有机溶剂如乙醇、异丙醇、乙二醇等，加入量应低于 30%，亦可加入表面活性剂如 Tween20、Triton X-100 等。
4、在位清洗
在位清洗（Cleaning In Place，CIP）是去除层析介质上强结合的、沉淀的或者变性的物质。杂质残留将会影响层析柱的层析性能。如果聚集严重，将会堵塞层析柱，加大反压并减慢流速。所以定期的在位清洗能够防止污染物在柱床聚集，
有助于保持介质的载量，流速等。在 CIP 时，可以反向冲洗层析柱。
层析柱在使用 3-5 个循环后需要进行清洗，帮助介质恢复良好的性能。对不同类 型的杂质和污染物建议清洗条件如下：
4.1 去除蛋白沉淀和其他杂质：用 1 M NaOH 浸泡层析柱 1 h 以上，然后用 5-10 CV 纯化水冲洗层析柱。
4.2 去除紧密结合的疏水蛋白、脂蛋白和脂质等疏水性杂质：用 4-10 CV 的 70% 乙醇或者 30%异丙醇冲洗层析柱（15-20 min），然后用 3-5 CV 纯化水冲洗层析 柱。
4.3 去除其他污染物：用 4 CV 的 0.5-1 M NaOH 以 40 cm/h 流速清洗层析柱，然 后用 5-10 CV 纯化水冲洗层析柱。 备注：使用有机溶剂清洗时，应采用梯度加入方式，避免在使用高浓度有机溶剂 时形成气泡。
清洗后如果立即使用，用结合缓冲液冲洗 3-10 CV 即可。
5、 消毒灭菌
消毒灭菌是为了将层析柱中的微生物污染降至最低。本品可使用 NaOH 溶液作为消毒剂。NaOH 溶液可有效去除病毒、细菌、酵母及内毒素，运行成本极低。消毒步骤为：
5.1 使用结合缓冲液将层析柱冲洗3-5CV。
5.2 使用 0.5 M -1M NaOH 溶液以 40 cm/h 冲洗层析柱 0.5-1 h。
5.3 使用 pH 在 7-8 之间的缓冲液按 40 cm/h 正向冲洗层析柱 5-10 CV 后即完成 消毒过程。
注意事项：层析柱污染程度严重时，使用混有 30-40%丙醇的 0.5 M NaOH进行清洗。高浓度NaOH或长时间NaOH处理会降低介质载量，消毒时要注意清洗浓度和时间。
6. 保存
未使用的介质应当保存在 2-8 ℃的干燥、通风、洁净环境中，确保容器口完全密闭，切记不可冷冻。
已装填的层析柱应当使用 20%乙醇或 2%苯甲醇浸泡密封保存，防止微生物污染。
为了防止乙醇挥发导致微生物滋生，建议间隔3个月更换新鲜的保存液。
备注：保存的介质在使用前，应进行 CIP 和至少用结合缓冲液平衡5CV。
7. 线性放大
在实验室规模优化后的纯化工艺，可以线性放大到中试或生产规模。在放大过程中需要注意以下事项：
保持停留时间不变，保证动态载量的稳定。
根据需要的结合能力选择层析柱容积，如果柱高改变需要注意是否对纯化步骤产生影响。
根据流速需求确定层析柱直径，根据已知的停留时间确定柱床高度，一般推荐 3-15 cm。
保证样品浓度均匀一致，洗脱条件一致。
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（High 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. Compared to Phenyl Agarose 6FF (Low Sub), it has a faster flow rate and higher ligand density.
Phenyl Agarose 6FF（High 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（High 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（High Sub）
| Parameters | Specifications |
| Matrix | 6% highly cross-linked agarose |
| Average particle size | 90 μm |
| Ligand | Phenyl |
| Ligand density | 45 μmol/mL |
| Recommended flow rate | < 400 cm/h |
| Recommended pressure | < 0.3 MPa (3 bar) |
| pH stability ① | 3-13（Long‑term）；2-14（Short‑term） |
| 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<sub>R</sub> / W<sub>h</sub> )<sup>2</sup> As = a / bWhere: L is the column height; V<sub>R</sub> is the retention volume; W<sub>h</sub> 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.
ZJ26F0738015
危险品化学品经营许可证（带存储）