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。
丁基琼脂糖凝胶 HP (高性能) 是以6%高度交联的琼脂糖微球为基质，平均粒径34μm，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，分辨率高、应用广，是生物大分子规模分离纯化常用的介质之一。丁基琼脂糖凝胶 HP (高性能) 具有出色的物理化学稳定性，特别适用于分离的中间纯化和精细纯化阶段。
丁基琼脂糖凝胶 HP (高性能) 综合使用性能优异，具备粒径均一、亲水性强、非特异性吸附低的特性，同时拥有高载量、高分辨率、高蛋白回收率，介质稳定性与实验重复性优异，通用性强、适用范围广且工艺易于线性放大。
阿拉丁的丁基琼脂糖凝胶 HP (高性能) 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 | 34 μm | 配基 | 丁基 | 配基密度 | 50 μmol/mL | 建议流速 | ＜150 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
装柱缓冲液可选择柱结合缓冲液，超声脱气15 min。
装柱过程中不得使用高粘度缓冲液。如果分离需要使用此类缓冲液，可以在装柱完成后以低流速在高粘度缓冲液中平衡层析柱。
1.2 层析介质准备
计算所需层析介质的量（压缩系数约为1.25），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%浓度的胶悬液。
1.3 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720cm/h， 并逐步降低流速到 360 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 × (VR / Wh )2
As = a / b
其中：L为柱高； VR 为保留体积； Wh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 0.22 / 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 0.01 M 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 moderately hydrophobic ligands as the stationary phase (e.g., Butyl, Octyl, Phenyl; hydrophobicity increases in the order Butyl < Octyl < Phenyl) and an aqueous salt-containing 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 HIC media is their surface‑bound hydrophobic ligands, which are alkyl chains of varying lengths or aromatic compounds.
Butyl Agarose HP (High Performance) is based on 6% highly cross‑linked agarose microspheres with an average particle size of 34 μm. The functional groups are covalently attached to the agarose microspheres via stable chemical bonds. The ligands contain no charged groups, making true hydrophobic interaction chromatography possible without interfering ionic effects. It offers high resolution and broad applicability, and is one of the commonly used media for large‑scale purification of biomacromolecules. Butyl Agarose HP (High Performance) exhibits excellent physicochemical stability and is particularly suitable for intermediate purification and final polishing steps.
Butyl Agarose HP (High Performance) combines outstanding overall performance, including uniform particle size, strong hydrophilicity, low non‑specific adsorption, as well as high binding capacity, high resolution, high protein recovery, excellent medium stability and experimental reproducibility, broad versatility, wide application range, and ease of linear scale‑up.
Aladdin’s Butyl Agarose HP (High Performance) 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 Butyl Agarose HP (High Performance)
| Parameter | Specification |
|---|
| Matrix | 6% highly cross‑linked agarose | | Average particle size | 34 μm | | Ligand | Butyl
| | Ligand density | 50 μmol/mL | | Recommended flow rate | <150 cm/h | | Recommended pressure | <0.3 MPa (3 bar) | | pH stability① | 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 Preparation of packing buffer
The packing buffer can be the same as the binding buffer; degas by ultrasonication for 15 min. Do not use high‑viscosity buffers during packing. If such buffers are required for the separation, equilibrate the column with the high‑viscosity buffer at a low flow rate after packing.
1.2 Preparation of chromatography medium
Calculate the required volume of medium (compression factor approximately 1.25) and weigh out the appropriate amount. Exchange the medium into the packing buffer using a vacuum filtration flask. Add a certain volume of packing buffer to the exchanged medium to prepare a slurry of approximately 50% concentration.
1.3 Preparation of the chromatography column
Inspect the column to ensure all parts are intact and clean. Install the bottom adaptor, tighten the O‑ring, and mount the column vertically on a stand. Use a spirit level to check and adjust the column so that it is perfectly vertical. Using a syringe filled with packing buffer, connect to the outlet at the bottom of the column, slowly inject the liquid to expel air bubbles from the bottom frit, remove the syringe and close with a stopper. Add approximately 2 cm of packing buffer to the column.
1.4 Packing (example: 16 mm diameter, 10 cm bed height)
Mix the slurry thoroughly and pour it slowly into the column using a glass rod. If the liquid level is below the top of the column, fill with packing buffer. Connect the adaptor to the chromatography system, start the pump to purge air from the tubing and top frit, then stop. Insert the adaptor into the column at a 45° angle, secure it and tighten the sealing ring, taking care to avoid introducing air bubbles.
Remove the stopper from the column bottom and place the outlet tubing into a waste container. Set the flow rate to 60 cm/h. When the medium interface no longer changes, increase the flow rate to 720 cm/h, then gradually reduce to 360 cm/h and maintain this flow for 45 min. Mark the interface with a pen, then stop the system. Close the bottom outlet with a stopper, disconnect the column top from the pump, slightly loosen the sealing ring on the adaptor, lower the adaptor to 3 mm below the gel surface, and retighten the sealing ring. Connect the top and bottom lines 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
Sample | 1.0% acetone | 0.8 M NaCl | Sample volume | 1.0% of column volume | 1.0% of column volume | Mobile phase | Pure water | 0.4 M NaCl | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
|
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 / b
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 and wash with binding buffer for at least 5 CV, until the pH and conductivity of the column effluent match those of the binding buffer. After equilibration, zero the UV detector.
Note: The recommended binding buffer is a buffer containing 1.7 M (NH₄)₂SO₄, filtered through 0.22 μm. The salt type, concentration of (NH₄)₂SO₄, pH, etc., should be selected according to the actual conditions.
3.2 Sample loading
Apply the sample solution (filtered through 0.22/0.45 μm) onto the HIC column. Recommended loading flow rate: 75–150 cm/h. Loading volume depends on the medium binding capacity, the concentration of binding molecules in the sample, and other chromatographic conditions.
Note: Protein binding to HIC media is affected by ligand type, ligand density, buffer ionic strength, salting‑out effects, temperature, etc. Appropriate chromatographic conditions should be selected when using HIC.
3.3 Column washing
Wash the column with binding buffer for 3–5 CV to remove impurities that do not bind under these conditions (until UV returns to near baseline). Alternatively, wash according to the impurity removal conditions established in preliminary experiments.
3.4 Elution
Elute the column with elution buffer. Recommended elution flow rate: 75–150 cm/h, and collect the target peak.
Note: The recommended elution buffer is a buffer without (NH₄)₂SO₄, filtered through 0.22 μm. Salt type, (NH₄)₂SO₄ concentration, pH, etc., should be chosen according to actual conditions.
3.5 Cleaning and storage
Wash with purified water for 5 CV, then with 20% ethanol for 5 CV, and store at 2–8 °C.
Note: The salting‑out strength of anions decreases in the order: PO₄³⁻ > SO₄²⁻ > CH₃COO⁻ > Cl⁻ > Br⁻ > NO₃⁻ > ClO₄⁻ > I⁻ > SCN⁻. For cations: NH₄⁺ > Rb⁺ > K⁺ > Na⁺ > Cs⁺ > Li⁺ > Mg²⁺ > Ba²⁺. Stronger salting‑out effect leads to stronger hydrophobic interactions.
Additional notes: HIC is usually performed after ammonium sulfate precipitation and ion‑exchange chromatography. HIC buffers are often neutral sodium phosphate or potassium phosphate buffers, e.g., 20 mM PB, 1.7 M (NH₄)₂SO₄, pH 7.0. Typically (NH₄)₂SO₄ concentrations are below 2 M and NaCl below 3 M. In process development, gradient elution is recommended (gradually increasing the proportion of elution buffer until salt concentration is minimal), collecting fractions during elution and analysing them. The gradient results can serve as a basis for subsequent optimisation. If the target protein binds too strongly to the medium, organic solvents such as ethanol, isopropanol, or ethylene glycol (addition amount <30%) or surfactants such as Tween 20 or Triton X‑100 may be added to the elution buffer.
4. Cleaning‑in‑Place (CIP)
CIP is used to remove strongly bound, precipitated, or denatured materials from the chromatography medium. Residues can impair column performance. Severe accumulation may block the column, increase back‑pressure, and reduce flow rate. Regular CIP helps prevent contaminant build‑up, maintain capacity, and preserve flow properties. During CIP, the column can be washed in the reverse direction.
After 3–5 cycles of use, the column should be cleaned to restore medium performance. Recommended cleaning conditions for different types of impurities are as follows:
4.1 Removal of protein precipitates and other impurities: Soak the column with 1 M NaOH for ≥1 h, then wash with 5–10 CV of purified water.
4.2 Removal of strongly 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 rinse 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 40 cm/h, then rinse with 5–10 CV of purified water.
Note: When using organic solvents for cleaning, introduce them in a gradient manner to avoid bubble formation caused by high concentrations of organic solvent.
If the column is to be used immediately after cleaning, equilibrate with 3–10 CV of binding buffer.
5. Sanitisation
Sanitisation is performed to minimise microbial contamination of the column. This product can be sanitised using NaOH solution, which effectively removes viruses, bacteria, yeast, and endotoxins at low operating cost. The sanitisation procedure is as follows:
5.1 Wash the column with 3–5 CV of binding buffer.
5.2 Wash with 0.01 M NaOH at 40 cm/h for 0.5–1 h.
5.3 Wash forward with 5–10 CV of buffer at pH 7–8 at 40 cm/h to complete the sanitisation.
Note: For heavily contaminated columns, a mixture of 30–40% isopropanol in 0.5 M NaOH may be used. High concentrations of NaOH or prolonged NaOH treatment may reduce medium capacity; therefore, pay attention to concentration and exposure time during sanitisation.
6. Storage
Unused medium should be stored in a dry, ventilated, clean environment at 2–8 °C with the container tightly sealed. Do not freeze.
Packed columns should be stored immersed in 20% ethanol or 2% benzyl alcohol to prevent microbial contamination. To prevent ethanol evaporation and microbial growth, it is recommended to replace the storage solution with fresh solution every 3 months.
Note: Before use, stored medium should be subjected to CIP and equilibrated with at least 5 CV of binding buffer.
7. Linear Scale‑up
Purification processes optimised at laboratory scale can be linearly scaled up to pilot or production scale. The following should be considered during scale‑up:
Keep the residence time constant to ensure stable dynamic binding capacity.
Choose the column volume according to the required binding capacity; if bed height is changed, evaluate the effect on purification steps.
Determine column diameter based on flow rate requirements; bed height is generally recommended to be 3–15 cm based on the known residence time.
Ensure uniform sample concentration and consistent elution conditions.
ZJ26F0938902
危险品化学品经营许可证（带存储）