离子交换层析法 (Ion Exchange Chromatography, IEC) 是以离子交换配基为固定相，依据流动相中的组分离子与固定相平衡离子可逆交换时结合力差异而进行分离的一种层析方法。离子交换层析法分辨率高、工作容量大且易于操作，现已成为分离纯化生化制品、蛋白质、多肽等物质中使用最频繁的纯化技术之一。离子交换层析介质主要由三部分组成：交联的网状骨架，即母体结构；固定在骨架上的功能基团；与功能基团带相反电荷、可移动、能进行交换的活动离子。具备分辨率高、处理容量大、操作简便等优势，普遍用于生物大分子、蛋白质、多肽等生化制品的分离纯化工作。
Q 琼脂糖凝胶 HP (高性能) 是以6%高度交联的琼脂糖微球作为基质，介质平均粒径为34μm，通过稳定化学键将专属功能基团固定在琼脂糖微球表面，属于强y阴离子型离子交换层析介质。该产品专为生物大分子规模化分离纯化研发，适配生物制品下游处理流程，可完美应用于中间纯化与精纯纯化等核心工序，是生物纯化领域实用性极强的专用层析介质。
Q 琼脂糖凝胶 HP (高性能) 综合性能优异，核心优势覆盖面广：产品载样容量高，能够满足规模化大批量纯化作业需求；微球粒径均匀统一，可有效提升分离精度，保障超高分离分辨率；纯化全过程物料损耗低，能实现较高产品回收率；介质整体理化稳定性出色，耐受多类缓冲体系与作业工况，实验与生产重复性极佳；介质基底亲水性优异，对目标生物大分子的非特异性吸附极低，减少物料无效损耗；同时产品操作门槛低，工艺适配性强，极易完成实验室到工业化生产的工艺放大，通用性出众，可适配多品类生物大分子的分离纯化场景。
阿拉丁的Q 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%高度交联的琼脂糖 | 配基 | 季氨基 | 平均粒径 | 34 μm | 离子载量 | 140-200 μmol Cl⁻/mL 介质 | 建议流速 | ＜150 cm/h | 建议压力 | ＜0.3 MPa（3 bar） | 工作 pH 范围① | 2-12 | pH 稳定性 | 2-12（长期）1-14（短期）② | 化学稳定性 | 常用缓冲液中稳定，如 1 M NaCl、1 M NaOH、6 M 盐酸胍、8 M 尿素、70% 乙醇、30%异丙醇等 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
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注：
① 工作 pH 范围，指在层析介质稳定前提下，实现分离纯化功能的 pH 范围；
② 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1.25），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 2 cm 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 60 cm/h，至介质界面无变化后，设置流速为 720 cm/h，并逐步降低流速到 360 cm/h，维持该流速 45 min，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 3 mm 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 |
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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 3-5 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。
3.2 加载样品
经 0.22 / 0.45 μm 过滤后的样品溶液加载在离子交换层析柱上，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。
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从层析柱中洗去（至 UV 280 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，目标紫外峰升起后开始收集洗脱样品。
3.5清洗和保存
用 1-2 M NaCl 清洗 5 CV，去除可逆结合的杂质，然后用纯化水冲洗至流出液电导为 0，最后用 20%乙醇冲洗 2-3 CV。层析柱置于2-8℃环境中保存。
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。
层析柱使用 3-5 个循环后需进行 CIP，以恢复介质性能。通常使用无机盐、酸、碱或有机溶剂等进行清洗，对于不同类型的杂质和污染物建议清洗条件如下：
4.1 去除离子交换作用吸附的杂质：用 2-3 CV 的 2 M NaCl 溶液清洗层析柱，然 后用 3-5 CV 纯化水冲洗层析柱。
4.2 去除蛋白沉淀、疏水性杂质：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。
4.3 去除强结合的疏水性杂质：用 3-5 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。
4.4 去除核酸：用中性缓冲液冲洗 1-2 CV 后，以 pH3.0 的 0.1 M 乙酸清洗 2-5 CV， 再用 1 M NaOH，反向清洗 15-30 min。 清洗后如果立即使用，用平衡缓冲液冲洗 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 NaOH 溶液对层析柱冲洗2-5CV。
5.3 使用 0.5-1 M NaOH 溶液对层析柱浸泡 1 h。
5.4 使用 pH 在 7-8 之间的结合缓冲液冲洗层析柱 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10-25 cm。
保证样品浓度均匀一致，洗脱条件一致。
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.
Q 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 anion 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.
Q 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 Q 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 Q Agarose HP(High Performance)
Parameters | Specifications | Matrix | 6% highly cross-linked agarose | Ligand | Quaternary | Average particle size | 34 μm | Ion capacity | 140–200 μmol Cl⁻/mL medium | Recommended flow rate | < 150 cm/h | Recommended pressure | < 0.3 MPa (3 bar) | Working pH range① | 2–12 | pH stability | 2–12 (longterm), 1–14 (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, 2–8 °C | Shelf life | 5 years |
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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 |
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| 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 |
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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 / bWhere: L is the column height; VR is the retention volume; Wh 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.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 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.
ZJ26F0838148
危险品化学品经营许可证（带存储）