琼脂糖凝胶4FF (Agarose 4FF)属于凝胶过滤层析介质，也被称作尺寸排阻层析（SEC）介质，该分离技术主要依据样品分子的大小、形状及分子量差异，完成物质的分离纯化，行业内常被用于样品脱盐作业，同时适配蛋白质、多肽、多糖、核酸、抗生素及天然产物等各类样品的分离纯化工作。凝胶过滤层析介质具有特定的分离范围，小分子使用低分离范围的介质；较大分子（如抗体）使用更高分离范围的介质。在分离原理上，该介质依托自身专属孔径实现分离，分为两种作业模式：待分离分子能够进入介质孔隙时，采用半排阻模式，上样体积建议控制在层析柱柱体积的0.5%-5%；若分子无法进入介质孔隙，则采用全排阻模式，上样体积可控制在柱体积30%以内。
本品经过高密度交联工艺制备而成，相较于传统层析介质，交联度更高，理化性能更加优异，不仅支持高流速作业，提升分离效率，还具备有机溶剂耐受能力，可直接应用于含有机试剂样品的分离场景。本品适用性极强，既能够直接用于多糖、核酸、病毒等生物大分子的纯化与检测；还可通过多样化化学修饰，衍生出多功能分离介质，作为离子交换、疏水、亲和、混合模式等多种层析方式的基质使用。
本品整体综合性能出众，介质颗粒粒径均匀统一，分离稳定性与实验重复性极佳；支持高流速运行，有效缩短层析分离时长；工艺适配性强，可轻松完成实验规模到工业化生产规模的放大升级；介质亲水性优异，样品非特异性吸附极低，最大程度保障样品完整性，提升样品回收率；同时产品通用性强，覆盖分子量分离范围广，可满足多行业、多类型生物样品的层析分离需求。
阿拉丁的琼脂糖凝胶4FF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 | 4%高度交联琼脂糖 | 平均粒径 | 90 μm | 球蛋白分离范围 (Mr) | 6×10⁴ – 2×10⁷ | 葡聚糖分离范围 (Mp) | 3×10⁴ – 5×10⁶ | 核酸分离范围 (bp) | 45-872 | 建议流速 | ＜250 cm/h | 建议压力 | ＜0.3 MPa（3 bar） | pH 稳定性 | 2-12（长期）2-14（短期）① | 化学稳定性 | 常用水溶性缓冲液中稳定，如：6 M 盐酸胍、8 M 尿素、2 M NaOH、 1 M 乙酸、70%乙醇、30%异丙醇、1%SDS 等。 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
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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%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 |
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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2-3CV 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 过滤浓缩后的样品溶液加载在凝胶过滤层析柱上，上样体积 取决于具体应用，若按半排阻分离方式，上样体积为 0.5-5% CV；按全排阻分离 方式，上样体积为 30% CV 以下。（备注：上样体积越小，分离效果越好）
3.3 洗脱
恒定流速下，使用平衡缓冲液冲洗层析柱，根据目标分子大小，收集对应的紫外 吸收峰。
3.4 清洗和保存
用平衡缓冲液清洗 2-3 CV，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。 备注：建议平衡缓冲液中加入 0.15 M NaCl，避免任何溶质与介质的离子相互作 用；缓冲液需经过 0.2 μm 过滤。
备注：在连续上样-洗脱循环之间，不需要进行柱平衡步骤。
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， 有助于保持介质的流速和分离效果等。
如果观察到层析柱反压升高、有色素附着、分辨率降低现象，请及时进行 CIP。 (备注：当层析柱反压升高时，在开始 CIP 前，先检查阀门、管道等是否有堵塞) 通常层析柱在使用 3-5 个循环后需要进行清洗，帮助介质恢复良好的性能。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2-3CV。
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。
Agarose 4FF is a gel filtration chromatography medium, also known as a size exclusion chromatography medium. This separation technique primarily separates and purifies substances based on differences in the molecular size, shape, and molecular weight of sample molecules. In the industry, it is commonly used for sample desalting and is suitable for the separation and purification of various samples including proteins, peptides, polysaccharides, nucleic acids, antibiotics, and natural products.Gel filtration chromatography media have specific separation ranges: small molecules require a medium with a low separation range, while larger molecules (e.g., antibodies) require a medium with a higher separation range. In terms of separation principle, this medium relies on its specific pore size to achieve separation and operates in two modes: when the target molecules can enter the pores of the medium, a semi-exclusion mode is used, with a recommended sample loading volume of 0.5%–5% of the column bed volume; if the molecules cannot enter the pores, a total exclusion mode is used, with a sample loading volume of up to 30% of the bed volume.
The product is manufactured using a high-density crosslinking process. Compared with traditional chromatography media, it features a higher degree of crosslinking and superior physicochemical properties. It not only supports high-flow-rate operation to improve separation efficiency but also exhibits tolerance to organic solvents, making it directly applicable for separating samples containing organic reagents. This product has strong applicability: it can be used directly for the purification and detection of biomacromolecules such as polysaccharides, nucleic acids, and viruses; it can also be modified through various chemical derivatizations to produce multifunctional separation media that serve as matrices for ion exchange, hydrophobic interaction, affinity, mixed-mode, and other chromatography methods.
The product offers excellent overall performance. The medium particles have uniform and consistent size, providing outstanding separation stability and experimental reproducibility. It supports high-flow-rate operation, effectively shortening the chromatography separation time. The process adaptability is strong, allowing easy scale-up from laboratory scale to industrial production scale. The medium exhibits excellent hydrophilicity and extremely low nonspecific adsorption to samples, maximizing sample integrity and improving sample recovery. Moreover, the product has broad applicability, covering a wide molecular weight separation range and meeting the chromatographic separation needs of various industries and diverse biological sample types.
Aladdin Agarose 4FF is stored in 20% ethanol, with a gel-to-preservation-solution volume ratio of 7:3. Our product specifications refer to the actual gel volume.
Table 1 Product Specifications of Agarose 4FF
Parameter | Specification | Matrix | 4% highly crosslinked agarose | Mean particle size | 90 μm | Separation range for globular proteins (Mr) | 6×10⁴ – 2×10⁷ | Separation range for dextrans (Mp) | 3×10⁴ – 5×10⁶ | Separation range for nucleic acids (bp) | 45–872 | Recommended flow rate | <250 cm/h | Recommended pressure | <0.3 MPa (3 bar) | pH stability | 2–12 (long-term), 2–14 (short-term)① | Chemical stability | Stable in common aqueous buffers, e.g., 6 M guanidine hydrochloride, 8 M urea, 2 M NaOH, 1 M acetic acid, 70% ethanol, 30% isopropanol, 1% SDS, etc. | Storage | 20% ethanol, 2–8°C | Shelf life | 5 years |
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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
Use purified water and degas by ultrasonication for 15 minutes.
1.2 Preparation of chromatography medium
Calculate the required medium volume (compression factor: approx. 1.15) and weigh the medium. Replace the storage solution with packing buffer via vacuum filtration, then add an appropriate amount of packing buffer to prepare a 50% gel slurry.
1.3 Preparation of chromatography column
Inspect the column to ensure all components are intact and clean. Install the bottom adapter and tighten the O-ring. Fix the column vertically on a stand and calibrate its verticality with a level gauge. Draw packing buffer with a syringe, connect it to the column outlet and slowly push the liquid to remove air bubbles from the bottom mesh. Replace the syringe with a stopper, and add packing buffer to a height of about 2 cm inside the column.
1.4 Column packing (Example: 16 mm inner diameter, 10 cm bed height)
Mix the gel slurry thoroughly and pour it slowly into the column along a glass rod. Top up with packing buffer if necessary. Connect the column regulator to the chromatography system, start the pump to remove air bubbles in pipelines and top mesh, then stop the system. Insert the regulator into the column at a 45° angle, fix it and tighten the sealing ring to avoid air entrapment.
Remove the bottom stopper and place the outlet tube into a waste container. Set the flow rate to 60 cm/h until the medium bed interface stabilizes. Adjust the flow rate to 600 cm/h, then gradually reduce it to 300 cm/h and maintain for 45 minutes. Mark the bed interface with a marker and stop the system. Replace the bottom stopper, disconnect the column top from the pump, slightly loosen the sealing ring of the regulator, lower the adapter to 3 mm below the gel surface, then retighten the ring. Connect both ends of the column to the system for 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 2–3 column volumes (CV) of binding buffer until the pH and conductivity of the column effluent match those of the binding buffer. After equilibration, zero the UV detector.
3.2 Sample loading
Load the sample solution, which has been concentrated and filtered through a 0.22/0.45 μm filter, onto the gel filtration column. The loading volume depends on the specific application. For semi-exclusion separation, load 0.5–5% CV; for total exclusion separation, load ≤30% CV. (Note: Smaller loading volumes yield better separation.)
3.3 Elution
At a constant flow rate, wash the column with equilibration buffer. Collect UV absorbance peaks corresponding to the target molecule size.
3.4 Cleaning and storage
Wash with 2–3 CV of equilibration buffer, then rinse 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. Note: It is recommended to add 0.15 M NaCl to the equilibration buffer to avoid any ionic interaction between solutes and the medium. The buffer should be filtered through a 0.2 μm filter.
Note: Column equilibration is not required between consecutive sample-loading–elution cycles.
4. Cleaning-In-Place (CIP)
Cleaning-in-place (CIP) removes strongly bound, precipitated, or denatured substances from the chromatography medium. Residual impurities can affect column performance. If accumulation is severe, it can clog the column, increase backpressure, and reduce flow rate. Regular CIP helps prevent contaminant build-up in the bed and maintains flow rate and separation performance.
If you observe increased backpressure, pigment attachment, or reduced resolution, perform CIP promptly. (Note: When backpressure increases, first check valves, tubing, etc., for blockages before starting CIP.) Typically, cleaning is recommended after 3–5 cycles to restore good medium performance. Common cleaning agents include inorganic salts, acids, bases, or organic solvents. Recommended cleaning conditions for different types of impurities and contaminants are as follows:
4.1 Removal of impurities bound via ion exchange: Wash the column with 2–3 CV of 2 M NaCl solution, then rinse 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 rinse with 5–10 CV of purified water.
4.3 Removal of strongly bound hydrophobic impurities: Wash the column with 3–5 CV of 70% ethanol or 30% isopropanol (15–20 min), then rinse 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 backwash with 1 M NaOH for 15–30 min. If used immediately after cleaning, rinse with 3–10 CV of equilibration buffer.
5. Sanitization and Sterilization
Disinfection/sterilization minimizes microbial contamination in the column. This product can be disinfected using NaOH solution. NaOH solution is effective against viruses, bacteria, yeast, and endotoxins, and has very low operating cost. Disinfection procedure:
5.1 Rinse the chromatography column with binding buffer for 2-3 CV.
5.2 Rinse the chromatography column with 0.5 M NaOH solution for 2-5 CV.
5.3 Soak the chromatography column in 0.5-1 M NaOH solution for 1 hour.
5.4 Rinse the chromatography column with binding buffer with pH between 7 and 8 for 5-10 CV to complete the sanitization process.
Precautions: 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.
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.
ZJ26F0738068
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