CM HR 离子交换树脂 (高分辨率) 是兼顾高分辨和高流速的强阴离子交换层析介质，由羧甲基（CM） 与高刚性琼脂糖凝胶微球键合而成。本产品的平均粒径为 35 μm，仍可达到 900 cm/h 以上的流速。得益于离子交换容量（配基密度）经过了优化， 在 2 min 停留时间下，本品对溶菌酶的动态结合载量大于 60 mg/mL。当纯 化规模不是很大，或者初始的样品比较干净时（例如上清表达且表达量较高的样品），也 可用本产品通过一步纯化达到高纯度。
阿拉丁CM HR 离子交换树脂（高分辨率）储存在20%乙醇中，凝胶和保护液的体积比为1:1，我司产品规格为实际凝胶的体积。
参数 | 指标 | 基质 | 高分辨、高刚性琼脂糖微球 | 功能基团 | 羧甲基， -CH₂COO⁻ | 平均粒径 | 35 μm | 离子交换容量 | 80~140 μmol H⁺ /mL | 动态结合载量① | ≥60 mg/mL | 推荐工作流速 | 50~150 cm/h | 最大流速② | 900cm/h | pH稳定性 | 6~10（工作 pH）；3~12（长期稳定）；2~14（短期稳定） | 化学稳定性 | 在以下溶液中稳定：常用的水相缓冲液、1 M 氢氧化钠、8 M 尿素、 6 M 盐酸胍、70%乙醇等 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
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注：
① DBC10%测试条件为：10%流穿，溶菌酶，2 min 停留时间
② 10 cm 柱高下的最大测 试流速。
离子交换层析是带电荷的目标分子与离子交换层析介质进行离子交换的分离纯化 过程，主要依赖电荷相互作用，利用带电分子的差异进行分离。因所有生物分子都是极 性的，在一定条件下均可带电荷，以蛋白质为例，当溶液 pH 低于蛋白质的等电点（PI） 时，蛋白质分子带正电荷，则可以与阳离子交换填料进行离子交换而结合。不同分子的 带电量不同，与层析介质的结合牢固程度不同，通过调控洗脱条件可以将不同分子先后 洗脱下来，从而达到分离纯化的目的。
图 1 是典型的阳离子交换层析过程示意图，阳离子交换层析是指层析介质可以与溶 液中的阳离子进行离子交换。CM 基团的电离度小，是弱酸性离子交换剂，因而本产品 被称为弱阳离子交换填料。当填料处于平衡状态时，CM 阴离子周边是带正电荷的对离 子，通常是 Na+。在与目标分子进行离子交换而结合的过程中，带正电的分子被结合， 不带电或者带负电的分子未被结合，达到了初步分离的效果。阳离子交换层析可以通过 提高流动相的离子强度或者升高 pH 的方式将目标分子洗脱。提高流动相的离子强度是 竞争洗脱的过程（图 1a），流动相的离子强度提高后，正电荷与目标分子竞争成为 CM 基团的对离子，根据电荷的多少和结合的牢固程度，不同的分子被先后洗脱下来，从而 达到纯化的效果。
另外一方面，目标分子所带的电荷与环境的 pH 相关，升高 pH 后， 带正电的目标分子所带电荷数量减少（图 1b），与介质结合的牢固程度降低，因此升高 pH 也是阳离子交换层析的一种洗脱方式。
图1：阳离子交换填料工作原理示意图
使用说明
1、色谱柱装填
以下阐述与层析系统连接时，填料的色谱柱装填方法。
（1）所有需要用到的材料的温度要与色谱操作的温度一样，液体最好做脱气处理。
（2）填料用量计算：通过沉降定量填料体积，需要的沉降填料体积=柱体积×压缩比（也称压缩因子）。沉降体积是指填料在20%乙醇保存液中自然沉降完全读取的稳定体积。CM HR 离子交换树脂（高分辨率）的压缩比为~1.10。为使达到压缩比，可通过柱头下压的方法，也可以通过高流速压柱。
（3）填料清洗：将填料悬液充分摇匀后量取相应体积，抽滤除去液体，并用约3 倍填料体积的纯化水洗涤，重复3 次，以除去保存液。
（4）装柱填料悬液准备：将填料转移到适当的容器中，加入适量的装柱液，制成50~75 v%的装柱填料悬液，使用前搅匀。
（5）装填前准备：在清洗干净的层析柱下端加入装柱液，以除去下垫片及层析柱下端的空气，在柱内保留少量的蒸馏水，拧紧下堵头，调整柱子使其垂直于地面。
（6）装填：将搅匀后的填料悬液一次性缓慢倒入层析柱内（必要时使用装柱器），为避免引入气泡，应使之沿层析柱内壁自然流下。将所有填料加入后，用装柱液将装柱器加满，拧紧装柱器上盖，将柱子与层析系统连接。
（7）压柱：使柱内填料自然沉降（或50~150 cm/h 的低流速下沉降），待填料沉降完全后（填料与液体的界面清晰），去除装柱器，装上上柱头，并将柱头下降至界面处；通过高流速（推荐流速见下表，注意柱压不超过0.3 MPa），继续压柱至界面清晰稳定，标记界面稳定时的柱高。停泵，打开柱头上的阀门/堵头，关闭柱底的阀门/堵头，下压柱头至标记位置下方与压缩比对应的位置，旋紧柱头，装柱完成。装柱完成后，需用高流速平衡柱内的填料。
装柱条件 | CM HR 离子交换树脂（高分辨率） | 压缩比 | 1.10 | 装柱流速 | 600-1500 cm/h，因柱子型号而异，以压缩因子为准 |
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2、柱效测定和评价
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 | 0.8~1.0 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、平衡与上样（Equilibration & Loading）
使用阳离子交换填料时，常用的缓冲液有磷酸盐、醋酸盐、柠檬酸盐等缓冲体系，浓度为~20 mM为宜。不含其他盐的上述缓冲液称为平衡缓冲液，记为缓冲液A。缓冲液A的特点为低盐，pH至少低于目标蛋白等电点一个单位。
在上样前，需用缓冲液 A 在操作流速下平衡层析柱，该过程通常需要 5~10 个柱体积的缓冲液 A，以电导、pH 等检测信号参数不变且与缓冲液 A 对应为准。
离子交换填料中最常用的纯化模式为“结合模式（binding）”，即目标蛋白与填料进行离子交换后结合，杂质则流穿。在有些场合，也会使用“流穿模式（flowthrough）”，即杂质与填料结合，目标蛋白流穿，该模式更多用于中度或者精纯阶段。
样品通常溶于上述缓冲液A，或者将样品溶液进行换液处理，置换成缓冲液A体系。上样量可按“mg目标蛋白/mL填料”来设定，通常为DBC10%的50~80%，例如CM HR 离子交换树脂（高分辨率）对溶菌酶的DBC10%为~60 mg/mL，纯化时的上样量可为30~48 mg/mL。除了DBC10%，也可以测试上样流穿液中的目标蛋白确定上样量。
上样后需要 3~10 个柱体积的缓冲液 A 再平衡层析柱。
4、冲洗 （Wash）
用3~10个柱体积的平衡液A冲洗上样后的层析柱，观察检测器的变化，直到电导、pH等参数不变，此时未交换的组分被清洗出去。
5、洗脱（Elution）
最常用的洗脱方式为提高洗脱液中NaCl的浓度（在缓冲液A中加入NaCl），推荐在0~500 mM范围内进行阶跃洗脱或者线性梯度洗脱。对于一些场合，也可以改变pH或者同时改变离子强度和pH进行洗脱，通过优化洗脱条件可以提高层析的效果。
6、再生（Regeneration）
可用更高离子强度的NaCl溶液（1~2 M NaCl）将结合牢固、未被洗脱的蛋白洗脱，对填料进行再生，本产品在再生后可使用多次。也可以改变pH或者同时改变离子强度和pH进行再生。再生后需要用缓冲液A平衡。
7. 在位清洗（CIP）
若有蛋白沉淀、脂蛋白或疏水蛋白与填料结合无法在上述过程中洗脱，则需在位清洗除去。可采用以下选择进行在位清洗：1 M NaOH、70%乙醇、30%异丙醇等。需用~2.5 CV的溶液进行在位清洗，接触时间~30 min。在位清洗后需要用缓冲液A平衡。
8、填料保存
填料的初始保存液为20%乙醇，使用过后可继续用20%乙醇保存。保存温度在2-8 ℃为宜，不可冻存。
CM HR Agarose (High Resolution) is a weak cation exchange chromatography medium that combines high resolution and high flow rates. It is composed of carboxymethyl (CM) groups coupled to high-rigidity agarose microspheres. This product has an average particle size of 35 μm yet can still achieve flow rates above 900 cm/h. Thanks to the optimized ion exchange capacity (ligand density), its dynamic binding capacity for lysozyme exceeds 60 mg/mL at a residence time of 2 min. When the purification scale is not very large, or when the initial sample is relatively clean (e.g., supernatant samples with high expression levels), this product can also be used for one-step purification to achieve high purity.
Aladdin CM HR Agarose (High Resolution) is stored in 20% ethanol, with a gel-to-preservation-solution volume ratio of 1:1. Our product specifications are based on the actual settled gel volume.
Parameter | Specification | Matrix | High-resolution, high-rigidity agarose microspheres | Functional Group | Carboxymethyl, -CH₂COO⁻ | Mean Particle Size | 35 μm | Ion Exchange Capacity | 80–140 μmol H⁺ /mL | Dynamic Binding Capacity<sup>①</sup> | ≥60 mg/mL | Recommended Working Flow Rate | 50–150 cm/h | Maximum Flow Rate<sup>②</sup> | 900 cm/h | pH Stability | 6–10 (working pH); 3–12 (long-term stability); 2–14 (short-term stability) | Chemical Stability | Stable in the following solutions: common aqueous buffers, 1 M NaOH, 8 M urea, 6 M guanidine hydrochloride, 70% ethanol, etc. | Storage | 20% ethanol, 2–8°C | Shelf Life | 5 years |
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Notes:
① DBC<sub>10%</sub> test conditions: 10% breakthrough, lysozyme, 2 min residence time.
② Maximum test flow rate at a column height of 10 cm.
Ion exchange chromatography is a separation and purification process in which charged target molecules undergo ion exchange with an ion exchange chromatography medium. It primarily relies on electrostatic interactions to separate charged molecules based on their differences in charge. Since all biomolecules are polar, they can carry charges under certain conditions. Taking proteins as an example, when the solution pH is below the protein's isoelectric point (pI), the protein molecules carry a positive charge and can bind to cation exchange media via ion exchange. Different molecules have different net charges and thus different binding affinities to the chromatography medium. By adjusting the elution conditions, different molecules can be eluted sequentially, thereby achieving separation and purification.
Figure 1 is a schematic diagram of a typical cation exchange chromatography process. Cation exchange chromatography refers to the process in which the chromatography medium exchanges ions with cations in the solution. The CM group has a low degree of ionization and is a weakly acidic ion exchanger; therefore, this product is referred to as a weak cation exchange medium. When the medium is in an equilibrated state, the CM anions are surrounded by positively charged counter-ions, typically Na⁺. During the binding process via ion exchange with the target molecules, positively charged molecules are bound, while uncharged or negatively charged molecules are not, achieving preliminary separation. Cation exchange chromatography can elute target molecules by increasing the ionic strength of the mobile phase or by increasing the pH. Increasing the ionic strength of the mobile phase is a competitive elution process (Figure 1a). As the ionic strength of the mobile phase increases, positively charged ions compete with the target molecules to become counter-ions for the CM groups. Depending on the amount of charge and binding strength, different molecules are eluted sequentially, achieving purification.
On the other hand, the net charge of the target molecule is related to the environmental pH. When the pH is increased, the number of positive charges on the target molecule decreases (Figure 1b), weakening its binding to the medium. Therefore, increasing the pH is also an elution method for cation exchange chromatography.
Figure 1: Schematic diagram of the working principle of a cation exchange medium.
图中文字翻译
微球及内部骨架的平衡状态
Equilibrium state of the microspheres and their internal matrix
上样结合目标分子
Sample loading and binding of target molecules
提高离子强度洗脱
Elution by increasing ionic strength
升高 pH 洗脱
Elution by increasing pH
Instructions for Use
1. Column Packing
The following describes the column packing method for the resin when connected to a chromatography system.
(1) All materials used should be at the same temperature as the chromatography operation. Liquids should preferably be degassed.
(2) Calculation of medium volume: The settled gel volume is used to quantify the medium. Required settled gel volume = column volume × compression ratio (also called compression factor). The settled gel volume refers to the stable volume read after the medium has settled completely in the 20% ethanol storage solution. The compression ratio of CM HR Agarose (High Resolution) is ~1.10. To achieve this compression ratio, either the column adapter can be lowered, or high flow rates can be used.
(3) Resin Washing: Thoroughly resuspend the resin slurry, measure the required volume, and vacuum filter to remove the liquid. Wash the resin three times with approximately 3 volumes of purified water each time to remove the storage solution.
(4) Preparation of the packing suspension: Transfer the medium to a suitable container, add an appropriate amount of packing solution to prepare a 50–75 v% packing suspension, and mix well before use.
(5) Pre-packing preparation: Add packing solution to the bottom of the cleaned chromatography column to remove air from the bottom frit and the lower part of the column, leaving a small amount of distilled water in the column. Tighten the bottom cap and adjust the column so that it is perpendicular to the ground.
(6) Packing: Pour the mixed medium suspension slowly into the chromatography column in one go (using a packing reservoir if necessary). To avoid introducing air bubbles, allow it to flow down naturally along the inner wall of the column. After all the medium has been added, fill the packing reservoir with packing solution, tighten the top cap of the reservoir, and connect the column to the chromatography system.
(7) Compression: Allow the resin in the column to settle naturally (or at a low flow rate of 50-150 cm/h). Once the resin is completely settled (a clear interface between the resin and the liquid is visible), remove the packing reservoir, attach the top adaptor, and lower the adaptor to the interface. Continue to compress the resin using a high flow rate (see recommended flow rates below, ensuring column backpressure does not exceed 0.3 MPa) until the interface is clear and stable. Mark the column height at the stable interface. Stop the pump. Open the valve/plug on the top adaptor, close the valve/plug at the column bottom, lower the adaptor to the position corresponding to the compression ratio below the marked position, and tighten the adaptor. Packing is complete. After packing, equilibrate the resin in the column using a high flow rate.
Packing Condition | CM HR Agarose (High Resolution) | Compression ratio | 1.10 | Packing flow rate | 600–1500 cm/h (varies by column type; the compression factor shall prevail) |
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2. Column Efficiency Test & Evaluation
Column efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).
Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.
Item | 1.0% Acetone | 0.8–1.0 M NaCl | Sample volume | 1.0% column volume | 1.0% column volume | Mobile phase | Pure water | 0.4 M NaCl | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
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Calculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:
- HETP = L / N
- N = 5.54 × (Vr / Wh)2
- As = a / b
Where:L = column height; Vr = retention volume; Wh = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.Generally, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.
3. Equilibration & Loading
For cation exchange media, commonly used buffers include phosphate, acetate, and citrate buffer systems, with a concentration of approximately 20 mM. The above buffer without additional salts is called the equilibration buffer, designated as Buffer A. Buffer A is characterized by low salt content and a pH at least one unit below the isoelectric point of the target protein.
Before loading, equilibrate the column with Buffer A at the operating flow rate. This process typically requires 5–10 column volumes of Buffer A, until the conductivity, pH, and other detector signals remain constant and correspond to those of Buffer A.
The most commonly used purification mode for ion exchange media is the "binding mode," where the target protein binds to the medium via ion exchange while impurities flow through. In some cases, the "flowthrough mode" is also used, where impurities bind to the medium and the target protein flows through. This mode is more often employed in intermediate or polishing purification stages.
The sample is typically dissolved in Buffer A, or the sample solution is buffer-exchanged into the Buffer A system. The loading amount can be set as "mg target protein / mL medium," usually at 50–80% of the DBC10%. For example, CM HR Agarose (High Resolution) has a DBC10% of ~60 mg/mL for lysozyme, so the loading amount for purification could be 30–48 mg/mL. In addition to DBC10%, the loading amount can also be determined by testing the target protein in the flowthrough fraction.
After loading, re-equilibrate the column with 3–10 column volumes of Buffer A.
4. Wash
Wash the loaded column with 3–10 column volumes of equilibration buffer (Buffer A). Monitor the detector signals until the conductivity, pH, and other parameters remain constant, indicating that unexchanged components have been washed out.
5. Elution
The most commonly used elution method is to increase the NaCl concentration in the elution buffer (Buffer A with NaCl added). A step elution or linear gradient elution within the range of 0–500 mM NaCl is recommended. In some cases, elution can also be achieved by changing the pH or by simultaneously altering both ionic strength and pH. Optimization of the elution conditions can improve the chromatography performance.
6. Regeneration
Use a higher ionic strength NaCl solution (1–2 M NaCl) to elute strongly bound proteins that were not eluted, thereby regenerating the medium. This product can be reused multiple times after regeneration. Alternatively, regeneration can also be achieved by changing the pH or simultaneously changing both ionic strength and pH. After regeneration, equilibrate with Buffer A.
7. Clean-in-Place (CIP)
If protein precipitates, lipoproteins, or hydrophobic proteins bind to the medium and cannot be removed during the above steps, CIP is required for removal. The following options can be used for CIP: 1 M NaOH, 70% ethanol, 30% isopropanol, etc. Use approximately 2.5 CV of the CIP solution with a contact time of about 30 min. After CIP, re-equilibrate the column with Buffer A.
8. Resin Storage
The initial storage solution for the medium is 20% ethanol. After use, it can continue to be stored in 20% ethanol. The recommended storage temperature is 2-8°C. Do not freeze.
ZJ26F0838144
危险品化学品经营许可证（带存储）