你知道液相色谱上有几种清洗液吗？一种？两种？还是三种？

Do you know how many cleaning solutions are used in liquid chromatography? A sort of? Two kinds? Or three?

液相色谱的清洗液种类繁多，根据不同的色谱柱类型和清洗需求，可以使用以下几种清洗液：

带着问题，让我们来慢慢地捋一下。首先，我们知道液相色谱仪器主要由泵、自动进样器、柱温箱、检测器这四个模块组成。接着，我们就来一个个模块看，它们有没有清洗液，用什么清洗，洗什么，需要注意什么？

With the question in mind, let’s walk through it slowly. First of all, we know that the liquid chromatography instrument mainly consists of four modules: pump, autosampler, column oven, and detector. Next, let’s look at the modules one by one to see if they have cleaning fluid, what should be used to clean them, what should be washed, and what should we pay attention to?

泵柱塞密封清洗液 Pump plunger seal cleaning fluid

泵在线清洗系统（即seal wash），主要的作用就是清洗输液泵的柱塞密封圈和柱塞杆，所以我们称它为泵柱塞密封清洗液。

The main function of the pump online cleaning system (ie seal wash) is to clean the plunger seal ring and plunger rod of the infusion pump , so we call it pump plunger seal cleaning fluid.

如上图所示，清洗垫、密封圈、泵头三者之间存在2个腔体。其中右边的腔体，通过柱塞杆的往复运动，流动相经单向阀进入到色谱系统，它属于高压流路。左边的腔体中，由泵密封圈清洗液流经清洗垫和密封圈之间起到清洗送液泵的柱塞密封圈和柱塞杆的作用，它属于常压流路。因此，通常都是使用蠕动泵来进行液体输送。

As shown in the picture above, there are two cavities between the cleaning pad, sealing ring and pump head. In the cavity on the right, through the reciprocating motion of the plunger rod, the mobile phase enters the chromatographic system through the one-way valve, which belongs to the high-pressure flow path. In the cavity on the left, the pump seal ring cleaning liquid flows between the cleaning pad and the seal ring to clean the plunger seal ring and plunger rod of the liquid delivery pump. It is a normal pressure flow path. Therefore, peristaltic pumps are usually used for liquid delivery.

可能有人要问了，我为什么要清洗柱塞杆和密封圈呢？那是因为柱塞杆在往复运动的过程中难免会携带一些流动相，当使用缓冲盐做流动相时，随着溶剂挥发，可能会有盐分析出，而析出的盐结晶则会加速柱塞密封圈及柱塞杆的磨损老化。所以为了延长柱塞封圈及柱塞杆的使用寿命，故推荐使用10%的异丙醇水溶液作为清洗液。

Someone may ask, why should I clean the plunger rod and sealing ring? That's because the plunger rod will inevitably carry some mobile phase during its reciprocating motion. When buffer salt is used as the mobile phase, as the solvent evaporates, salt may precipitate out, and the precipitated salt crystallization will accelerate the plunger. The sealing ring and plunger rod are worn and aged. Therefore, in order to extend the service life of the plunger seal and plunger rod, it is recommended to use 10% isopropyl alcohol aqueous solution as the cleaning fluid.

紧着着又是一个问题，为什么要使用10%的异丙醇水溶液呢？其他的行不行？首先，咱们的目的是洗掉析出的盐，那么自然是水的溶解效果更好。但是长期使用纯水，我们都知道容易长菌，而加入一定量的有机溶剂则能有效地抑制细菌的繁殖生长。这与我们实验过程中，为什么推荐在A相（即水相）中添加少量（如5%以上）的有机相是一样的道理，它可以延长流动相的有效期。

Next is another question, why use 10% isopropyl alcohol aqueous solution? Is the other one okay? First of all, our purpose is to wash away the precipitated salt, so naturally the water will dissolve better. However, when pure water is used for a long time, we all know that bacteria are prone to grow, and adding a certain amount of organic solvents can effectively inhibit the reproduction and growth of bacteria. This is the same reason why it is recommended to add a small amount (such as more than 5% ) of organic phase to phase A (water phase) during our experiment. It can extend the validity period of the mobile phase.

其次，不用异丙醇，用其他溶剂可以吗？当然是可以的。但是异丙醇相对于常用的甲醇、乙腈来说，它的毒性更低，价格也便宜，同时它的润滑作用和对盐及有机相的溶解能力也都比其他有机溶剂更好。此处还需要提醒大家注意的是：

Secondly, instead of isopropyl alcohol , can I use other solvents? Of course it is possible . However, isopropyl alcohol is less toxic and cheaper than commonly used methanol and acetonitrile. At the same time, its lubricating effect and ability to dissolve salts and organic phases are better than other organic solvents. What also needs to be reminded here is:

1、正相色谱中，由于流动相中不使用水，因此也不会出现盐析的问题，所以正相系统中不需要使用清洗液。这一点对于不是固定正相仪器，经常需要切换的伙伴来说，尤其需要注意下。不然，可就是好心办坏事了。

1. In normal phase chromatography, since water is not used in the mobile phase, there will be no problem of salting out, so there is no need to use a cleaning solution in the normal phase system. This is especially important for partners who do not have fixed normal-phase instruments and often need to switch. Otherwise, it would be a good intention but a bad thing.

2、对于泵密封圈清洗液，建议大家尽量每天观察和勤更换，最长不要超过1周。否则，本来是用来清洗的溶液，反而成了污染的另一个来源。

2. For pump seal ring cleaning fluid, it is recommended that you observe and replace it frequently every day, no more than 1 week at most. Otherwise, the solution originally used for cleaning becomes another source of contamination.

3、对于一些老旧的仪器，可能配备的是手动清洗装置（即注射器），建议每次运行完后手动清洗，注射清洗液10~20mL。

3. For some old instruments, they may be equipped with a manual cleaning device (i.e. syringe). It is recommended to clean manually after each operation and inject 10~20mL of cleaning solution.

进样系统清洗液 Sampling system cleaning fluid

自动进样器是样品的引入装置，它通常由进样针、计量泵、定量环和六通阀组成。如下图所示：

The autosampler is a sample introduction device, which usually consists of a syringe, a metering pump, a quantitative loop and a six-way valve. As shown below:

在这张图中，我们又发现了一个清洗液，即进样器针头清洗液。它的主要作用就是清洗进样针的针头。

In this picture, we find another cleaning fluid, the injector needle cleaning fluid. Its main function is to clean the needle of the syringe.

以岛津液相为例，岛津的自动进样器清洗主要由一个低压阀和计量泵组成。清洗进样针时，针会移动到清洗口，由清洗液持续冲洗进样针的外壁，以去除可能残留在针外壁的污染物。

Taking Shimadzu Liquid Phase as an example, Shimadzu's automatic sampler cleaning mainly consists of a low-pressure valve and metering pump. When cleaning the syringe, the needle will move to the cleaning port, and the cleaning solution will continuously flush the outer wall of the syringe to remove any contaminants that may remain on the outer wall of the needle.

但是在岛津的这个系统中，由于计量泵也在里面，所以清洗液也参与了样品进样量的计量。这里也就顺便解答了大家的一个问题，那就是进样器清洗液是需要脱气的。因为如果清洗液中有气泡会导致吸样量不准。

But in this Shimadzu system, since the metering pump is also inside, the cleaning fluid also participates in the measurement of the sample injection volume. Here we also answer a question everyone has, that is, the injector cleaning fluid needs to be degassed. Because if there are bubbles in the cleaning solution, the sample amount will be inaccurate.

那么进样器针头的清洗液推荐用什么呢？进样器清洗液的目的是减小样品残留，因此原则上最好是使用不含盐的流动相。但存在的问题就是做不同的品种时就需要配不同的清洗液，那就会变得比较麻烦，尤其是对于不是固定品种的研发实验室来说。

So what is the recommended cleaning solution for the injector needle? The purpose of the injector cleaning solution is to reduce sample residue, so in principle it is best to use a salt-free mobile phase . But the problem is that when making different varieties, different cleaning solutions need to be prepared, which becomes more troublesome, especially for R&D laboratories that do not have fixed varieties.

对反相系统而言，考虑到样品要么偏向于易溶于水相，要么偏向于易溶于有机相，所以大部分人就用中国特色来处理了，采用折中方案使用50%的甲醇水溶液作为自动进样器的针头清洗液了，基本上可以解决大部分的残留问题。但是针对一些特殊情况还是需要特殊考虑的。比如：

For the reversed-phase system, considering that the sample is either soluble in the water phase or soluble in the organic phase, most people use Chinese characteristics to deal with it, using a compromise solution of 50% methanol aqueous solution. As a needle cleaning solution for autosamplers , it can basically solve most of the residual problems. However, some special circumstances still require special consideration. for example:

1、当流动相采用较高浓度的盐溶液时，如超过50mmol/L时，为防止清洗液与流动相接触后产生盐析，建议清洗液中的有机溶剂浓度进一步降低。

1. When the mobile phase uses a higher concentration of salt solution, such as more than 50mmol/L, in order to prevent salting out after the cleaning solution comes into contact with the mobile phase, it is recommended that the concentration of the organic solvent in the cleaning solution be further reduced.

2、当检测波长使用低波长，如210nm及以下，考虑到甲醇的截止波长为210nm，建议使用乙腈代替甲醇。

2. When the detection wavelength is low, such as 210nm and below, considering that the cutoff wavelength of methanol is 210nm, it is recommended to use acetonitrile instead of methanol.

3、当样品为一些酸性或碱性，以及离子化合物等容易残留的样品时，可以考虑在清洗液中少量添加一些甲酸、三氟乙酸等来作为清洗液，增强样品的清洗效果。

3. When the sample is acidic or alkaline, as well as ionic compounds that are easy to remain, you can consider adding a small amount of formic acid, trifluoroacetic acid, etc. to the cleaning solution as a cleaning solution to enhance the cleaning effect of the sample.

4、对于正相系统，则建议是直接使用流动相作为清洗液，因为这并不会增加多少工作量。

4. For the normal phase system, it is recommended to directly use the mobile phase as the cleaning solution, because this will not increase the workload much.

柱温箱和检测器 Column oven and detector

再来看看这两个模块，有没有清洗液呢。 Let's take a look at these two modules to see if there is any cleaning fluid.

柱温箱的作用主要是对流动相进行预加热并控制色谱柱的外部工作环境温度，该模块可以理解为只是包裹在色谱柱外部的控温装置，所以并不涉及需要清洗的地方。检测器的作用则是将经色谱柱分离后流经检测器的待测物质转化为电信号。该模块也只有一条主流路，运行过程中一直在冲洗，所以也没有清洗的地方。

The main function of the column thermostat is to preheat the mobile phase and control the external working environment temperature of the chromatographic column. This module can be understood as just a temperature control device wrapped around the outside of the chromatographic column, so it does not involve areas that require cleaning. The function of the detector is to convert the substance to be measured that flows through the detector after being separated by the chromatographic column into an electrical signal. This module also has only one main channel, which is constantly being flushed during operation, so there is no place for cleaning.