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你知道液相色譜上有幾種清洗液嗎？一種？?jī)煞N？還是三種？

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

液相色譜的清洗液種類繁多，根據(jù)不同的色譜柱類型和清洗需求，可以使用以下幾種清洗液：

帶著問(wèn)題，讓我們來(lái)慢慢地捋一下。首先，我們知道液相色譜儀器主要由泵、自動(dòng)進(jìn)樣器、柱溫箱、檢測(cè)器這四個(gè)模塊組成。接著，我們就來(lái)一個(gè)個(gè)模塊看，它們有沒(méi)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

泵在線清洗系統(tǒng)（即seal wash），主要的作用就是清洗輸液泵的柱塞密封圈和柱塞桿，所以我們稱它為泵柱塞密封清洗液。

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.

如上圖所示，清洗墊、密封圈、泵頭三者之間存在2個(gè)腔體。其中右邊的腔體，通過(guò)柱塞桿的往復(fù)運(yùn)動(dòng)，流動(dòng)相經(jīng)單向閥進(jìn)入到色譜系統(tǒng)，它屬于高壓流路。左邊的腔體中，由泵密封圈清洗液流經(jīng)清洗墊和密封圈之間起到清洗送液泵的柱塞密封圈和柱塞桿的作用，它屬于常壓流路。因此，通常都是使用蠕動(dòng)泵來(lái)進(jìn)行液體輸送。

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.

可能有人要問(wèn)了，我為什么要清洗柱塞桿和密封圈呢？那是因?yàn)橹麠U在往復(fù)運(yùn)動(dòng)的過(guò)程中難免會(huì)攜帶一些流動(dòng)相，當(dāng)使用緩沖鹽做流動(dòng)相時(shí)，隨著溶劑揮發(fā)，可能會(huì)有鹽分析出，而析出的鹽結(jié)晶則會(huì)加速柱塞密封圈及柱塞桿的磨損老化。所以為了延長(zhǎng)柱塞封圈及柱塞桿的使用壽命，故推薦使用10%的異丙醇水溶液作為清洗液。

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.

緊著著又是一個(gè)問(wèn)題，為什么要使用10%的異丙醇水溶液呢？其他的行不行？首先，咱們的目的是洗掉析出的鹽，那么自然是水的溶解效果更好。但是長(zhǎng)期使用純水，我們都知道容易長(zhǎng)菌，而加入一定量的有機(jī)溶劑則能有效地抑制細(xì)菌的繁殖生長(zhǎng)。這與我們實(shí)驗(yàn)過(guò)程中，為什么推薦在A相（即水相）中添加少量（如5%以上）的有機(jī)相是一樣的道理，它可以延長(zhǎng)流動(dòng)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，不用異丙醇，用其他溶劑可以嗎？當(dāng)然是可以的。但是異丙醇相對(duì)于常用的甲醇、乙腈來(lái)說(shuō)，它的毒性更低，價(jià)格也便宜，同時(shí)它的潤(rùn)滑作用和對(duì)鹽及有機(jī)相的溶解能力也都比其他有機(jī)溶劑更好。此處還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、正相色譜中，由于流動(dòng)相中不使用水，因此也不會(huì)出現(xiàn)鹽析的問(wèn)題，所以正相系統(tǒng)中不需要使用清洗液。這一點(diǎn)對(duì)于不是固定正相儀器，經(jīng)常需要切換的伙伴來(lái)說(shuō)，尤其需要注意下。不然，可就是好心辦壞事了。

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、對(duì)于泵密封圈清洗液，建議大家盡量每天觀察和勤更換，最長(zhǎng)不要超過(guò)1周。否則，本來(lái)是用來(lái)清洗的溶液，反而成了污染的另一個(gè)來(lái)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、對(duì)于一些老舊的儀器，可能配備的是手動(dòng)清洗裝置（即注射器），建議每次運(yùn)行完后手動(dòng)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.

進(jìn)樣系統(tǒng)清洗液 Sampling system cleaning fluid

自動(dòng)進(jìn)樣器是樣品的引入裝置，它通常由進(jìn)樣針、計(jì)量泵、定量環(huán)和六通閥組成。如下圖所示：

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:

在這張圖中，我們又發(fā)現(xiàn)了一個(gè)清洗液，即進(jìn)樣器針頭清洗液。它的主要作用就是清洗進(jìn)樣針的針頭。

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

以島津液相為例，島津的自動(dòng)進(jìn)樣器清洗主要由一個(gè)低壓閥和計(jì)量泵組成。清洗進(jìn)樣針時(shí)，針會(huì)移動(dòng)到清洗口，由清洗液持續(xù)沖洗進(jìn)樣針的外壁，以去除可能殘留在針外壁的污染物。

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.

但是在島津的這個(gè)系統(tǒng)中，由于計(jì)量泵也在里面，所以清洗液也參與了樣品進(jìn)樣量的計(jì)量。這里也就順便解答了大家的一個(gè)問(wèn)題，那就是進(jìn)樣器清洗液是需要脫氣的。因?yàn)槿绻逑匆褐杏袣馀輹?huì)導(dǎo)致吸樣量不準(zhǔn)。

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.

那么進(jìn)樣器針頭的清洗液推薦用什么呢？進(jìn)樣器清洗液的目的是減小樣品殘留，因此原則上最好是使用不含鹽的流動(dòng)相。但存在的問(wèn)題就是做不同的品種時(shí)就需要配不同的清洗液，那就會(huì)變得比較麻煩，尤其是對(duì)于不是固定品種的研發(fā)實(shí)驗(yàn)室來(lái)說(shuō)。

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.

對(duì)反相系統(tǒng)而言，考慮到樣品要么偏向于易溶于水相，要么偏向于易溶于有機(jī)相，所以大部分人就用中國(guó)特色來(lái)處理了，采用折中方案使用50%的甲醇水溶液作為自動(dòng)進(jìn)樣器的針頭清洗液了，基本上可以解決大部分的殘留問(wèn)題。但是針對(duì)一些特殊情況還是需要特殊考慮的。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、當(dāng)流動(dòng)相采用較高濃度的鹽溶液時(shí)，如超過(guò)50mmol/L時(shí)，為防止清洗液與流動(dòng)相接觸后產(chǎn)生鹽析，建議清洗液中的有機(jī)溶劑濃度進(jìn)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、當(dāng)檢測(cè)波長(zhǎng)使用低波長(zhǎng)，如210nm及以下，考慮到甲醇的截止波長(zhǎng)為210nm，建議使用乙腈代替甲醇。

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、當(dāng)樣品為一些酸性或堿性，以及離子化合物等容易殘留的樣品時(shí)，可以考慮在清洗液中少量添加一些甲酸、三氟乙酸等來(lái)作為清洗液，增強(qiáng)樣品的清洗效果。

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、對(duì)于正相系統(tǒng)，則建議是直接使用流動(dòng)相作為清洗液，因?yàn)檫@并不會(huì)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.

柱溫箱和檢測(cè)器 Column oven and detector

再來(lái)看看這兩個(gè)模塊，有沒(méi)有清洗液呢。 Let's take a look at these two modules to see if there is any cleaning fluid.

柱溫箱的作用主要是對(duì)流動(dòng)相進(jìn)行預(yù)加熱并控制色譜柱的外部工作環(huán)境溫度，該模塊可以理解為只是包裹在色譜柱外部的控溫裝置，所以并不涉及需要清洗的地方。檢測(cè)器的作用則是將經(jīng)色譜柱分離后流經(jīng)檢測(cè)器的待測(cè)物質(zhì)轉(zhuǎn)化為電信號(hào)。該模塊也只有一條主流路，運(yùn)行過(guò)程中一直在沖洗，所以也沒(méi)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.