Basic maintenance of liquid chromatography

People need health care, car maintenance, equipment is the same, usually do a good job in daily maintenance, can greatly reduce the failure rate, reduce repair and shutdown costs. This topic will discuss the maintenance precautions of the instrument from the perspective of hardware and use, mainly taking Agilent 1260 liquid chromatography as an example, but in fact, in many places, each instrument will have the same.
In terms of the structure of the instrument, according to the sequence of mobile phase passing through: solvent bottle - degasser - pump - column temperature box (chromatographic column) - detector - waste liquid, we will talk about the matters needing attention in the use of each part of the instrument according to this order.
First of all, it's the solvent bottle. It seems that the solvent bottle is just a few glass bottles. It seems that there is nothing to say about it. However, it looks simple. If you don't pay attention to the use of it, it will cause a lot of trouble
one point one The first problem is pollution: pay special attention to the bottles containing water or buffer solution. Although most of the water used in the liquid phase has been sterilized, the bacteria are very tenacious. They will become active under appropriate temperature and light. If phosphate and other additives are added to the mobile phase, they will be even more powerful. Therefore, what should be done for solvent bottles It is very important to change the mobile phase frequently, and to change it often. In addition, another way to prevent bacterial growth is to add a certain concentration of sodium azide into the water. However, it is highly toxic and controlled, which is not easy to obtain and has a high risk. Therefore, in my opinion, it is more convenient to replace the mobile phase directly.
1.2 the water phase should be changed frequently. What about the organic phase? You don't have to worry about bacterial reproduction, do you? Yes, there is no need to worry about the organic phase at all. However, if it is a bottle containing acetonitrile, you should pay special attention to the "polymerization" of acetonitrile. Under natural conditions, especially under light conditions, acetonitrile molecules will form small groups hand in hand with their partners. The small groups will gradually grow until they can be seen by the eyes, and some flocculent precipitates will appear in the bottle, This is the "polymerization" of acetonitrile. In order to prevent polymerization, special attention should be paid to acetonitrile. It is better to use a dark bottle with direct sunlight on the other side. In addition, when changing acetonitrile, the rest of the bottle bottom must be poured out and replaced with a new one.
one point three After finishing the solution, we have to pay attention to the bottle itself. Usually there are several small holes on the cap of the bottle for the mobile phase pipeline to pass through, but I don't know why we need to open more holes... I have seen some users block this small hole, which is a good way to prevent organic solvent volatilization, reduce laboratory pollution and prevent dust from entering the mobile phase bottle. However, if this Some small holes are completely blocked. If the bottle cap is tightened tightly, the liquid absorption of the mobile phase may not be smooth, because the bottle is completely isolated from atmospheric pressure... In order to solve this problem, you can consult the manufacturer to buy a kind of "safety bottle cap", which can prevent volatilization and dust, and do not have to worry about blocking the air completely.
one point four The last one is the filter head in the solvent bottle. Its function is to prevent the particles and impurities in the solution bottle from entering into the flow path system of the instrument. The pore diameter of the filter is 0.45um. The materials are glass sintered quartz and stainless steel. If this thing is dirty, it will cause poor liquid absorption of the mobile phase. It can be cleaned. Glass is usually made of dilute nitric acid bubble overnight Stainless steel can be directly ultrasonic cleaned.
Next, degassing is to remove the dissolved gas in the mobile phase, so we have to start with the gas
two point one How to make the degasser run efficiently, the best way is to "help it". In fact, the mobile phase is degassed in advance. After the flow matching is good, now degassing in the ultrasonic machine for a few minutes can greatly reduce the work load of the degasser, because the degassing efficiency of degassing is certain, if the dissolved gas in the solution exceeds the deaerator's processing capacity When the flow rate is high (especially at high flow rate), the gas will not be cleaned. At that time, bubbles may appear during mixing, which will affect the pressure fluctuation and noise of the instrument.
2.2 in addition to being afraid that the degasser is not clean, it is also afraid of blocking. Is degassing opportunity blocked? Usually not, but after a long holiday, it's hard to say that the main culprit for blocking the degasser is the microorganisms, bacteria and algae in the mobile phase. If they stay in the degasser with the mobile phase, after a few days and a week, they will probably develop into a model, and the pipeline of the degasser may be full of "algae" and then it will be blocked When the degasser is not used for a long time, such as the National Day holiday, the solution in the water phase pipeline must be replaced with methanol to prevent "grass growing". Acetonitrile is also not recommended because it will "polymerize".
two point three Plugging and leakage are a pair of happy enemies. The places that can be blocked on the liquid chromatography may also leak, and the degasser is no exception. Generally, there are two possibilities for liquid leakage: one is that the joint is loose, so long as it is tightened again; the other is more unfortunate, and the vacuum chamber is damaged, which needs to be overhauled. Generally, the flow rate of mobile phase exceeds the tolerance of degassing function Usually, the maximum flow rate is exceeded when purging, or the mobile phase is sucked directly through the pipeline by the syringe for convenience.
With the footprints of the mobile phase, to the pump, the pump is the most important component of liquid chromatography, and is usually the most expensive one. It should be used with care
three point one In many cases, the problem of the pump is reflected in the pressure, and the pressure fluctuation is the most common type of problem. The normal pressure fluctuation of the pump is usually less than 2%, which is smooth, and the abnormal fluctuation is usually caused by bubbles and salt: if the bubbles in the mobile phase are not removed by the degasser and reach the pump, then the pressure fluctuation will be caused. Usually, we can re PU The fluctuation caused by salt is mainly due to the addition of buffer salt with high concentration in the mobile phase. When the salt containing mobile phase is mixed with the organic phase, the salt will precipitate slightly, resulting in abnormal pressure fluctuation. To solve this problem, we can consider appropriate reduction of salt concentration, or use methanol to replace acetonitrile around the organic phase This kind of mobile phase must be used for various reasons. It can be considered to add a certain proportion of water into the organic phase, such as 10-20, and then appropriately increase the end point of the gradient.
three point two In the maintenance of the pump, it is often done to replace the filter white head on the purge valve. In fact, the difficulty is not to replace the white head itself, but to determine when to replace it. The standard of judgment is that when the flow rate of pure water is 5ml / min, if the pressure exceeds 10bar, it is necessary to consider replacement. The replaced white head can also roughly judge the use condition of the instrument : if the white head is white, it seems not dirty, but it is blocked, it may be caused by the precipitation of salt in the mobile phase. This instrument has been enduring the high salt mobile phase for a long time. If the replaced one is gray black, this is the most common condition, which is caused by the wear of the pump head sealing gasket. If the white head cutter is replaced frequently, it will be prompted If the sealing gasket of the instrument is worn too fast, it may be necessary to upgrade the plunger rod cleaning equipment; if the color of the white head is yellow, green and other strange colors, then it is time for the instrument to talk about hygiene, which is caused by microorganisms in the mobile phase.
3.3 in the process of using the pump, sometimes the mobile phase will be replaced. The replacement refers to the process of replacing the reverse phase solvent to the normal phase solvent or vice versa. The most important thing in this process is the compatibility of the mobile phase. The commonly used solvents in the forward and reverse phase chromatography are immiscible. Therefore, in the process of replacement, the system must be thoroughly washed with isopropanol to ensure that all the components in the pipeline are in good condition The original solvent was replaced by isopropanol.
4. If you go on, you will come to the injector. The injector is divided into two categories: automatic and automatic. Although the working modes of the two are different, the main points of using them are basically the same
four point one The most common problem of the autosampler is dirty, which is called cross contamination in professional terms. The cause of cross contamination is very direct. The sample remains on the inner / outer surface of the injection probe and enters the chromatographic system with the next injection. To solve the cross contamination, it is very simple, that is, washing. If the sample concentration is high or the adsorption is strong, the autosampler will have the function of washing the needle For samples, this function must be turned on. Due to the principle of instrument design, Agilent's automatic sampler does not need to clean the inner wall of the injection probe; for the manual injector, pay attention to the inner and outer surface of the manual syringe. If you forget to turn on the needle washing function, the contamination may have been left on the needle holder or the flow valve. At this time, these two parts should be cleaned, and the two parts should be removed for ultrasonic cleaning according to the maintenance steps in the instrument manual
4.2 another problem with the autosampler is the reproducibility of peak area. Usually, this is because there is a problem when the automatic sampler sucks the sample. First, observe whether the liquid level of the sample is high enough to ensure that the injector can absorb the sample. If this problem is eliminated, it is necessary to check the setting of the automatic sampler. For some samples with high viscosity, the absorption speed of the automatic sampler should be reduced, which can be changed in the instrument setting; of course, there are also There are some more rare problems: the sample is adsorbed on the sample bottle, so we can consider changing to a sample bottle of medium material; there is another case, because the bottle cap of the sample bottle is screwed too tightly, causing the bottle to be isolated from the air, which makes the sampling inaccurate. At this time, we can consider to loosen the bottle cap properly.
5 column incubator. There are chromatographic columns in the column incubator. In fact, the separation takes place on the chromatographic column. There is not much to say about the maintenance and use points of the column incubator itself. As for the chromatographic column, it is a long time to talk, so we should open another special topic.
In my opinion, the column incubator is actually the most substantial part in the whole liquid chromatography system. As long as the joint is screwed properly, it seems that it will not go wrong in ten thousand years. The only thing to note is that Agilent's column incubator has two temperature control zones, with 3ul on the left and 6ul on the right, indicating that the volumes in the two temperature zones are different. Generally, we need to use 3 Ul to reduce the delay and diffusion of the instrument.
There are many kinds of detectors, and each has its own characteristics. It's hard to say. So we'd better look for the most commonly used ultraviolet (VWD) / diode array (DAD) detector here. These two kinds of detectors are both UV detectors. Although the optical path design is different, the principle is the same.
six point one A very important part of the detection is the light source, commonly known as the lamp. The lamp is a consumable, just like a bulb at home, it will be broken to a certain extent. However, the lamp in the detector may not be on like the lamp bulb at home. Because the lamp in the detector requires the emission energy. Once the energy is attenuated to a certain extent, the baseline noise will change Large, sensitivity reduction and other issues affecting the use of some columns. Usually, the life of the lamp is 1000 or 2000 hours according to the model. When the time limit is reached, we should pay special attention to the energy status of the lamp. We can judge by the test of "lamp energy test" in the instrument maintenance software. The test results will evaluate the energy of three wavelength bands of low, medium and high respectively. Once the result of a certain wavelength segment shows failure, it means To change the light, usually the first failure is the low wavelength.
six point two Another important part of the detector is the detector, also known as the flow cell. Most of the time, the most concerned problem is that the detection cell is blocked. Because the detection cell is not very pressure resistant, once it is blocked, it is likely to cause damage. Fortunately, the detection cell is not easy to be blocked, because almost all the particulate impurities will be blocked by the chromatographic column Most of the things that block the detector are not from the sample, which is likely to be "produced" later. For example, the salt containing mobile phase remains in the detection cell, resulting in salting out, or acetonitrile polymerizing in the detection cell, or some children's shoes are not careful and accidentally press down the waste liquid pipeline... So let's ask for more happiness
6.3 as for the detection cell, there will be another small problem, that is, where the bubbles come from, which may be the dissolved gas in the mobile phase, or it may be caused by the drying of the detection cell when the instrument has not been used for a long time. This is reflected in the performance of the instrument, which is that the baseline noise is obviously increased, burr appears, etc. in this case, the system pressure can be observed first If the pressure is stable and normal, it can be judged that the fluctuation is not caused by the pump, then it may be caused by the bubble in the detection cell. There is no good way to deal with this bubble, but there is a small way to prevent it, that is, use a small section of peek tube with fine inner diameter to hold a little pressure at the outlet of the detector Large degree of prevention of bubbles, but pay attention not to be too long, about 3-5 cm long enough.
Finally, there is waste liquid. In fact, the waste liquid itself has nothing to say. As long as you pay attention to safety, you must use a safe solvent bottle and a secondary container. My laboratory has encountered a painful lesson. The waste liquid overflows the bottle and corrodes a floor!
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