Common trouble blocking treatment of high performance liquid chromatography

The phenomenon of "blocking" in high-performance liquid chromatography is that the column pressure is abnormally increased, and the direct cause is that the flow path is not smooth. The main location of the blockage is at the front end of the column. The main reason is that there are impurities in the mobile phase. The main source of impurities is bacteria. One of the reasons for "blocking" is bacterial contamination when preparing the mobile phase. Even some solid particulate impurities can be removed from the filter head at the forefront of the liquid phase flow system. What is really causing problems is the bacteria in the water. The newly prepared pure water will grow in the room for a few days, and these bacteria, although invisible to the naked eye, are enough to block the voids of the column packing particles, causing the column to be quickly scrapped. This is the cause of bacterial contamination during the preparation of the mobile phase. The solution to this problem is simple: to ensure the reliability of the water.

After the phenomenon of "blocking" occurs, it is necessary to find out the cause, mainly what position has been "blocked". Note that in most cases, there will only be a blockage in the entire system. The method of checking the plugging is to open the segment from the tail to the reverse direction, and carefully observe the pressure value. If the pressure of a certain component (except the column) is large and different when it is installed and removed, the change judgment can be developed.

There are several common problems:

(1) The model selected when replacing parts is incorrect, the interface is not very matched, and deformation occurs when tightening, which makes the pipeline blocked.

(2) The sample treatment liquid is not cleaned, and the blockage between the six-way valve and the column will be poor for a long time.

(3) When using the manual six-way valve, some people may not be able to rotate due to small hand strength, thus causing a dead block in the flow path, and the pressure rises rapidly beyond the warning value.

(4) When using the metal pipe to make the waste pipe, it should be noted that it is best to put some water in the waste bottle first, and put the outlet end of the waste pipe under the liquid surface. If it is located in the liquid phase and the experiment uses a higher concentration of buffered saline solution, it may crystallize at the outlet end and cause blockage during shutdown.

Let’s talk about the “leakage” problem below. There are two types of “leakage”: leaking and leaking.

1. Leakage

The flow path from the mobile phase bottle to the waste bottle is a fully enclosed system with high internal pressure but an external leak. If a component leaks, it is the fault, there are two reasons for leakage:

(1) Improper contact hardware:

When replacing parts such as flow tubes or changing columns, the changed joint interfaces do not match, causing leakage. The PEEK connector is a better solution. It is not only versatile, but also can be leaked by hand screwing. Even if the interface itself is matched, if it is improperly operated, it will leak. Not good, too tight or too loose.

(2) Improper use of the instrument:

If the pump is leaking, the most common cause is the buffering of the salt at the piston position. There are two reasons for the precipitation: First, when the buffer solution is suddenly added with pure methanol, the error is easily avoided. Try not to use pure methanol and pure water. As long as there is a 10% ratio to each other, this problem will not occur. Another reason is that when the buffered saline solution is used as the mobile phase, the methanol water is not rinsed after the end of the experiment, so that the micro-osmotic mobile phase is dried to form crystals. Leakage of the detector is a very troublesome thing, and it is generally a problem with the absorption tank, and the replacement cost is quite high. However, it does not mean that it must be replaced immediately. It can also be seen from the actual experimental results.

2. Air leak

The leakage is leaking from the inside to the outside, and the leak is the inside of the flow path where the external gas enters the liquid chromatograph. In the following, the causes of the bubbles and the corresponding solutions are analyzed on a component-by-part basis in the direction of the flow path:

(1) When the filter head is pumping, there are irregular but continuous small bubbles in the flow tube. At this time, it is considered whether the mobile phase has degassed. If it has been deaerated, it is necessary to pay attention to the contamination of the filter head. This will cause this phenomenon. The treatment method is relatively simple. Unscrew the filter head and soak it in dilute nitric acid. After ultrasonicing for half an hour, wash it and put it back.

(2) Transparent flow tube

Refers to the section of tubing between the filter head and the transfer pump. This part is often not a bit of a bubble, but often the entire tube is full of air and the operator is ignorant, so that the pump has been working for a long time and found that there is not much liquid in the mobile phase bottle. This is why we often say that the liquid chromatograph should be turned on at least once a week. If it is not used for a long time, the liquid in this section will be completely dried, and the air-filled pipeline and the liquid-filled pipeline will not be distinguished by careful observation. This situation is dangerous for the pump because the pump is designed to deliver liquid rather than gas. The internal liquid acts as an oil for the piston. If there is some buffer salt remaining on the piston rod, it is easy. Pulling, causing irreversible effects. The solution is to use external force to fill the pipeline with liquid, as follows:

1. Find the joint of the flow pipe into the transfer pump;

2. Unscrew it;

3. Align the flat cut of the tubing with the tip of a clean ear wash;

4. Suction liquid, see the liquid level rise from the mobile phase bottle, and stop the action when it is about 5 cm away from the ear wash ball;

5. Quickly screw the connector back onto the transfer pump.

6. Turn on the machine, open the drain valve, and start the transfer pump;

7. When the solution flowing out of the drain pipe has no air bubbles, close the drain valve and the instrument works normally.

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