"Block" & "Leakage" in HPLC System

One of the most common failures of liquid chromatographs is "block" and "leakage". These two points are discussed separately below. The mobile phase is methanol as an example, and the column is C18 as an example.

The phenomenon of "block" is the abnormal increase of column pressure, and the direct cause is the poor flow path. The main location of the blockage is at the front of the column, and the main reason is the impurities in the mobile phase, and the main source of impurities is bacteria.

1. "Block"

Bacterial contamination during the preparation of the mobile phase

First of all, we have to realize that general domestic methanol actually does not need additional filtration treatment, and it is no problem to use directly. Even if there are some solid particulate impurities, they can be removed from the filter head at the very front of the liquid phase flow path system, and what really causes problems is the bacteria in the water.

Newly prepared pure water left indoors for a few days will grow bacteria, and these bacteria, although not visible to the naked eye, are sufficient to clog 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 it is very simple, is to ensure the reliability of the water.

In principle, as long as you buy pure water of acceptable quality, you can do without filtration. The recommended filtration is for the salt-containing mobile phase, which also needs to be filtered again after mixing with the organic phase.

Bacterial contamination when using the mobile phase

This means that the mobile phase does not grow bacteria at the beginning, but it becomes contaminated with bacteria when it is used. This is mainly caused by a bad usage habit when using multiplex liquid chromatography.

To take the simplest example, there are two ways to use a 50% methanol-water mobile phase. One way is to mix it together before starting the machine, and the other way is to put pure methanol in flow path A and pure water inflow path B. In terms of pure experimental results, the latter has obvious advantages: firstly, it is simple and does not require the experimenter to make another calculation to mix the ratios, and secondly, the ratios are accurate and can give experimental results with excellent retention time repeatability.

However, it has a fatal flaw, that is, pure water in the mobile phase bottle for a few days will grow bacteria (in many cases not only with pure water as the mobile phase, but with a buffered salt solution, itself a high-quality fertilizer, bacteria grow more rapidly), once there is bacteria column is broken very quickly. So this way requires the operator to use newly prepared pure water for each experiment, and moreover requires that the aqueous phase be changed after each experiment and replaced by methanol to rinse clean, which in practice many people are not aware of, that, they are aware of but there is always one or two omissions in multiple uses. Still, often these one or two are enough to have a fatal impact. Because the blockage of the liquid chromatography column is irreversible.

Improper operation

Common problems include the following.

(1) When replacing the parts, the wrong type is selected and the interface is not well matched, resulting in deformation when tightening and blocking the pipeline.
(2) The sample processing fluid is not clean, which will form a blockage between the six-way valve and the column for a long time.
(3) In the use of a manual six-way valve, some people may not turn in place due to the small hand strength, which causes the flow path to form a dead plug, and the pressure rises rapidly above the alarm value.

The reasons for "plugging" have been told a lot, now introducing the method of checking the plug.

After the phenomenon of "plugging" occurs, you need to find out the cause, mainly what is the location of the "plug". Note that in most cases, there is only one place where the whole system is blocked.

The way to check the blockage is to disassemble the system from the end to the front in the reverse direction, and carefully observe the pressure value, if a certain part (except the column) is installed and removed when the pressure is very different, you can develop a change judgment. As for the blockage of the column, it can be judged by whether the pressure of changing the same size column is consistent.

2. "Leakage"

The flow path of the liquid chromatograph from the mobile phase bottle to the waste bottle is a completely closed system with high internal pressure, but no external leakage. If a component is leaking, that is where the fault lies.

Fluid leakage

(1) Improper contact with the hardware

When replacing parts such as flow path tubes or changing columns, the connector interface of the replacement does not match, causing fluid leakage. Be aware that many of the column joints from different companies are different, and even the same company in different periods of production of liquid phase column joints are very different. Of course, the choice of PEEK joints is a better solution, not only is it versatile, but it can be guaranteed not to leak by hand twisting.

Even if the interface itself is a match, if the operation is not appropriate will also leak, one improper is a poor grasp of strength, screwing too tight or too loose; another improper is a fatal error: slip wire, which is often not too strong hands, screws rarely screwed workers make mistakes, slip wire consequences are not only leakage of liquid so simple, often resulting in the scrapping of important parts. To solve this problem can only rely on the basic skills to experiment, that is, screwing.

(2) Improper use of instruments

If the transfer pump leakage, the most common reason is the piston position in the buffer salt precipitation caused. There are two reasons for the precipitation, one is the use of buffer salt solution suddenly added pure methanol and precipitation, this error is easy to avoid, which is to try not to use pure methanol and pure water. As long as there is a 10% ratio between each other, this problem will not occur. The other reason is that when using the buffered salt solution (regardless of the amount of methanol content) as the mobile phase, the experiment is not changed after the end of the methanol-water rinse so that the mobile phase of micro-osmosis dry formation of crystals caused.

Air leakage

Liquid leakage is from the inside to the outside, while gas leakage is caused by the gas from the outside entering the inside of the flow path of the liquid chromatograph and forming bubbles. The following is a component-by-component analysis of the causes of air bubbles and the corresponding solutions according to the direction of the flow path.

(1) Filter head

When pumping liquid, there are irregular but continuous small bubbles in the flow path tube, when considering whether the mobile phase has degassed (need to be especially reminded that even with the vacuum degasser is also to first ultrasonic degassing, at least to reduce the working pressure of the degasser and improve efficiency), if it has degassed, it is important to note that the pollution of the filter head can also cause this phenomenon. Treatment is relatively simple, unscrew the filter head soaked in dilute nitric acid (need special instructions: only the glass filter head can be soaked in dilute nitric acid, the metal filter head can not be soaked in dilute nitric acid), ultrasound for half an hour, wash and put back.

(2) Transparent flow path tube

Refers to the section of the pipeline between the filter head and the transfer pump. This part is often not a little bubble, but often the whole tube is full of air and the operator is unaware of it so the transfer pump work for half a day to find the liquid in the mobile phase bottle is not less. This is also the reason why we often say that the liquid chromatograph should be turned on at least once a week (we must have the concept of "micro-permeation" to do the liquid phase). If not used for a long time, the liquid in this section of the pipeline will be completely dry, and the pipeline filled with air and the pipeline filled with liquid can not be distinguished unless you look closely.

This situation is very dangerous for the pump because the pump is designed to transport liquid rather than gas, the internal liquid for the piston to play the role of oil, if the piston rod still has some buffer salt, it is very easy to strain, causing irreversible effects.

For this situation, to highlight the "prevention-oriented" such as liquid chromatography personnel to be relatively fixed and stable, work with an appropriate mix of resources, the instrument is not used for a long time to save 10-20% methanol, regular replacement flushing on. If not used for a long time at least 2 hours a week to flush the mobile phase. It is important to develop good working habits.

What if this problem inadvertently occurs? My suggestion is to use external force to fill the line with liquid. Specifically as follows.

1、 Find the connector of the flow line pipe into the transfer pump.
2、 Unscrew it.
3、 Use the tip of a clean earwash ball to align with the flat cut of the line.
4、 Suck the liquid and watch the liquid level rise from the mobile phase bottle to about 5 cm from the earwash ball to stop the action.
5、 Quickly screw the connector back into the transfer pump (this process may have a little mobile phase leakage, which is normal).
6、 Turn on the machine, open the drain valve, and start the transfer pump.
7、When there are no bubbles in the solution flowing out of the drainage tube, close the drainage valve again and the instrument works normally.

(3) transfer pump and column

These parts in the air bubbles are generally not afraid, just flush out.

(4) Detector

It should be said that the entire flow path as long as there is a bubble will be strongly reflected in the signal on the detector, the detector inside the bubble can generally be flushed away, but there are also difficult to flush out the residual bubble situation.

If there are residual bubbles in the detector, there will be a characteristic and obvious expression, that is, in the baseline will occasionally appear at intervals straight up and down the signal is huge signal peaks. At this time to see whether the ordinary flow can be flushed away, if not, the only way is to remove the column, the detector directly connected to the outlet of the transfer pump, increase the flow rate several times flushing, then certainly can flush away the bubbles.