Maintenance tips for liquid chromatography

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Liquid chromatograph components: mobile phase solvent bottle → high-pressure pump → injector → column → detector → waste liquid, we follow this order to explain one by one how to do maintenance?

I. Maintenance of mobile phase solvent bottles

The solvent bottle is the starting point of the mobile phase and usually holds either the aqueous solution or the organic phase solution.

1. Aqueous phase solution, for aqueous phase solution, the primary problem is to prevent contamination. For solvent bottles we have to do a very important job is to change the mobile phase diligently, often change often new.

2.Organic phase solution, for organic phase solution, you can not worry about the problem of bacterial reproduction. However, the organic phase is easy to polymerise, especially acetonitrile is very easy to polymerise under suitable light conditions, and some flocculent polymerisation precipitates will appear in the bottle. To prevent polymerisation, acetonitrile should be contained in a brown solvent bottle, protected from direct sunlight, and the remaining solution at the bottom of the bottle should be discarded when replacing the acetonitrile.

3. Cleaning and filtration, the solvent bottle in the filter head, its role is to prevent the solution bottle particles in the impurities into the instrument flow system, it is usually divided into the material of glass sintered quartz and stainless steel, if inadvertently clogged will cause the mobile phase suction fluid is not smooth, so it must be cleaned, the glass is usually made of dilute nitric acid soak, and stainless steel can be directly cleaned ultrasonic.

II. Maintenance of high-pressure pumps

The pump is the core of liquid chromatography, the pump will transport the mobile phase from the solvent bottle to the liquid phase flow path system, and to maintain the stability of flow and pressure under high pressure. Normal state of the high-pressure pump is the basis for accurate analysis of liquid chromatography, so we must pay attention to the maintenance of the pump on weekdays.

1.Pump pressure fluctuations, in many cases, the pump problem is reflected in the pressure, pressure fluctuations is a common type of problem. Usually we can be solved by re-cleaning the flow path and degassing the mobile phase again.

2.Filter white head maintenance, in the pump maintenance there is a common work is to replace the filter white head on the cleaning valve, usually judge the standard is pure water to 5mL/min flow rate when cleaning, if the pressure is more than 1MPa is considered to be replaced.

III. Maintenance of the injector

There are two main types of feeders, manual and automatic, and although they work in different modes, the main points of use are basically the same.

1.To prevent cross-contamination, autosampler is a common problem of cross-contamination, cross-contamination is a direct cause, the sample remains on the inner and outer surfaces of the injection needle, and with the next injection into the chromatographic system. To solve the cross-contamination, mainly by cleaning.

2.Fine operation, the operation of the manual injector points are more or less the same, you should use the liquid chromatography LC special injection needle, injection needle should be inserted into the end, not use the needle to stay in the injector, before and after the use of timely cleaning.

IV. Maintenance of chromatographic column

The chromatographic column is the key to the separation of compounds. A well-maintained column has a high plate count and a smooth baseline.

1. Avoid sharp changes in pressure and temperature and any mechanical vibration. Sudden changes in temperature or dropping the column from a high place will affect the filling condition of the column; a sudden increase or decrease in column pressure will also impulse the packing in the column, so the adjustment of the flow rate should be carried out slowly, and the rotation of the valve should not be too slow when the valve is feeding the samples (as mentioned before).

2. The composition of the solvent should be changed gradually, especially in reversed-phase chromatography, which should not be changed directly from an organic solvent to all water and vice versa.

3.Column backflush, NanoSpectrum's small molecule separation liquid chromatography columns ChromCore series and EcoPak series can be backflushed; NanoSpectrum's chiral columns UniChiral series can be backflushed, and the backflush can remove the impurities left in the head of the column. However, NanoSpectrum's biomolecule liquid chromatography columns BioCore series columns should not be backflushed, or backflush will rapidly reduce the column efficiency.

4.choose to use the appropriate mobile phase (especially pH), in order to avoid the destruction of the stationary phase. Sometimes you can connect a pre-column in front of the injector, when the analytical column is bonded silica gel, the pre-column is silica gel, which can make the mobile phase ‘saturated’ by silica gel before entering the analytical column, to avoid the dissolution of silica matrix in the analytical column.

5. Avoid injecting samples with complex matrices, especially biological samples, directly into the column. It is necessary to pre-treat the samples or connect a guard column between the injector and the column. A guard column is usually a short column filled with a similar stationary phase. The guard column can and should be replaced frequently.

6. Frequently flush the column with strong solvents to remove impurities retained in the column. When cleaning, the replacement of the mobile phase in the flow path system should be a gradual transition with a miscible solvent, and the volume of each mobile phase should be about 20 times the volume of the column, i.e., 50-75 ml is required for routine analysis.

7. When preserving the column, the column should be filled with acetonitrile or methanol, and the column joints should be tightened to prevent the solvent from evaporating and drying. It is forbidden to leave the buffer solution in the column to stand overnight or longer.

8. If the pressure rises during the use of the column, one possibility is that the sintered filter is blocked, and the filter should be replaced or taken out for cleaning; another possibility is that the macromolecules enter into the column, so that the column head is contaminated; if the column efficiency is lowered or the peaks are deformed, the column head may be collapsed, and the dead volume is enlarged.

V. Detector maintenance

1.The light source part

Detector is a very important part of the light source, the light source on the emission of energy requirements, once the energy attenuation to a certain extent, there will be a series of impact on the use of baseline noise, sensitivity reduction and other issues, so the light source is a consumable. Usually the life of the UV lamp is 2000h, when the time limit is reached, we should pay special attention to the energy status of the lamp, you can through the instrument maintenance software comes with the ‘lamp energy test’ function to determine the test results will be evaluated separately in the low, medium and high three wavelength bands of energy, once a certain wavelength band test results show that the lamp has failed, it means that it needs to be replaced. Once the test result of a wavelength band shows failure, it means that the lamp needs to be replaced.

2.Detection cell

Another important part of the detector is the detection cell, also called the flow cell. A common concern is that the detector cell gets clogged because it is usually not very pressure resistant, so once it is clogged it is likely to cause damage. In fact, the detector cell is usually less prone to clogging because almost all particulate impurities are blocked by the column, so anything that clogs the detector is not coming from the sample, but is likely to be ‘generated’ later, for example, if the salt-containing mobile phase is left in the detector cell and causes salt to precipitate out.

VI. Disposal of waste liquids

Most of the waste liquid of liquid chromatograph LC contains organic solvents, which will cause some harm to human body and pollution to the environment, and it is strictly prohibited to dump it at will, and it should be disposed of in time. At present, most laboratories in China send the waste liquid to special waste liquid treatment station for centralised harmless treatment.