About the HPLC Column Working Process, Use Notes and Clean Methods

1. High performance liquid chromatography column

The high performance liquid chromatography column system consists of several parts such as reservoir, pump, sampler, chromatographic column, detector, recorder, and so on. The mobile phase in the reservoir is pumped into the system by the high-pressure pump, and the sample solution enters the mobile phase through the sampler and is loaded into the chromatographic column (stationary phase) by the mobile phase.

Since the components in the sample solution have different distribution coefficients, when moving relative to each other in the two phases, each component will have a faster-moving speed after repeated adsorption-desorption distribution processes. The difference is separated into individual components and flowed out from the column in turn.

2. How the HPLC column filter the mobile phase and sample?

The mobile phase is usually made of water or a buffer solution mixed with an organic solvent. In principle, all mobile phases passing through the chromatographic column should be filtered, but in actual operation, it should be different according to the specific situation.

When the organic solvent is chromatographically pure water, quartz sub-boiling water, or other ultra-pure water, when the quality of the water and the organic solvent can be ensured and the quality of the organic solvent is not contaminated, filtration is of no more practical significance. When the mobile phase contains buffer salts, filtration is necessary. When the buffer solution is left for a period of time (depending on the ambient temperature, it generally does not exceed 48 hours in summer and generally does not exceed one week in winter). It should be re-filtered before use.

The prepared sample test solution must be filtered through a 0.45μm (0.22μm is better) pore filter membrane before being injected into the flow path system. Pay attention to distinguish between the water system and the oil system. The two cannot be mixed to prevent the filter membrane from dissolving and causing pollution.

The container with the mobile phase and the online filter in the chromatographic system should be cleaned and replaced regularly to prevent the filter from failing to filter due to overload.

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3. What we should pay attention to when using the HPLC column?

When you get a new HPLC column, you must read its instructions first, then measure the efficiency of the column, check the efficiency of the column regularly, keep the chromatogram obtained on the new column, record the conditions, and regularly check the band broadening of the instrument.

If applied to online monitoring, online physical and chemical protection should be provided to the chromatographic column, such as installing online filters, self-installed packing HPLC guard columns, and pre-installed guard columns. The function of the online filter is to prevent particles from accumulating at the head of the column. The advantage is that it basically does not affect the column efficiency. It is commonly used when high column efficiency is required.

The frequently replaced consumables are the filter element and the gasket to protect the column. The function is to prevent the column from being chemically contaminated. The disadvantage is that most guard columns affect the column efficiency of the chromatographic column, especially when using micro-columns, the guard columns with ordinary self-packed packing are commonly used. Some new guard columns are characterized by high quality, high efficiency packing, increased analytical column efficiency, large load capacity for dirty samples, and can extend the life of the guard column.

The connector can be tightened by hand, tools not required. It is time saving can use a variety of different fillers, which is mainly suitable for dirty, complex sample backgrounds, such as biochemical samples, environmental samples, and food, etc., or the situations where operators do not want to do too much garden phase extraction, and to reduce the efficiency of the column, etc.

4. Why the HPLC column fails?

4.1 Poorly packed column
For the poorly packed chromatographic columns, the compression of the packed bed usually causes the column head to collapse, after a short time of application, resulting in a sudden drop in column efficiency. The initial evaluation indicators of the chromatographic column, such as the number of plates and the asymmetry factor, often cannot well characterize the stability of the chromatographic column bed. This sentiment can only be found in practical applications.

4.2 Pressure factor
Sudden pressure fluctuations, mechanical impacts, or sudden temperature changes during the use of high performance liquid chromatography columns will affect the column bed, resulting in poor peak shape and reduced column efficiency. During the sampling process, too slow rotation of the injection valve can cause pressure fluctuations, and cracks in the chromatographic column bed, and the same problem occurs during column switching. Using well-packed chromatographic columns and operating at lower column pressures can greatly reduce column damage caused by pressure fluctuations.

4.3 Sieve plate clogged
Chromatographic column inlet frit clogging is one of the common problems of high performance liquid chromatographic columns, which can cause problems such as increased column pressure, tailing of chromatographic peaks, and decreased the number of plates. Generally, the particulate impurities in the analysis sample may block the entrance of the chromatographic column.

Therefore, the analysis needs to be filtered or centrifuged first. The milky or turbid sample should be treated with a 0.25 m filter membrane, or a syringe filter can be used. The abrasion of the injector and pump seals will also bring in particles. Using an in-line filter of 0.25 µm or 0.45 µm between the injection valve and the chromatographic column can usually avoid such problems.

4.4 Strong retention of sample components
Strongly adsorbed sample components are adsorbed on the column head packing, which can seriously affect the life of the liquid high performance liquid chromatography column. Complex samples such as extracts of biological tissues or body fluids, natural products, etc., can easily cause contamination of the stigma. Relatively pure samples generally do not have this problem. In chromatographic column applications, severe tailing or splitting of chromatographic peaks is often a manifestation of the adsorption of strongly retained contaminants at the head of the column.

5. How to clean the HPLC column?

General reversed-phase chromatography, buffer salts are often used. White salt is more harmful to the chromatographic column. If the salt gets stuck in the column, the column is useless. So it needs to be washed away. Please remember it needs to be washed away every time. If there is no salt in the mobile phase of your column, there is no need to rinse.

Washing method: The reversed-phase column is stored in pure methanol or pure acetonitrile, but the salt is insoluble in the pure organic phase. Therefore, methanol water or acetonitrile water is used in the intermediate transition.

What exactly is used depends on your mobile phase. For example, we can use the mobile phase of phosphate buffer-acetonitrile (70:30), so when washing, use 10%-15% acetonitrile water, wash 1.0ml/min, at least 30min. If you continue to use it the next day, you don’t need to seal it with pure methanol or pure acetonitrile. If it is not used the next day, use a pure organic phase to seal the column and flush it for 30 minutes at the same flow rate of 1.0ml/min. Besides, it does not mean that it will be broken if it is not cleaned. It will shorten the life service.