How to prolong the service life of liquid chromatography col

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1、 Preparation before use
1. Before use, carefully read the instruction manual of chromatographic column, understand the types of chromatographic column, and select appropriate chromatographic column.
In the selection of chromatographic column, the polarity of the sample, the number of compounds and the structural characteristics should be fully considered. According to the properties of the compounds, the suitable chromatographic column and analytical conditions were selected. Different types of chromatographic column use different mobile phase, the use of the wrong mobile phase will reduce the column efficiency and damage the column. If the analysis of polar polysaccharide components, hydrophilic reversed phase fillers should be used. For the chromatographic column used for the first time, the chromatographic column should be flushed and activated at low flow rate according to the manufacturer's instructions. After activation, the covalent bond strength of chromatographic packing is enhanced, the column efficiency is improved and the service life is prolonged.
2. Sample preparation and pretreatment
Our experience is that the cleaner the sample is purified, the longer the column life is. The components of many samples, especially biological samples, are very complex, which can cause great damage to the chromatographic column. Therefore, the sample should be pretreated during sample preparation, including the selection of preparation solvent, sample filtration, etc.
2.1 selection of sample preparation solvent
The solubility of sample, the solubility of mobile phase and the applicability of chromatographic packing should be considered. This kind of solvent should have a large solubility for the sample and be soluble with the mobile phase. The elution intensity should be lower than the initial mobile phase in the mobile phase or gradient elution, so as not to affect the sample separation. At present, it is forbidden to use DMSO, tetrahydrofuran and chloroform to dissolve samples in many chiral chromatographic columns. These solvents will destroy the structure of stationary phase and shorten the service life of chromatographic column. In addition, the sample preparation solvent should also be suitable for other components of the chromatographic system, such as high-pressure pump, injector, etc.
2.2 sample preparation solvent filtration
Before sample injection, the sample solution should be filtered. For example, 0.22 μ m microporous membrane should be used to remove insoluble particles, so as to avoid blocking the column filter and packed bed. If the conditions permit, it is better to filter the injection solution by solid phase extraction column with the same packing as the chromatographic column, which can reduce the dead adsorbed substances or macromolecular samples easily blocked on the chromatographic column. For example, the small polar oils in biological samples are easy to precipitate and adsorb in C18 reversed-phase chromatographic column, resulting in decreased column efficiency and increased column pressure. After filtration with SPE column, the dead adsorbed components attached to the column can be effectively reduced, and the column will not be polluted and its service life can be guaranteed.
2.3 others, such as solution concentration, injection volume, etc
Some properties of analytes can also affect the service life of chromatographic column. Strong acid, strong basic substances and protein biomacromolecules can interact with the stationary phase packing, or form irreversible adsorption layer, change the surface characteristics of the packing, change the performance of chromatographic column, and finally lead to separation failure. In addition, the separation performance and service life of chromatographic column will be affected by excessive sample injection and overload.
2、 Maintenance during use
1. Use of mobile phase and selection of analytical methods
The purity of mobile phase, the choice of solvent and the use of appropriate analytical methods are closely related to the performance and life of chromatographic column.
1.1 selection of mobile phase
The selected flow is compatible with the chromatographic column and the sample to be analyzed, that is, the sample, sample solution and mobile phase are mutually soluble. The mobile phase can dissolve the sample and avoid precipitation; at the same time, it is required that the mobile phase does not react with the sample and can not dissolve or react with the chromatographic column.
The mobile phase of chromatographic grade should be selected for chromatographic analysis. Generally, analytical pure solvents contain trace impurities, such as polyethylene glycol in organic solvents, inorganic iron ions (Fe +) and so on. It is better to use the reagent with chromatographic purity or higher purity to minimize the damage caused by impurities in the solvent.
1.2 mobile phase filtration
The mobile phase was prepared with chromatographic pure reagent. Before use, the mobile phase should be filtered by 0.45 μ m or smaller pore size filter membrane and ultrasonic degassing treatment to reduce dust, microorganism and other impurities blocking the chromatographic column, especially the water-soluble mobile phase, which is easy to cause microbial growth and block the chromatographic column. The mobile phase should be prepared and used immediately, and the storage time should not exceed 2 days.
1.3 selection of pH of mobile phase and buffer salt
The mobile phase with extreme pH will destroy the covalent bond in the packing and "dissolve" the silica gel, resulting in the loss of the stationary phase, thus reducing the column efficiency and shortening the service life. Generally, the pH of the stationary phase based on silica gel should be in the range of 2.5 ~ 7. In the environment of pH > 7 or pH < 2 for a long time, the silica gel will gradually dissolve or the surface bonding functional groups will gradually lose. If the mobile phase with high or low pH must be used, the suitable chromatographic packing should be selected.
1.4 control of flow rate
At present, the flow rate of UPLC with particle size of 1.8 μ m is 0.3 ~ 0.5ml/min. The flow rate of HPLC with particle size of 5 μ m is less than 1.5ml/min. The flow rate of semi preparative column with particle size of 10 μ m is controlled at 3ml / min. If the flow rate is too high and the pressure is increased, the packing will collapse and collapse.
2. Operation of chromatographic instruments
When the analytical instrument is started up every time, the pump starts too fast, the flow rate and column pressure rise instantaneously, the column bed is impacted, resulting in disorder, resulting in voids, affecting the service life of the chromatographic column. Therefore, the flow rate and column pressure should be increased gradually at the beginning of operation experiment.
3. Use of protective column
"Protective column" is a short type chromatographic column with the same packing as the used liquid chromatographic column, which can effectively block the macromolecules and insoluble particles that are easy to damage the chromatographic column, and filter the substances that are easy to settle on the chromatographic column to produce life and death adsorption, so as to prolong the service life of the chromatographic column.
4. Control of column temperature
The temperature tolerance of different types of column is different. Generally, the chromatographic column temperature is maintained between 10 ~ 40 ℃, which can give full play to the performance of chromatographic column. Beyond the temperature range of chromatographic column, especially higher than the range of column temperature, the adsorption of chemical substances in the mobile phase will be increased, and the structure of stationary phase of chromatographic column will be changed; in addition, the column bed may collapse, change the peak shape, reduce the column efficiency and cause irreversible damage.
3、 Cleaning and preservation after use
After the column is used for a period of time, there will always be some impurities accumulated in the column. The substances with weak retention value can be washed out from the chromatographic column quickly without interference; the impurities with medium retention strength can be washed out slowly, but they will cause certain interference to the analysis; strong retention impurities usually gather in the column head or chromatographic column, which are difficult to be eluted, and may even be in phase with the filler A new pseudo stationary phase was formed and the separation performance of the column was changed. It is usually characterized by high column pressure, uneven baseline, double peaks and low separation performance. After cleaning these contaminated chromatographic columns, some or even most of the separation capacity can be restored. Therefore, careful and regular cleaning after use can not only prolong the service life of chromatographic column, save resources, but also greatly reduce the cost of analysis. Taking the silica gel matrix chromatographic column as an example, the cleaning and regeneration of common chromatographic column are briefly described.
1. Cleaning and regeneration of chromatographic column
The chromatographic column should be washed with strong mobile phase before and after use. Usually, when using silica gel, alumina and polar bonding chromatography column, after each use, long time rinse with dichloromethane or hexane and other solvents can be used for a long time. The bonded silica gel column, ion exchange column and gel column can be washed with a high proportion of water (methanol water mixed solvent) and then washed with 100% methanol. In addition, the reversed-phase washing with low flow rate can effectively remove the impurities blocked on the column head or sieve plate, as well as the strong adsorbed substances accumulated in the column head. Some chromatographic columns were used in turn after many methods failed to deal with the pollution, which not only reduced the column pressure drop, but also restored the column efficiency. For example, the use time of the chromatographic column was prolonged.
If the above conventional cleaning method can not remove the pollutants, it is necessary to use stronger eluent, for example, the washing sequence of reverse phase materials is: 100% methanol → 100% acetonitrile → acetonitrile ∶ isopropanol (75 ∶ 25, V / V) → 100% isopropanol. Or a lower concentration of dilute acid or alkali can be used to remove pollutants that cannot be eluted by organic solvents. For example, 0.05mol/l sulfuric acid and mobile phase solution can achieve good results; or 1% ammonium hydroxide or 50% dimethylformamide aqueous solution can have good cleaning effect on the pollutants accumulated on the column head.
If the mobile phase contains buffer solution (usually salt solution), the column should be washed with water instead of buffer solution and mixed with organic phase (20 times the volume of column) and then washed with 100% organic solvent. If 100% organic solvent is used to wash directly, the buffer solution will precipitate and the column quality will be damaged. Similarly, if acid and alkali solutions are added into the mobile phase, the column volume should be washed 20 times with high proportion of water (water: methanol 10:90) according to the above method to prevent the dissolution of silica gel matrix filler caused by strong acid and alkali solution.
The contamination of reversed-phase chromatographic columns by proteins has become a common problem, especially in the separation of biological samples such as untreated animal tissues. In general, pure organic solvents such as acetonitrile or methanol can not clean chromatographic column effectively, so some special cleaning methods are needed. First try to wash with high proportion of strong polar solvent mobile phase, such as acetonitrile: isopropanol (1:2, V / V); or use 0.1% trifluoroacetic acid aqueous solution or 0.1% acetic acid aqueous solution. In addition, 1% sodium dodecyl sulfate (SDS) and gradient washing with 5% - 95% acetonitrile / water (containing 0.1% TFA) can also be used to remove protein pollutants.
If the chromatographic column can not achieve the desired effect after cleaning under the above conditions, it is necessary to take the stationary phase out of the column, clean and regenerate, and then refill. The specific operation is as follows: after the stationary phase is removed from the chromatographic column, the fine broken particles are removed by flotation with methanol; then ultrasonic cleaning is performed with dimethylformamide, acetone and methanol; finally, the stationary phase is dried and the column is filled again. The performance of the column treated by this method can be significantly improved.
2. Preservation of chromatographic column
The chromatographic column should be stored in 100% organic solvent as much as possible. The chromatographic column cannot be stored in water or solvent with high water content, which will cause microorganism breeding and affect the column life. If the reversed-phase chromatographic column is not used for a long time, it is better to use 90% ~ 95% organic solvent mixed with aqueous solution for preservation, so as to prevent the chromatographic column from drying up and shortening the service life caused by faults.
In addition, the chromatographic column should be handled with care to avoid collapse and fault of chromatographic column packing caused by violent collision and shorten its service life.
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